(en)The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diabetes mellitus and/or conditions related to diabetes. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

1.ApplicationNumber: US-47295304-A
1.PublishNumber: US-2005176061-A1
2.Date Publish: 20050811
3.Inventor: ROSEN CRAIG A.
RUBEN STEVEN M.

4.Inventor Harmonized: ROSEN CRAIG A(US)
RUBEN STEVEN M(US)

5.Country: US
6.Claims:
(en)The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diabetes mellitus and/or conditions related to diabetes. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

7.Description:
(en)FIELD OF INVENTION
The present invention relates to human secreted proteins/polypeptides, and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus and conditions related thereto. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
BACKGROUND OF THE INVENTION
Over the past few decades, an increasing percentage of the population has become diabetic. Diabetes mellitus is categorized into two types: Type I, known as Insulin-Dependent Diabetes Mellitus (IDDM), or Type II, known as Non-Insulin-Dependent Diabetes Mellitus (NIDDM). IDDM is an autoinmmune disorder in which the insulin-secreting pancreatic beta cells of the islets of Langerhans are destroyed. In these individuals, recombinant insulin therapy is employed to maintain glucose homeostasis and normal energy metabolism. NIDDM, on the other hand, is a polygenic disorder with no one gene responsible for the progression of the disease.
In NIDDM, insulin resistance eventually leads to the abolishment of insulin secretion resulting in insulin deficiency. Insulin resistance, at least in part, ensues from a block at the level of glucose uptake and phosphorylation in humans. Diabetics demonstrate a decrease in expression in adipose tissue of insulin-receptor substrate 1 (“IRS1”) (Carvalho et al., FASEB J 13(15):2173-8 (1999)), glucose transporter 4 (“GLUT4”) (Garvey et al., Diabetes 41(4):465-75 (1992)), and the novel abundant protein M gene transcript 1 (“apM1”) (Statnick et al., Int J Exp Diabetes 1(2): 81-8 (2000)), as well as other as of yet unidentified factors. Insulin deficiency in NIDDM leads to failure of normal pancreatic beta-cell function and eventually to pancreatic-beta cell death.
Insulin affects fat, muscle, and liver. Insulin is the major regulator of energy metabolism. Malfunctioning of any step(s) in insulin secretion and/or action can lead to many disorders, including for example the dysregulation of oxygen utilization, adipogenesis, glycogenesis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, and maintenance of the basal metabolic rate. This malfunctioning results in diseases and/or disorders that include, but are not limited to, hyperinsulinemia, insulin resistance, insulin deficiency, hyperglycemia, hyperlipidemia, hyperketonemia, and diabetes.
Numerous debilitating diabetes-related secondary effects include, but are not limited to, obesity, forms of blindness (cataracts and diabetic retinopathy), limb amputations, kidney failure, fatty liver, coronary artery disease, and neuropathy.
Some of the current drugs used to treat insulin resistance and/or diabetes (e.g., insulin secratogogues—sulfonylurea, insulin sensitizers—thiazolidenediones and metformin, and alpha-glucosidase and lipase inhibitors) are inadequate due to the dosage amounts and frequency with which they have to be administered as a result of poor pharmacokinetic properties, the lack of effective control over blood sugar levels, and potential side effects, among other reasons. Diabetes Therapeutic proteins in their native state or when recombinantly produced exhibit a rapid in vivo clearance. Typically, significant amounts of therapeutics are required to be effective during therapy. In addition, small molecules smaller than the 20 kDa range can be readily filtered through the renal tubules (glomerulus) leading to dose-dependent nephrotoxicity. Therefore, there is a need for improvement in treatment (e.g., a need for prolonging the effects of therapeutics of diabetes and/or diabetes related conditions).
SUMMARY OF THE INVENTION
The present invention encompasses human secreted proteinsipolypeptides, and isolated nucleic acid molecules encoding said proteinsipolypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus and conditions related thereto. Antibodies that bind these polypeptides are also encompassed by the present invention; as are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention also encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.


DETAILED DESCRIPTION
Polynucleotides and Polypeptides of the Invention
Description of Table 1A
Table 1A summarizes information concerning certain polypnucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC Deposit No:Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Eipress vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH1OB, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH1OB, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC Deposit No.Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC deposit No.Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC Deposit No.Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC Deposit No.Z.
Description of Table 1B (Comprised of Tables 1B.1 and 1B.2)
Table 1B.1 and Table 1B.2 summarize some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifiers (SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column of Tables 1B. 1 and 1B.2 provide the gene numbers in the application for each clone identifier. The second column of Tables 1B.1 and 1B.2 provide unique clone identifiers, “Clone ID:”, for cDNA clones related to each contig sequence disclosed in Table 1A and/or Table 1B. The third column of Tables 1B.1 and 1B.2 provide unique contig identifiers, “Contig ID:” for each of the contig sequences disclosed in these tables. The fourth column of Tables 1B.1 and 1B.2 provide the sequence identifiers, “SEQ ID NO:X”, for each of the contig sequences disclosed in Table 1A and/or 1B.
Table 1B.1
The fifth column of Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineates the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B.1 as SEQ ID NO:Y (column 6). Column 7 of Table 1B.1 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wis.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1B.1 as “Predicted Epitopes”. In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8 of Table 1B.1 (“Cytologic Band”) provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000. World Wide Web URL: http:/www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in Table 1B.1, column 9 labeled “OMIM Disease Reference(s)”. A key to the OMIM reference identification numbers is provided in Table 5.
Table 1B.2
Column 5 of Table 1B.2, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first code number shown in Table 1B.2 column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested. The second number in column 5 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33 P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
Description of Table 1C
Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
Description of Table 1D
Table 1D: In preferred embodiments, the present invention encompasses a method of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A, Table 1B, and Table 1C, in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate the disease or disorder.
As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to treat the associated disease.
Table 1D provides information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1D also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, and 1C. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. Table 1D describes the use of FMAT technology, inter alia, for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound flurophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for highthroughput screening using flourometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
Table 1D also describes the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).
Description of Table 2
Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, “Clone ID:”, corresponding to a cDNA clone disclosed in Table 1A or Table 1B. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Table 1B and allowing for correlation with the information in Table 1B. The third column provides the sequence identifier, “SEQ ID NO:X”, for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”) as further described below. The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, “Score/Percent Identity”, provides a quality score or the percent identity, of the hit disclosed in columns five and six. Columns 8 and 9, “NT From” and “NT To” respectively, delineate the polynucleotides in “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
Description of Table 3
Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to contig sequences disclosed in Table 1B. The second column provides the sequence identifier, “SEQ ID NO:X”, for contig sequences disclosed in Table 1A and/or Table 1B. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourth column provides a unique integer ‘a’ where ‘a’ is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a unique integer ‘b’ where ‘b’ is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a +14. For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
Description of Table 4
Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B.2, Column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
Description of Table 5
Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.govlomim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8, as determined using the Morbid Map database.
Description of Table 6
Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application. These deposits were made in addition to those described in the Table 1A.
Description of Table 7
Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
The first column shows the first four letters indicating the Library from which each library clone was derived. The second column indicates the catalogued tissue description for the corresponding libraries. The third column indicates the vector containing the corresponding clones. The fourth column shows the ATCC deposit designation for each libray clone as indicated by the deposit information in Table 6.
Definitions
The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or MnRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genornic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof (e.g., the polypeptide delinated in columns fourteen and fifteen of Table 1A); a nucleic acid sequence contained in SEQ ID NO:X (as described in column 5 of Table 1A and/or column 3 of Table 1B) or the complement thereof; a cDNA sequence contained in Clone ID: (as described in column 2 of Table 1A and/or Table 1B and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 (EXON From-To) of Table 1C or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
In the present invention, “SEQ ID NO:X” was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc. (HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1B, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID:). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library. Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID: to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, “HTWE.” The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example “HTWEP07”. As mentioned below, Table 1A and/or Table 1B correlates the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1A, 1B, 6, 7, and 9 to determine the corresponding Clone ID, which library it came from and which ATCC deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 7 and 8 of Table 1A or the complement thereof, the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID: (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1C or the complement thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCI; 0.2M NaH 2 PO 4 ; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
“SEQ ID NO:X” refers to a polynucleotide sequence described in column 5 of Table 1A, while “SEQ ID NO:Y” refers to a polypeptide sequence described in column 10 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 6 of Table 1A. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. The polynucleotide sequences are shown in the sequence listing immediately followed by all of the polypeptide sequences. Thus, a polypeptide sequence corresponding to polynucleotide sequence SEQ ID NO:2 is the first polypeptide sequence shown in the sequence listing. The second polypeptide sequence corresponds to the polynucleotide sequence shown as SEQ ID NO:3, and so on.
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidehains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formnation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
“SEQ ID NO:X” refers to a polynucleotide sequence described, for example, in Tables 1A, Table 1B, or Table 2, while “SEQ ID NO:Y” refers to a polypeptide sequence described in column 11 of Table 1A and or of Table 1B. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1B. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. “Clone ID:” refers to a cDNA clone described in column 2 of Table 1A and/or 1B.
“A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity (e.g. activity useful in treating, preventing and/or ameliorating diabetes mellitus), antigenicity (ability to bind [or compete with a polypeptide for binding] to an anti-polypeptide antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
The polypeptides of the invention can be assayed for functional activity (e.g. biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay secreted polypeptides (including fragments and variants) of the invention for activity using assays as described in the examples section below.
“A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
Tables
Table 1A
Table 1A summarizes information concerning certain polypnucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC Deposit No:Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DHIOB, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC Deposit No.Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC deposit No.Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC Deposit No.Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC Deposit No.Z.
TABLE 1A NT 5′ NT AA First Last ATCC SEQ 5′ NT 3′ NT of First SEQ AA AA First AA Last Deposit ID Total of of 5′ NT AA of ID of of of AA Gene cDNA No: Z and NO: NT Clone Clone of Start Signal NO: Sig Sig Secreted of No. Clone ID Date Vector X Seq. Seq. Seq. Codon Pep Y Pep Pep Portion ORF 1 H6EDM64 203959 Uni-ZAP XR 11 2610 1275 2377 1448 1448 205 1 6 Apr. 26, 1999 2 H6EEU40 203917 Uni-ZAP XR 12 951 1 951 175 175 206 1 27 28 47 Apr. 8, 1999 3 HACAB68 203917 Uni-ZAP XR 13 1300 1 1300 135 135 207 1 26 27 78 Apr. 8, 1999 4 HACBJ56 203979 Uni-ZAP XR 14 888 1 888 250 208 1 9 10 25 Apr. 29, 1999 5 HADMB15 203979 pBluescript 15 330 1 330 238 209 1 11 12 20 Apr. 29, 1999 6 HAGDW20 203917 Uni-ZAP XR 16 1284 1 1284 238 238 210 1 16 17 17 Apr. 8, 1999 7 HAGEQ79 203917 Uni-ZAP XR 17 785 1 785 515 515 211 1 11 Apr. 8, 1999 8 HAJBV67 PTA-181 pCMVSport 18 2536 1 2536 605 605 212 1 19 20 359 Jun. 7, 1999 3.0 9 HAJCH70 203917 pCMVSport 19 2182 1 2182 284 284 213 1 32 33 38 Apr. 8, 1999 3.0 10 HAOAG15 203979 pSport1 20 5143 7 4802 8 214 1 22 23 1167 Apr. 29, 1999 11 HATCD80 203917 Uni-ZAP XR 21 1809 95 1809 296 296 215 1 23 24 37 Apr. 8, 1999 12 HATCI03 203917 Uni-ZAP XR 22 934 1 934 271 271 216 1 17 Apr. 8, 1999 13 HBAGD86 203917 pSport1 23 1713 293 1596 521 521 217 1 18 19 19 Apr. 8, 1999 14 HBHAA05 203917 Uni-ZAP XR 24 690 1 690 110 218 1 16 17 58 Apr. 8, 1999 15 HBHAA81 203959 Uni-ZAP XR 25 1647 1 1647 28 28 219 1 24 25 203 Apr. 26, 1999 16 HBIAA59 203917 Uni-ZAP XR 26 2392 1612 2392 1877 1877 220 1 15 16 136 Apr. 8, 1999 17 HBJAC40 203979 Uni-ZAP XR 27 1767 184 1729 329 221 1 13 Apr. 29, 1999 18 HBJCR46 203917 Uni-ZAP XR 28 3208 2270 3202 589 589 222 1 1 2 733 Apr. 8, 1999 19 HBJDW56 203917 Uni-ZAP XR 29 637 1 637 121 223 1 8 Apr. 8, 1999 20 HBJEL16 203979 Uni-ZAP XR 30 750 1 750 115 115 224 1 24 25 36 Apr. 29, 1999 21 HBJIG20 PTA-181 Uni-ZAP XR 31 637 1 637 321 225 1 16 17 77 Jun. 7, 1999 22 HBJKD16 203979 Uni-ZAP XR 32 1629 1 1629 78 78 226 1 18 19 31 Apr. 29, 1999 23 HBMTY48 203917 Uni-ZAP XR 33 1891 1 1891 660 660 227 1 36 37 94 Apr. 8, 1999 24 HBMUH74 PTA-181 Uni-ZAP XR 34 726 1 726 344 344 228 1 13 14 28 Jun. 7, 1999 25 HBQAB79 203917 Lambda ZAP 35 1331 1 1331 190 190 229 1 11 Apr. 8, 1999 II 26 HBXCX15 203917 ZAP Express 36 1219 1 1219 1148 230 1 1 Apr. 8, 1999 27 HCDCY76 203917 Uni-ZAP XR 37 1392 628 1392 860 231 1 17 18 35 Apr. 8, 1999 28 HCEDR26 203917 Uni-ZAP XR 38 1419 1 1419 177 177 232 1 26 27 55 Apr. 8, 1999 29 HCEEU18 203917 Uni-ZAP XR 39 1229 1 1229 209 209 233 1 30 31 43 Apr. 8, 1999 30 HCEGX05 203917 Uni-ZAP XR 40 1305 1 1305 237 237 234 1 15 Apr. 8, 1999 31 HCFLN88 203917 pSport1 41 1434 1 1434 101 101 235 1 16 17 25 Apr. 8, 1999 32 HCHAB84 203979 pSport1 42 1359 62 1359 304 236 1 23 24 147 Apr. 29, 1999 33 HCMSX51 203917 Uni-ZAP XR 43 2253 334 2190 539 237 1 31 32 80 Apr. 8, 1999 34 HCNCO11 203917 Lambda ZAP 44 746 1 746 101 101 238 1 14 Apr. 8, 1999 II 35 HCNSD29 PTA-181 pBluescript 45 1728 1031 1633 1145 1145 239 1 19 20 31 Jun. 7, 1999 36 HCQBH72 203917 Lambda ZAP 46 1796 776 1796 31 31 240 1 25 26 47 Apr. 8, 1999 II 37 HCQCC96 203979 Lambda ZAP 47 2166 632 1455 782 782 241 1 20 21 45 Apr. 29, 1999 II 38 HCQCJ56 203917 Lambda ZAP 48 1287 1 1287 728 242 1 1 Apr. 8, 1999 II 39 HCUDD64 203917 ZAP Express 49 402 150 389 256 256 243 1 35 36 49 Apr. 8, 1999 40 HCWAE64 203917 ZAP Express 50 471 1 471 410 244 1 5 Apr. 8, 1999 41 HDPDJ58 203960 pCMVSport 51 1997 1 1997 279 279 245 1 20 Apr. 26, 1999 3.0 42 HDPFU43 203960 pCMVSport 52 1904 1 1889 220 220 246 1 28 29 52 Apr. 26, 1999 3.0 43 HDPGE24 203960 pCMVSport 53 2625 1 2625 173 173 247 1 11 12 73 Apr. 26, 1999 3.0 44 HDPIU94 203960 pCMVSport 54 2196 21 2196 208 208 248 1 21 22 23 Apr. 26, 1999 3.0 45 HDPIY31 PTA-793 pCMVSport 55 1978 1 1978 268 268 249 1 16 17 35 Sep. 27, 1999 3.0 46 HDPOC24 203960 pCMVSport 56 1777 302 1725 418 418 250 1 23 24 133 Apr. 26, 1999 3.0 47 HDPPD93 203960 pCMVSport 57 701 1 701 28 28 251 1 12 Apr. 26, 1999 3.0 48 HDPPQ30 203960 pCMVSport 58 1063 1 1063 220 220 252 1 22 23 38 Apr. 26, 1999 3.0 49 HDQHM36 PTA-181 pCMVSport 59 1547 1 1547 129 129 253 1 18 19 48 Jun. 7, 1999 3.0 50 HDTFX18 203960 pCMVSport 60 678 1 678 164 164 254 1 16 17 20 Apr. 26, 1999 2.0 51 HE2CM39 203960 Uni-ZAP XR 61 566 1 566 10 255 1 13 Apr. 26, 1999 52 HE2HC60 203960 Uni-ZAP XR 62 1569 236 1569 273 273 256 1 16 17 39 Apr. 26, 1999 53 HE2PO93 203960 Uni-ZAP XR 63 1323 638 1323 770 770 257 1 27 28 42 Apr. 26, 1999 54 HE6FU11 203979 Uni-ZAP XR 64 2000 1 1994 145 145 258 1 26 27 226 Apr. 29, 1999 55 HE6FV29 203960 Uni-ZAP XR 65 1526 1 1526 210 210 259 1 18 19 33 Apr. 26, 1999 56 HE8TY46 PTA- Uni-ZAP XR 66 2204 1400 2204 1413 1413 260 1 18 19 187 1838 May 9, 2000 57 HE9EA10 203960 Uni-ZAP XR 67 2114 1 2111 212 261 1 28 29 78 Apr. 26, 1999 58 HEBCY54 203960 Uni-ZAP XR 68 1189 1 1189 172 172 262 1 24 25 118 Apr. 26, 1999 59 HEBFR46 203979 Uni-ZAP XR 69 1304 1 1304 200 200 263 1 26 27 29 Apr. 29, 1999 60 HEBGE07 203960 Uni-ZAP XR 70 1867 1 1867 106 106 264 1 25 26 42 Apr. 26, 1999 61 HEGAU15 203960 Uni-ZAP XR 71 1125 1 1125 59 59 265 1 30 31 34 Apr. 26, 1999 62 HETCI16 203979 Uni-ZAP XR 72 2285 73 2285 237 237 266 1 27 28 40 Apr. 29, 1999 63 HFCEI04 203960 Uni-ZAP XR 73 887 1 887 136 267 1 17 18 42 Apr. 26, 1999 64 HFCFE20 203960 Uni-ZAP XR 74 1205 1 1205 216 216 268 1 18 Apr. 26, 1999 65 HFPCZ55 203960 Uni-ZAP XR 75 2735 341 2735 676 676 269 1 24 25 44 Apr. 26, 1999 66 HFPDR62 203960 Uni-ZAP XR 76 2644 1 2644 414 414 270 1 28 29 35 Apr. 26, 1999 67 HFPDS07 203960 Uni-ZAP XR 77 3115 2302 3114 2546 2546 271 1 23 24 25 Apr. 26, 1999 68 HFVHW43 203960 pBluescript 78 1233 1 1233 92 92 272 1 30 31 39 Apr. 26, 1999 69 HFXAV37 203960 Lambda ZAP 79 1520 40 1520 163 273 1 13 14 36 Apr. 26, 1999 II 70 HFXFZ46 203960 Lambda ZAP 80 1378 1 1378 258 258 274 1 6 Apr. 26, 1999 II 71 HGBHP91 203960 Uni-ZAP XR 81 1054 1 1054 50 275 1 14 15 52 Apr. 26, 1999 72 HHEAK45 203960 pCMVSport 82 2014 87 1935 813 276 1 3 Apr. 26, 1999 3.0 73 HHEOW19 PTA-793 pCMVSport 83 1589 1 1589 183 183 277 1 18 19 64 Sep. 27, 1999 3.0 74 HHFFS40 203960 Uni-ZAP XR 84 1816 1 1816 37 37 278 1 18 19 47 Apr. 26, 1999 75 HHGCS78 203960 Lambda ZAP 85 575 46 575 290 290 279 1 17 18 24 Apr. 26, 1999 II 76 HHPSA85 203960 pBluescript 86 1147 1 1147 157 157 280 1 28 29 38 Apr. 26, 1999 77 HHSBI06 203959 Uni-ZAP XR 87 1049 27 803 690 281 1 5 Apr. 26, 1999 78 HHSBI65 203917 Uni-ZAP XR 88 1444 1 1431 62 62 282 1 17 18 55 Apr. 8, 1999 79 HHSGL28 203960 Uni-ZAP XR 89 1093 1 1093 453 453 283 1 6 Apr. 26, 1999 80 HISAT67 203959 pSport1 90 2154 1061 2142 1239 1239 284 1 35 36 56 Apr. 26, 1999 81 HJBCU75 203957 pBluescript 91 1009 1 1009 61 61 285 1 5 Apr. 26, 1999 SK- 82 HJMAA03 203957 pCMVSport 92 665 1 665 527 286 1 9 Apr. 26, 1999 3.0 83 HKABU43 203959 pCMVSport 93 1919 581 1919 755 755 287 1 20 21 281 Apr. 26, 1999 2.0 84 HKACI79 PTA-181 pCMVSport 94 1181 1 1181 207 207 288 1 14 15 50 Jun. 7, 1999 2.0 85 HKIXC44 203957 pBluescript 95 788 343 750 572 572 289 1 26 27 36 Apr. 26, 1999 86 HKTAB41 203957 Uni-ZAP XR 96 797 1 797 172 172 290 1 10 Apr. 26, 1999 87 HLDQU79 203959 pCMVSport 97 1488 1 1488 99 99 291 1 23 24 348 Apr. 26, 1999 3.0 87 HLDQU79 203959 pCMVSport 191 3179 163 1474 75 75 385 1 29 30 348 Apr. 26, 1999 3.0 88 HLHAP05 203957 Uni-ZAP XR 98 1842 12 1842 45 45 292 1 14 Apr. 26, 1999 89 HLHCS23 203957 Uni-ZAP XR 99 1427 1 1427 25 25 293 1 24 25 34 Apr. 26, 1999 90 HLICE88 203957 pCMVSport 1 100 840 401 824 708 294 1 2 Apr. 26, 1999 91 HLMGP50 203957 Lambda ZAP 101 1063 1 1063 214 214 295 1 10 Apr. 26, 1999 II 92 HLMMX62 203957 Lambda ZAP 102 268 1 268 185 185 296 1 17 18 28 Apr. 26, 1999 II 93 HLQCX36 203957 Lambda ZAP 103 1243 1 1243 89 89 297 1 16 17 52 Apr. 26, 1999 II 94 HLWAF06 203957 pCMVSport 104 2564 1 2564 192 192 298 1 18 19 30 Apr. 26, 1999 3.0 95 HLWDB73 203957 pCMVSport 105 1611 1 1611 95 95 299 1 27 28 35 Apr. 26, 1999 3.0 96 HLYDF73 203957 pSport1 106 626 1 626 363 300 1 11 12 23 Apr. 26, 1999 97 HLYGB19 203959 pSport1 107 2967 1527 2966 1863 1863 301 1 14 Apr. 26, 1999 98 HLYGY91 203957 pSport1 108 640 1 640 211 211 302 1 20 21 42 Apr. 26, 1999 99 HMDAB29 203957 Uni-ZAP XR 109 1190 1 1190 97 97 303 1 17 18 26 Apr. 26, 1999 100 HMDAD44 203957 Uni-ZAP XR 110 1204 1 1204 135 135 304 1 8 Apr. 26, 1999 101 HMEDI90 203957 Lambda ZAP 111 2276 362 2219 622 305 1 12 13 17 Apr. 26, 1999 II 102 HMIBF07 203957 Uni-ZAP XR 112 1738 1 1738 229 229 306 1 6 Apr. 26, 1999 103 HMICP65 203979 Uni-ZAP XR 113 2048 1 2048 249 249 307 1 16 17 30 Apr. 29, 1999 104 HMSHC86 203957 Uni-ZAP XR 114 1724 1 1724 37 37 308 1 20 21 93 Apr. 26, 1999 105 HMUAN45 203918 pCMVSport 115 2709 1 2709 239 239 309 1 25 26 227 Apr. 8, 1999 3.0 106 HMVBC31 203957 pSport1 116 2556 1327 2546 1437 1437 310 1 32 33 40 Apr. 26, 1999 107 HMWBL03 203957 Uni-ZAP XR 117 2596 80 2596 137 137 311 1 1 2 397 Apr. 26, 1999 108 HNFGR08 203957 Uni-ZAP XR 118 1436 1 1436 314 312 1 17 18 43 Apr. 26, 1999 109 HNGAK51 203957 Uni-ZAP XR 119 915 1 915 248 248 313 1 23 24 32 Apr. 26, 1999 110 HNGDX18 PTA-181 Uni-ZAP XR 120 1425 1 1425 237 237 314 1 30 31 243 Jun. 7, 1999 110 HNGDX18 PTA-181 Uni-ZAP XR 192 1411 1 1411 231 231 386 1 18 19 132 Jun. 7, 1999 111 HNGGP65 203957 Uni-ZAP XR 121 541 1 541 181 181 315 1 15 16 68 Apr. 26, 1999 112 HNGIV64 203957 Uni-ZAP XR 122 1047 1 1047 221 316 1 8 Apr. 26, 1999 113 HNGMW45 203959 Uni-ZAP XR 123 1530 1 1530 452 452 317 1 26 27 43 Apr. 26, 1999 114 HNGPJ25 203959 Uni-ZAP XR 124 853 129 853 544 544 318 1 20 21 25 Apr. 26, 1999 115 HNHEN82 203918 Uni-ZAP XR 125 573 1 573 78 319 1 13 14 17 Apr. 8, 1999 116 HNHFE71 203959 Uni-ZAP XR 126 903 1 903 598 598 320 1 21 Apr. 26, 1999 117 HNHKV56 203959 Uni-ZAP XR 127 1653 1 1653 294 294 321 1 31 32 66 Apr. 26, 1999 118 HOACG07 203959 Uni-ZAP XR 128 1298 772 1249 778 778 322 1 21 22 123 Apr. 26, 1999 119 HODBB70 203918 Uni-ZAP XR 129 604 1 604 173 323 1 7 8 27 Apr. 8, 1999 120 HOEBK60 203959 Uni-ZAP XR 130 2218 1449 2216 1714 1714 324 1 39 40 43 Apr. 26, 1999 121 HOFAA78 203959 pSport1 131 1356 1 1356 48 325 1 25 26 71 Apr. 26, 1999 122 HOFNB74 203959 pCMVSport 132 1036 1 1036 138 138 326 1 24 25 39 Apr. 26, 1999 2.0 123 HORBS82 203959 Uni-ZAP XR 133 1125 1 1125 21 327 1 19 20 39 Apr. 26, 1999 124 HOSDO75 PTA-181 Uni-ZAP XR 134 902 1 902 88 88 328 1 28 Jun. 7, 1999 125 HOUDR07 203959 Uni-ZAP XR 135 1911 1 1911 170 170 329 1 27 28 65 Apr. 26, 1999 126 HPEAD23 203959 Uni-ZAP XR 136 582 1 582 188 188 330 1 13 14 93 Apr. 26, 1999 127 HPFCI36 PTA-181 Uni-ZAP XR 137 879 1 879 94 94 331 1 17 18 19 Jun. 7, 1999 128 HPFDI37 PTA-181 Uni-ZAP XR 138 352 1 352 38 38 332 1 17 Jun. 7, 1999 129 HPIAA80 203959 Uni-ZAP XR 139 919 312 919 314 333 1 13 14 37 Apr. 26, 1999 130 HPJCW58 203918 Uni-ZAP XR 140 1165 1 1165 177 177 334 1 19 20 28 Apr. 8, 1999 131 HPRBH85 203959 Uni-ZAP XR 141 1673 558 1648 684 684 335 1 18 19 134 Apr. 26, 1999 132 HPRCA64 203959 Uni-ZAP XR 142 2805 1701 2757 1810 1810 336 1 22 23 39 Apr. 26, 1999 133 HPRCD35 PTA-181 Uni-ZAP XR 143 709 1 689 265 337 1 16 17 35 Jun. 7, 1999 134 HPTRM02 203959 pBluescript 144 1760 658 1680 885 885 338 1 16 17 80 Apr. 26, 1999 135 HRAAD30 PTA-181 pCMVSport 145 1496 1 1496 220 339 1 19 20 25 Jun. 7, 1999 3.0 136 HRADF49 PTA-181 pCMVSport 146 2704 1 2684 169 169 340 1 39 40 253 Jun. 7, 1999 3.0 137 HRDDQ39 203959 Uni-ZAP XR 147 776 1 773 215 341 1 17 18 46 Apr. 26, 1999 138 HRDEX93 203959 Uni-ZAP XR 148 1681 711 1638 649 649 342 1 20 21 72 Apr. 26, 1999 139 HROEA08 PTA-181 Uni-ZAP XR 149 281 1 281 50 50 343 1 25 26 33 Jun. 7, 1999 140 HRTAP63 203979 pBluescript 150 2576 891 2576 959 959 344 1 28 29 42 Apr. 29, 1999 SK- 141 HSAVW42 203959 Uni-ZAP XR 151 595 1 595 129 129 345 1 16 17 22 Apr. 26, 1999 142 HSAYC41 203959 Uni-ZAP XR 152 214 1 214 106 106 346 1 16 17 36 Apr. 26, 1999 143 HSLHX15 203959 Uni-ZAP XR 153 655 1 655 485 485 347 1 20 21 41 Apr. 26, 1999 144 HSNBM34 203959 Uni-ZAP XR 154 2186 1391 1765 1508 348 1 14 15 62 Apr. 26, 1999 145 HSSEF77 203959 Uni-ZAP XR 155 1053 1 1053 184 349 1 25 26 60 Apr. 26, 1999 146 HT4FV41 PTA-181 Uni-ZAP XR 156 1764 1 1764 39 350 1 16 17 137 Jun. 7, 1999 147 HT5FX79 203959 Uni-ZAP XR 157 682 59 682 228 351 1 17 18 50 Apr. 26, 1999 148 HT5GR59 203959 Uni-ZAP XR 158 1743 1 1743 135 135 352 1 23 24 31 Apr. 26, 1999 149 HTAEI78 203918 Uni-ZAP XR 159 1623 1 1623 632 632 353 1 4 Apr. 8, 1999 150 HTEAG62 203959 Uni-ZAP XR 160 2221 57 2221 1017 1017 354 1 20 21 22 Apr. 26, 1999 151 HTEDJ28 203959 Uni-ZAP XR 161 1247 1 1247 287 355 1 18 19 45 Apr. 26, 1999 152 HTEDS12 203918 Uni-ZAP XR 162 1587 1 1587 260 260 356 1 24 25 36 Apr. 8, 1999 153 HTEHA56 203959 Uni-ZAP XR 163 1402 529 1400 280 357 1 5 6 88 Apr. 26, 1999 154 HTEJD29 203959 Uni-ZAP XR 164 1324 1 1324 101 101 358 1 23 Apr. 26, 1999 155 HTENR63 PTA-792 Uni-ZAP XR 165 1591 1 1591 132 132 359 1 20 21 56 Sep. 27, 1999 156 HTGGM44 203959 Uni-ZAP XR 166 3016 1 2761 179 179 360 1 18 19 84 Apr. 26, 1999 157 HTHBZ06 203959 Uni-ZAP XR 167 623 193 619 318 318 361 1 1 Apr. 26, 1999 158 HTLBT80 203959 Uni-ZAP XR 168 2101 817 1881 912 912 362 1 27 28 129 Apr. 26, 1999 159 HTLDU78 203918 Uni-ZAP XR 169 1318 1 1318 219 219 363 1 8 Apr. 8, 1999 160 HTLFA13 203918 Uni-ZAP XR 170 1160 1 1160 209 364 1 8 9 31 Apr. 8, 1999 161 HTLGI89 203959 Uni-ZAP XR 171 2377 1205 2377 1802 1802 365 1 16 17 37 Apr. 26, 1999 162 HTNBK13 203959 pBluescript 172 1160 295 1148 534 534 366 1 16 17 21 Apr. 26, 1999 SK- 163 HTOAM11 203918 Uni-ZAP XR 173 1200 1 1200 89 89 367 1 24 25 34 Apr. 8, 1999 164 HTOHO21 203918 Uni-ZAP XR 174 727 1 727 439 368 1 5 6 63 Apr. 8, 1999 165 HTPCO75 PTA-181 Uni-ZAP XR 175 1467 1 1467 73 369 1 23 24 40 Jun. 7, 1999 166 HTSFJ32 203918 pBluescript 176 1257 517 1257 93 93 370 1 18 Apr. 8, 1999 167 HTTCB60 PTA-181 Uni-ZAP XR 177 1504 1 1504 84 84 371 1 17 18 266 Jun. 7, 1999 168 HTTEE41 203959 Uni-ZAP XR 178 1973 864 1968 1171 372 1 8 Apr. 26, 1999 169 HTWEH94 203918 pSport1 179 1361 1 1361 66 66 373 1 43 44 81 Apr. 8, 1999 170 HTXFA72 PTA-181 Uni-ZAP XR 180 1861 1 1861 192 192 374 1 17 18 29 Jun. 7, 1999 171 HTXKF95 203959 Uni-ZAP XR 181 884 79 875 330 330 375 1 28 29 78 Apr. 26, 1999 172 HUKDF20 203918 Lambda ZAP 182 1105 1 1105 214 214 376 1 20 21 33 Apr. 8, 1999 II 173 HUSCJ14 PTA- Lambda ZAP 183 3342 1 3342 74 74 377 1 30 31 196 1838 II May 9, 2000 174 HUVDJ48 203918 Uni-ZAP XR 184 1827 1 1827 196 196 378 1 5 Apr. 8, 1999 175 HBDAB91 203917 pSport1 185 1007 320 1007 671 671 379 1 19 20 29 Apr. 8, 1999 175 HBDAB91 203917 pSport1 193 687 1 687 351 351 387 1 19 20 29 Apr. 8, 1999 176 HELGG84 203960 Uni-ZAP XR 186 1109 12 1109 147 147 380 1 16 17 22 Apr. 26, 1999 176 HELGG84 203960 Uni-ZAP XR 194 1109 12 1109 147 147 388 1 16 17 22 Apr. 26, 1999 177 HILCA24 203960 pBluescript 187 1982 153 1982 191 191 381 1 29 30 327 Apr. 26, 1999 SK- 177 HILCA24 203960 pBluescript 195 1980 151 1976 189 189 389 1 29 30 327 Apr. 26, 1999 SK- 178 HYABC84 203959 pCMVSport 188 1478 833 1306 1080 1080 382 1 28 29 62 Apr. 26, 1999 3.0 178 HYABC84 203959 pCMVSport 196 1338 768 1238 1015 1015 390 1 28 29 62 Apr. 26, 1999 3.0 179 HMCAZ04 203917 Uni-ZAP XR 189 1301 1 1301 97 97 383 1 20 21 35 Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAP XR 197 1735 407 1671 498 498 391 1 20 21 35 Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAP XR 198 1733 406 1670 106 106 392 1 25 26 450 Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAP XR 199 1733 405 1670 497 497 393 1 20 21 35 Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAP XR 200 1733 405 1670 106 106 394 1 25 26 450 Apr. 8, 1999 180 HE8FD92 203979 Uni-ZAP XR 190 3977 1986 3960 2141 2141 384 1 25 26 43 Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAP XR 201 1995 1 1978 157 157 395 1 25 26 43 Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAP XR 202 4102 2114 4085 2268 2268 396 1 25 26 43 Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAP XR 203 4907 2918 4890 2 397 1 1 2 471 Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAP XR 204 2908 918 2891 1074 1074 398 1 25 26 43 Apr. 29, 1999

Table 1B (Comprised of Tables 1B.1 and 1B.2)

The first column in Table 1B.1 and Table 1B.2 provides the gene number in the application corresponding to the clone identifier. The second column in Table 1B.1 and Table 1B.2 provides a unique “Clone ID:” for the cDNA clone related to each contig sequence disclosed in Table 1B.1 and Table 1B.2. This clone ID references the cDNA clone which contains at least the 5′ most sequence of the assembled contig and at least a portion of SEQ ID NO:X as determined by directly sequencing the referenced clone. The referenced clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. The third column in Table 1B.1 and Table 1B.2 provides a unique “Contig ID” identification for each contig sequence. The fourth column in Table 1B.1 and Table 1B.2 provides the “SEQ ID NO:” identifier for each of the contig polynucleotide sequences disclosed in Table 1B.
Table 1B.1
The fifth column in Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence “SEQ ID NO:X” that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1B.1, column 6, as SEQ ID NO:Y. Where the nucleotide position number “To” is lower than the nucleotide position number “From”, the preferred ORF is the reverse complement of the referenced polynucleotide sequence. The sixth column in Table 1B.1 provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by “ORF (From-To)”. Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. Column 7 in Table 1B.1 lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1B. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
Column 8 in Table 1B.1 provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a “cluster”; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequence(s) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.
A modified version of the computer program BLASTN (Altshul, et al., J. Mol. Biol. 215:403-410 (1990), and Gish, and States, Nat. Genet. 3:266-272) (1993) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the ‘Query’). A sequence from the UniGene database (the ‘Subject’) was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated in Table 1B under the heading “Cytologic Band”. Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIM™ and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000;. If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 9, Table 1B.1, labelled “OEM Disease Reference(s). Table 5 is a key to the OMIM reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
Table 1B.2
Column 5, in Table 1B.2, provides an expression profile and library code:count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1B, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention. The first number in Table 1B.2, column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4. The second number in column 5 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “R” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33 P dCTP, using oligo (dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
TABLE 1B.1 SEQ AA ID SEQ OMIM Gene cDNA Clone Contig NO: ORF ID Cytologic Disease No: ID ID: X (From-To) NO: Y Predicted Epitopes Band Reference(s): 1 H6EDM64 841331 11 1448-1468 205 11q13 102200, 106100, 131100, 131100, 131100, 133780, 147050, 153700, 161015, 164009, 168461, 168461, 168461, 180721, 180840, 191181, 193235, 209901, 232600, 259700, 259770, 600045, 600319, 600528, 601884 2 H6EEU40 757048 12 175-318 206 11q12.1 106100, 147050, 259700, 259770, 600045, 601884 3 HACAB68 584773 13 135-371 207 Leu-6 to Ser-12. 4 HACBJ56 847112 14 250-327 208 Arg-14 to Ile-24. 5 HADMB15 847116 15 238-300 209 6 HAGDW20 637489 16 238-291 210 7 HAGEQ79 828055 17 515-550 211 8 HAJBV67 866415 18 605-1684 212 Arg-24 to Trp-44, 10q23.33 157640, 174900, 236730, 600512 Leu-87 to Ser-93, Arg-119 to Trp-125, Pro-206 to Lys-211, Glu-280 to Trp-286. 9 HAJCH70 827275 19 284-400 213 10 HAOAG15 852204 20 8-3511 214 Asp-26 to Leu-32, 1q21 104770, 107670, 110700, 135940, 145001, Trp-62 to Asp-72, 146790, 152445, 152445, 159001, 174000, Gln-95 to His-101, 179755, 182860, 182860, 182860, 191315, Thr-158 to Thr-164, 230800, 230800, 266200, 600897, 601105, Phe-222 to Glu-227, 601412, 601652, 602491 Asn-234 to Thr-245, Gly-256 to Glu-266, Gly-277 to Glu-283, Arg-310 to Ser-317, Ser-327 to Phe-333, Ser-360 to Ser-366. 11 HATCD80 826098 21 296-409 215 12 HATCI03 580805 22 271-324 216 Lys-8 to Trp-13. 13 HBAGD86 838799 23 521-580 217 14 HBHAA05 603174 24 110-286 218 15 HBHAA81 846465 25 28-639 219 3p21.32 116806, 168468, 182280, 600163 16 HBIAA59 806303 26 1877-2287 220 Arg-34 to Ser-39, Pro-45 to Ile-55. 17 HBJAC40 841235 27 329-370 221 16p13.3 141750, 141800, 141800, 141800, 141800, 141850, 141850, 141850, 141850, 141850, 156850, 186580, 191092, 600140, 600273, 601313, 601785 18 HBJCR46 815649 28 589-2787 222 Met-1 to Ala-8, 15q12 103581, 146150, 182279, 203200, 203200, Phe-42 to Asp-57, 227220, 601623, 601800, 601889, 602117 Tyr-105 to Thr-110, His-121 to Cys-127, Asp-154 to Lys-181, Arg-186 to Pro-210, Ala-233 to Asp-252, Ser-296 to Ser-306, Pro-313 to Ser-320, Gln-331 to Gly-346, Ser-355 to Thr-360, Cys-386 to Phe-395, Ser-400 to Glu-425, Thr-440 to Thr-446, Pro-449 to Cys-466, Glu-470 to Thr-509, Ser-512 to Asp-533, Ala-544 to Arg-550, Arg-562 to Glu-571, Lys-587 to Thr-594, Asp-713 to Glu-733. 19 HBJDW56 520401 29 121-147 223 20 HBJEL16 847030 30 115-225 224 1q23.1- 107300, 131210, 136132, 145001, 173610, q23.2 249270, 601652 21 HBJIG20 866159 31 321-554 225 22 HBJKD16 853358 32 78-173 226 2p14 203800 23 HBMTY48 637521 33 660-944 227 Glu-35 to Pro-50. 24 HBMUH74 866160 34 344-430 228 12p11.22 112410, 135700, 168470, 200990 25 HBQAB79 810542 35 190-225 229 4q31.1 189800, 600983 26 HBXCX15 637542 36 72-77 230 27 HCDCY76 837972 37 860-967 231 Pro-20 to Phe-25. 11q14-q21 133780, 203100, 203100, 245000 28 HCEDR26 771144 38 177-344 232 29 HCEEU18 688041 39 209-340 233 30 HCEGX05 827060 40 237-284 234 Pro-4 to Phe-11. 20q13 600281, 600281 31 HCFLN88 610000 41 101-178 235 7q11.23 116860, 129900, 233700, 600079 32 HCHAB84 834326 42 304-747 236 Asn-47 to Leu-52, Tyr-134 to Trp-143. 33 HCMSX51 788643 43 539-781 237 Leu-57 to Glu-66. 8p21 152760, 180100, 185430, 602629 34 HCNCO11 775086 44 101-145 238 35 HCNSD29 862314 45 1145-1240 239 2q23.3 36 HCQBH72 637548 46 31-174 240 37 HCQCC96 845066 47 782-919 241 38 HCQCJ56 832157 48 728-733 242 39 HCUDD64 835082 49 256-402 243 Met-1 to Ser-6, 19p13.3 108725, 120700, 133171, 136836, 145981, Gln-32 to Asn-39. 147141, 164953, 188070, 600957, 601238, 601846, 602216, 602477 40 HCWAE64 535893 50 410-427 244 41 HDPDJ58 587265 51 279-341 245 42 HDPFU43 790189 52 220-378 246 22q12.1 123620, 188826, 600850, 601669 43 HDPGE24 801947 53 173-394 247 44 HDPIU94 813352 54 208-279 248 8p21.1 138300, 240400, 602629 45 HDPIY31 886159 55 268-375 249 20q13.33 46 HDPOC24 777493 56 418-819 250 Pro-36 to Cys-42, 9q34.12 Pro-44 to Cys-54, Arg-100 to Gly-105. 47 HDPPD93 637588 57 28-66 251 48 HDPPQ30 684292 58 220-336 252 49 HDQHM36 852328 59 129-275 253 50 HDTFX18 801957 60 164-226 254 51 HE2CM39 553651 61 10-51 255 52 HE2HC60 753265 62 273-392 256 Thr-26 to Gln-31. 1q42.2 106150, 106150, 214500, 600996, 601975, 602759 53 HE2PO93 771655 63 770-898 257 3p21.3 116806, 120120, 120120, 120120, 120436, 120436, 120436, 138320, 168468, 182280, 600163 54 HE6FU11 827236 64 145-825 258 55 HE6FV29 588454 65 210-311 259 56 HE8TY46 899528 66 1413-1976 260 11p11.2- 133701, 168500, 171650, 176930, 176930, p11.12 600623, 600811, 600958 57 HE9EA10 827796 67 212-448 261 Arg-6 to Trp-11. 58 HEBCY54 600355 68 172-528 262 Arg-18 to Lys-26, 8p22-p21 148370, 152760, 180100, 185430, 238600, Gly-35 to Ala-42, 238600, 238600, 238600, 600143, 601385, Gln-61 to Gly-67. 602629 59 HEBFR46 847064 69 200-289 263 Met-1 to Thr-6. 60 HEBGE07 798096 70 106-234 264 61 HEGAU15 834379 71 59-163 265 62 HETCI16 844543 72 237-359 266 Met-1 to Trp-9. 63 HFCEI04 692438 73 136-264 267 Asn-21 to Gly-28. 64 HFCFE20 701985 74 216-272 268 10q26 176943, 176943, 176943, 176943, 176943, 258870, 263700, 601969, 601969, 602084 65 HFPCZ55 840840 75 676-810 269 11p15 108985, 186921, 602092 66 HFPDR62 839400 76 414-521 270 67 HFPDS07 821646 77 2546-2623 271 2q32-q34 100690, 100730, 118800, 123660, 135600, 142989, 156232, 157655, 178600, 186860, 201460, 205100, 262000, 278250, 600258, 601277, 601318 68 HFVHW43 570948 78 92-211 272 69 HFXAV37 626595 79 163-273 273 70 HFXFZ46 600361 80 258-278 274 71 HGBHP91 693011 81 50-208 275 72 HHEAK45 765278 82 813-824 276 6p21.33 248611 73 HHEOW19 886174 83 183-377 277 Ala-41 to Pro-57. 1q42 106150, 106150, 145260, 173870, 173870, 600759, 600996, 601744, 601975 74 HHFFS40 824059 84 37-180 278 5p14.1 123000 75 HHGCS78 634605 85 290-364 279 17q11.1 182138, 600881, 601954 76 HHPSA85 658695 86 157-273 280 77 HHSBI06 639097 87 690-707 281 78 HHSBI65 801910 88 62-229 282 Ala-16 to Val-35. 8q24.3 188450, 188450, 188450 79 HHSGL28 801912 89 453-473 283 80 HISAT67 843549 90 1239-1409 284 2p23.3 176830, 176830, 182601, 229800, 602134 81 HJBCU75 638329 91 61-78 285 82 HJMAA03 824062 92 527-556 286 83 HKABU43 838573 93 755-1600 287 Ile-69 to Ala-74, Ala-122 to Ser-129, Thr-160 to Glu-170, Lys-197 to Arg-202. 84 HKACI79 853361 94 207-359 288 Ser-37 to Gly-43. 85 HKIXC44 716213 95 572-682 289 86 HKTAB41 695732 96 172-204 290 87 HLDQU79 740755 97 99-1142 291 Leu-68 to Lys-74, Tyr-109 to Lys-115, Gln-200 to Val-205, Lys-207 to Lys-214, Glu-237 to Ile-244, Ala-271 to Thr-279, Ser-317 to Ser-329, Gln-342 to Gly-348. HLDQU79 837599 191 75-1121 385 88 HLHAP05 638476 98 45-89 292 Gln-4 to Leu-14. 89 HLHCS23 560663 99 25-129 293 90 HLICE88 840321 100 708-716 294 4q28 107250, 134820, 134820, 134820, 134830, 134850, 134850, 181600, 189800, 266300 91 HLMGP50 647603 101 214-246 295 92 HLMMX62 688051 102 185-268 296 Gln-20 to Lys-28. 93 HLQCX36 584786 103 89-247 297 Pro-35 to Ser-40. 94 HLWAF06 658701 104 192-284 298 95 HLWDB73 838453 105 95-202 299 1q32.2 145260, 600759, 601975 96 HLYDF73 566869 106 363-434 300 97 HLYGB19 838083 107 1863-1907 301 2p23.3 176830, 176830, 182601, 229800, 602134 98 HLYGY91 658703 108 211-339 302 99 HMDAB29 584789 109 97-177 303 100 HMDAD44 566854 110 135-161 304 101 HMEDI90 840077 111 622-675 305 Ser-7 to Thr-13. 102 HMIBF07 603528 112 229-249 306 103 HMICP65 847403 113 249-341 307 104 HMSHC86 840402 114 37-318 308 Arg-32 to Gln-37, Arg-68 to Phe-73. 105 HMUAN45 833072 115 239-922 309 Pro-33 to Gly-45, 11q13.5 133780, 266150, 276903, 276903, 276903 Cys-121 to Gly-131, Ala-155 to His-166, Gly-180 to Gln-185. 106 HMVBC31 825598 116 1437-1559 310 Ser-33 to Tyr-39. 1p36.21 120550, 120570, 120575, 153454, 256700 107 HMWBL03 822861 117 137-1327 311 Met-1 to Leu-11, Val-13 to Lys-19, Thr-30 to Asp-39, Thr-49 to Gly-68, Ala-78 to Gly-111, Pro-140 to Thr-163, Ser-169 to Ser-185, Glu-197 to Lys-204, Lys-210 to Asp-215, Glu-220 to Ser-231, Ser-255 to Leu-266, Thr-269 to Asp-288, Cys-300 to Val-309, Phe-331 to Cys-339, Ser-362 to Ile-373. 108 HNFGR08 825417 118 314-445 312 109 HNGAK51 603910 119 248-346 313 110 HNGDX18 1145071 120 237-965 314 Ser-21 to Ser-39, Gln-45 to Gln-61, Cys-124 to Ser-139. HNGDX18 866177 192 231-629 386 Ser-21 to Ser-39, Gln-45 to Gln-61, Cys-124 to Gly-130. 111 HNGGP65 597449 121 181-387 315 112 HNGIV64 561572 122 221-247 316 113 HNGMW45 838613 123 452-583 317 114 HNGPJ25 834942 124 544-621 318 115 HNHEN82 836157 125 78-131 319 116 HNHFE71 834487 126 598-663 320 117 HNHKV56 800877 127 294-494 321 118 HOACG07 792928 128 778-1149 322 Pro-32 to Ser-42, 20p13 192340, 234200 Cys-51 to Gly-83, Gly-87 to Ser-93. 119 HODBB70 520196 129 173-256 323 120 HOEBK60 789396 130 1714-1845 324 Lys-5 to Thr-10, Gln-36 to Gly-43. 121 HOFAA78 836646 131 48-263 325 Trp-1 to Arg-7, 19q13.33 134790, 600040 Pro-65 to Gly-70. 122 HOFNB74 762821 132 138-257 326 Ser-30 to Ser-36. 12q12- 181430, 600194, 600231, 600808, 601284, 12q14.3 601769, 601769, 602116 123 HORBS82 638293 133 21-140 327 Gly-30 to Ser-35. 124 HOSDO75 862049 134 88-174 328 Phe-2 to Ser-8, 11q13.4 133780, 266150 Phe-21 to Ser-26. 125 HOUDR07 745404 135 170-367 329 Pro-27 to Arg-34. 19p13.3 108725, 120700, 133171, 136836, 145981, 147141, 164953, 188070, 600957, 601238, 601846, 602216, 602477 126 HPEAD23 773409 136 188-469 330 Ala-54 to Lys-59. 127 HPFCI36 855966 137 94-153 331 10q23.31 157640, 174900, 236730, 600512 128 HPFDI37 862056 138 38-91 332 22q11.21 123620, 151410, 600850 129 HPIAA80 829972 139 314-427 333 130 HPJCW58 612866 140 177-263 334 Leu-16 to Gly-21. 131 HPRBH85 695752 141 684-1088 335 Glu-121 to Leu-126. 3q21.1 106165, 117700, 117700, 150210, 169600, 180380, 180380, 180380, 203500, 232050, 276902, 600882, 601199, 601199, 601199, 601471, 601682 132 HPRCA64 824074 142 1810-1929 336 2q32 100690, 142989, 156232, 178600, 278250, 600258 133 HPRCD35 853551 143 265-372 337 Asp-16 to Gln-27. 134 HPTRM02 812879 144 885-1127 338 His-48 to Ser-61, 7 Ala-66 to Val-72. 135 HRAAD30 866187 145 220-297 339 2p23.3 176830, 176830, 182601, 229800, 602134 136 HRADF49 866481 146 169-930 340 Pro-85 to Asp-99, 2q36.1 120070, 120131, 120131, 138030, 259900 Arg-163 to Arg-170, Gln-183 to Thr-189, Pro-201 to Ser-209, Ser-216 to Gly-222. 137 HRDDQ39 840405 147 215-355 341 Gly-27 to Pro-35. 138 HRDEX93 816046 148 649-867 342 1p34 130500, 133200, 138140, 168360, 171760, 171760, 176100, 176100, 178300, 230000, 255800 139 HROEA08 866190 149 50-151 343 2q31.1 100690, 142989, 156232, 178600, 600258 140 HRTAP63 780698 150 959-1087 344 Asn-2 to Trp-13. 2p23.3 176830, 176830, 182601, 229800, 602134 141 HSAVW42 637660 151 129-197 345 3p22.3 182280, 227646, 261510, 600163, 601154 142 HSAYC41 688057 152 106-213 346 Lys-23 to Lys-36. 11q12.1 106100, 147050, 259700, 259770, 600045, 601884 143 HSLHX15 777861 153 485-610 347 Arg-28 to Arg-35. 144 HSNBM34 635131 154 1508-1696 348 Ala-17 to Thr-26, 17p13-p11 100710, 138190, 254210, 271900, 600179, Gly-49 to Gln-62. 600977, 601202, 601777 145 HSSEF77 658725 155 184-366 349 Arg-22 to Lys-27, 2p12 147200, 178640, 216900 Leu-30 to Asn-39. 146 HT4FV41 853400 156 39-452 350 Ala-15 to Gln-22, 19p13.3 108725, 120700, 133171, 136836, 145981, Gly-36 to Gly-41, 147141, 164953, 188070, 600957, 601238, Arg-47 to Pro-63, 601846, 602216, 602477 Pro-85 to His-98. 147 HT5FX79 794169 157 228-380 351 Glu-1 to Ser-9, Ser-23 to Ser-35. 148 HT5GR59 801930 158 135-230 352 8p21.3 602629 149 HTAEI78 637684 159 632-646 353 8p21.1 138300, 240400, 602629 150 HTEAG62 812332 160 1017-1085 354 151 HTEDJ28 762845 161 287-424 355 Thr-34 to Leu-41. 8p22-q11 148370, 180100, 238600, 238600, 238600, 238600, 600143, 601385, 602629 152 HTEDS12 838621 162 260-370 356 Ala-29 to Thr-36. 17q21.33 109270, 109270, 109270, 109270, 109270, 120150, 120150, 120150, 148065, 148080, 154275, 171190, 185800, 221820, 249000, 253250, 600119, 600119, 600525, 601844 153 HTEHA56 806461 163 280-546 357 His-10 to Ala-20. 19q13.43 154 HTEJD29 695798 164 101-172 358 155 HTENR63 877952 165 132-302 359 Pro-22 to Lys-28. 4q24 157147, 248510 156 HTGGM44 842856 166 179-433 360 3q23 106165, 110100, 117700, 117700, 150210, 169600, 180380, 180380, 180380, 203500, 276902, 601199, 601199, 601199, 601682 157 HTHBZ06 832477 167 318-323 361 12q24.31 181405 158 HTLBT80 840045 168 912-1301 362 Ser-107 to Ser-116. 20q11.21- 102700, 102700, 602025 q13.11 159 HTLDU78 637702 169 219-245 363 160 HTLFA13 535937 170 209-304 364 161 HTLGI89 835069 171 1802-1915 365 162 HTNBK13 831967 172 534-599 366 22q12 123620, 133450, 133450, 600850, 601669 163 HTOAM11 664508 173 89-193 367 164 HTOHO21 732808 174 439-630 368 Ile-35 to Cys-42. 15q24-q25 118485, 231680, 272800, 272800, 272800, 276700, 602685 165 HTPCO75 853645 175 73-195 369 166 HTSFJ32 637720 176 93-149 370 Leu-12 to Cys-18. 17p13.1 191170, 191170 167 HTTCB60 853401 177 84-884 371 Ser-83 to Asp-88, Val-166 to Gly-181, Pro-193 to Ala-199, Glu-235 to Gln-250. 168 HTTEE41 840950 178 1171-1197 372 12q15 181430, 600698, 600698, 600698, 600698, 600808, 602116 169 HTWEH94 561680 179 66-311 373 170 HTXFA72 853410 180 192-281 374 171 HTXKF95 834438 181 330-566 375 Met-1 to Pro-6, Gly-73 to Thr-78. 172 HUKDF20 566823 182 214-315 376 173 HUSCJ14 894699 183 74-661 377 Phe-166 to Arg-174, Ser-191 to Tyr-196. 174 HUVDJ48 564853 184 196-213 378 175 HBDAB91 864374 185 671-760 379 Lys-21 to Gln-29. HBDAB91 789532 193 351-440 387 Lys-21 to Gln-29. 176 HELGG84 851137 186 147-215 380 HELGG84 674456 194 147-215 388 177 HILCA24 869856 187 191-1174 381 Gln-52 to Arg-57, 5p15.2 123000, 602568 Glu-74 to Leu-84, Val-104 to Asp-110, Gly-157 to Gly-163, Asn-185 to Ser-195, Arg-245 to Asp-250, Pro-302 to Pro-310, Thr-316 to Tyr-322. HILCA24 782450 195 189-1172 389 Gln-52 to Arg-57, Glu-74 to Leu-84, Val-104 to Asp-110, Gly-157 to Gly-163, Asn-185 to Ser-195, Arg-245 to Asp-250, Pro-302 to Pro-310, Thr-316 to Tyr-322. 178 HYABC84 865064 188 1080-1268 382 Pro-3 to Ala-8. 20q11.22 HYABC84 789854 196 1015-1203 390 Pro-3 to Ala-8. 179 HMCAZ04 668249 189 97-204 383 Met-1 to Pro-7. 15q15 177070, 177070, 182500, 218000, 227220, 243500, 600839, 601800 HMCAZ04 887445 197 498-605 391 Met-1 to Pro-7. HMCAZ04 867910 198 106-1455 392 Pro-76 to Phe-81, Gln-95 to Pro-102, Leu-121 to Ile-128, Asp-131 to Ser-137, Thr-174 to Trp-179, Arg-217 to Lys-224, Val-257 to Asn-262, Asn-277 to Glu-283, His-325 to Asn-330, Lys-365 to Thr-377, Pro-404 to Arg-411. HMCAZ04 858210 199 497-604 393 Met-1 to Pro-7. HMCAZ04 839783 200 106-1455 394 Pro-76 to Phe-81, Gln-95 to Pro-102, Leu-121 to Ile-128, Asp-131 to Ser-137, Thr-174 to Trp-179, Arg-217 to Lys-224, Val-257 to Asn-262, Asn-277 to Glu-283, His-325 to Asn-330, Lys-365 to Thr-377, Pro-404 to Arg-411. 180 HE8FD92 901142 190 2141-2272 384 HE8FD92 888274 201 157-288 395 HE8FD92 869847 202 2268-2399 396 HE8FD92 856544 203 2-1414 397 Asp-11 to Tyr-16. HE8FD92 843825 204 1074-1205 398










TABLE 1B.2








SEQ ID
Tissue Distribution Library Code: Count

Gene No:
cDNA Clone ID
Contig ID:
NO: X
(see Table 4 for Library Codes)











1
H6EDM64
841331
11
AR277:22, AR060:22, AR055:21, AR283:18, AR282:18, AR104:16, AR185:16, AR089:16, AR299:16, AR219:14, AR240:14,





AR316:13, AR096:12, AR218:12, AR039:11, AR300:11, AR313:11 H0333:6, H0556:5, H0255:5, H0618:4, L0783:4, S0358:3,





H0549:3, S0222:3, H0318:3, H0052:3, H0553:3, H0135:3, L0769:3, L3905:3, H0547:3, H0521:3, H0555:3, H0423:3, H0716:2,





H0341:2, H0402:2, H0592:2, H0253:2, 50474:2, H0620:2, H0181:2, H0617:2, H0059:2, L0761:2, L0764:2, L0809:2, L5622:2,





H0520:2, H0682:2, S0330:2, H0436:2, L0751:2, L0747:2, L0750:2, L0755:2, S0436:2, L0596:2, L0601:2, H0624:1, H0686:1,





H0295:1, T0049:1, H0657:1, H0656:1, H0484:1, H0483:1, S0356:1, S0442:1, S0354:1, S0360:1, S0410:1, H0729:1, H0742:1,





S0045:1, S0476:1, H0619:1, S0300:1, L0717:1, S0220:1, H0370:1, H0455:1, H0586:1, H0587:1, H0559:1, L0623:1, T0082:1,





H0581:1, H0183:1, H0205:1, H0327:1, H0050:1, H0687:1, H0615:1, T0006:1, H0424:1, H0213:1, H0606:1, H0166:1, S0366:1,





H0090:1, H0087:1, H0264:1, H0488:1, H0413:1, H0100:1, H0625:1, H0561:1, H0130:1, H0633:1, H0647:1, S0426:1, H0529:1,





L0371:1, L0796:1, L0637:1, L5566:1, L0648:1, L0364:1, L0649:1, L0774:1, L0375:1, L0378:1, L0659:1, L0636:1, L5623:1,





L4501:1, L0663:1, H0693:1, H0593:1, S0126:1, H0522:1, S0027:1, S0028:1, L0740:1, L0780:1, L0758:1, H0445:1, S0011:1,





H0136:1, S0196:1 and H0352:1.

2
H6EEU40
757048
12
AR277:63, AR283:52, AR219:45, AR282:44, AR104:43, AR218:41, AR316:39, AR089:37, AR313:37, AR299:35, AR055:33,





AR240:33, AR096:30, AR185:29, AR300:28, AR039:27, AR060:27 L0741:8, H0677:7, L0439:6, H0052:5, H0494:5, L0747:5,





S0007:4, H0543:4, H0009:3, L0771:3, L0775:3, L0663:3, L0665:3, L0438:3, H0547:3, H0521:3, H0436:3, L0742:3, L0748:3,





L0751:3, S0436:3, H0556:2, H0255:2, S0420:2, S0358:2, S0046:2, L0717:2, S0222:2, H0333:2, H0559:2, H0318:2, H0581:2,





H0545:2, H0620:2, H0024:2, H0266:2, H0617:2, H0529:2, L0662:2, L0653:2, L0659:2, L0809:2, L0664:2, H0690:2, H0555:2,





L0743:2, L0755:2, L0757:2, L0759:2, L0588:2, H0352:2, H0624:1, L0685:1, L0740:1, H0295:1, S0134:1, H0583:1, H0656:1,





H0341:1, S0212:1, H0484:1, L3659:1, S0418:1, S0356:1, S0442:1, S0410:1, H0729:1, H0735:1, H0339:1, H0619:1, S0278:1,





H0257:1, H0069:1, H0744:1, H0327:1, H0023:1, S0051:1, T0010:1, H0031:1, H0181:1, H0032:1, H0169:1, S0364:1, H0135:1,





H0163:1, H0090:1, H0063:1, H0087:1, H0551:1, H0264:1, H0488:1, H0623:1, H0100:1, S0438:1, S0440:1, L0369:1, L0770:1,





L0769:1, L5565:1, L0761:1, L0667:1, L0772:1, L0641:1, L0644:1, L0764:1, L0773:1, L0363:1, L0768:1, L0794:1, L0766:1,





L0381:1, L0803:1, L0774:1, L0375:1, L0806:1, L0512:1, L0517:1, L0666:1, H0144:1, H0702:1, S0148:1, L0352:1, H0519:1,





L0593:1, H0435:1, H0658:1, H0539:1, S0406:1, H0478:1, H0631:1, S0028:1, S0206:1, L0744:1, L0740:1, L0745:1, L0749:1,





L0750:1, L0758:1, H0445:1, S0434:1, L0594:1, S0194:1, H0542:1 and H0423:1.

3
HACAB68
584773
13
L0748:4, H0457:3 and S6022:1.

4
HACBJ56
847112
14
AR251:7, AR310:6, AR265:6, AR053:6, AR060:6, AR182:6, AR055:6, AR312:5, AR309:5, AR273:5, AR282:5, AR061:5,





AR206:5, AR241:5, AR194:5, AR186:5, AR270:4, AR213:4, AR052:4, AR266:4, AR218:4, AR089:4, AR274:4, AR253:4,





AR269:4, AR291:4, AR248:4, AR296:4, AR183:4, AR240:4, AR104:4, AR293:4, AR205:4, AR284:4, AR289:3, AR313:3,





AR184:3, AR299:3, AR247:3, AR185:3, AR316:3, AR231:3, AR298:3, AR033:3, AR243:3, AR268:3, AR290:3, AR283:3,





AR295:3, AR300:3, AR246:3, AR277:3, AR232:3, AR175:3, AR096:3, AR219:3, AR177:3, AR234:3, AR233:3, AR275:3,





AR237:3, AR238:3, AR285:3, AR227:3, AR202:3, AR263:3, AR292:3, AR204:2, AR286:2, AR314:2, AR267:2, AR280:2,





AR229:2, AR258:2, AR039:2, AR294:2, AR256:2, AR259:2, AR281:1, AR315:1, AR226:1, AR244:1, AR249:1 H0661:1,





S0045:1, H0550:1, S0280:1, S0010:1, H0028:1, L0764:1, L0803:1, L0805:1, L0665:1, S0053:1, H0670:1, L0748:1, L0731:1 and





L0581:1.

5
HADMB15
847116
15
AR104:19, AR218:19, AR219:16, AR089:11, AR313:8, AR055:8, AR060:7, AR299:6, AR282:5, AR300:5, AR039:5, AR240:5,





AR316:5, AR185:5, AR277:4, AR283:4, AR096:3 L0595:2, L0442:1, L0005:1, L3653:1, H0390:1, H0081:1, H00241,





L0770:1, L5566:1, L0651:1, L0565:1, L0439:1, L0747:1, L0752:1, H0445:1, L0592:1 and L0599:1.

6
HAGDW20
637489
16
AR313:22, AR039:22, AR277:20, AR299:17, AR089:17, AR300:14, AR185:14, AR218:13, AR096:13, AR240:13, AR219:13,





AR104:13, AR316:13, AR282:12, AR283:11, AR060:11, AR055:11 S0010:1 and H0616:1.

7
HAGEQ79
828055
17
AR104:37, AR283:37, AR277:26, AR055:20, AR185:20, AR316:18, AR299:17, AR282:16, AR313:16, AR219:16, AR240:16,





AR089:16, AR218:14, AR060:14, AR096:13, AR039:12, AR300:10 L0805:6, L0809:4, L0803:3, L0779:3, L0794:2, L0776:2,





L0438:2, L0439:2, L0745:2, L0747:2, S0436:2, S0408:1, T0082:1, S0010:1, H0052:1, T0010:1, H0598:1, L0770:1, L0774:1,





L0783:1, L0788:1, L0665:1, L0742:1, L0777:1, L0753:1, L0755:1, L07591 and LOS92:1.

8
HAJBV67
866415
18
AR039:11, AR219:10, AR316:9, AR218:9, AR089:9, AR270:8, AR282:8, AR283:8, AR269:8, AR248:7, AR299:7, AR183:7,





AR309:7, AR277:7, AR096:7, AR104:7, AR313:7, AR281:7, AR265:6, AR249:6, AR253:6, AR315:6, AR300:6, AR290:6,





AR292:6, AR202:6, AR312:6, AR280:6, AR175:6, AR231:5, AR182:5, AR185:5, AR268:5, AR060:5, AR294:5, AR194:5,





AR238:5, AR247:4, AR243:4, AR053:4, AR267:4, AR295:4, AR291:4, AR055:4, AR285:4, AR213:4, AR052:4, AR184:4,





AR293:4, AR296:3, AR240:3, AR033:3, AR310:3, AR314:3, AR275:3, AR246:3, AR205:3, AR286:3, AR271:3, AR234:3,





AR192:3, AR298:3, AR263:3, AR232:3, AR256:3, AR251:3, AR259:2, AR284:2, AR229:2, AR289:2, AR226:2, AR274:2,





AR237:2, AR258:2, AR179:2, AR177:2, AR233:2, AR273:2, AR227:2, AR204:2, AR186:1, AR061:1, AR241:1 L0754:9,





S0444:6, S0442:5, S0358:5, H0622:5, H0624:4, H0040:4, L0659:4, H0144:4, H0521:4, H0171:3, L3499:3, L2630:3, H0046:3,





H0658:3, H0555:3, H0436:3, L0758:3, S0434:3, H0543:3, S0418:2, S0360:2, S0222:2, L2499:2, H0013:2, H0156:2, H0575:2,





H0615:2, H0674:2, H0616:2, H0551:2, H0412:2, H0623:2, S0440:2, H0647:2, S0422:2, H0529:2, L0666:2, L2263:2, S0374:2,





S0380:2, S0146:2, L0740:2, L0731:2, L0759:2, S0436:2, L0362:2, H0556:1, L3644:1, S0114:1, T0049:1, L0002:1, L2910:1,





S0282:1, L2300:1, S0356:1, S0354:1, S0408:1, S0410:1, L3709:1, H0637:1, H0742:1, H0722:1, S0046:1, S0132:1, S0300:1,





L2744:1, L0717:1, H0411:1, H0431:1, H0586:1, H0587:1, L2491:1, L2647:1, H0036:1, H0004:1, S0010:1, H0318:1, H0581:1,





H0251:1, H0596:1, L0471:1, H0024:1, H0014:1, H0375:1, H0266:1, S0003:1, S0214:1, H0688:1, T0023:1, H0553:1, L0055:1,





H0032:1, S0036:1, H0090:1, H0591:1, H0038:1, T0067:1, H0477:1, H0488:1, H0059:1, H0561:1, L0369:1, L3905:1, L0662:1,





L0766:1, L0388:1, L0774:1, L0775:1, L0606:1, L0661;1, L0526;1, L0809:1, L0665:l, L2653:1, L3665:1, L3811:1, H0519:1,





S0126:1, H0683:1, H0684:1, H0659:1, H0672:1, H0710:1, H0522:1, S3014:1, S0028:1, L0752:1, H0595:1, S0394:1, L0596:1,





L0589:1, L0485:1, L0595:1, H0667:1, S0242:1, S0194:1, H0423:1, H0422d, S0384:1, H0506:1 and H0352:1.

9
HAJCH70
827275
19
H0561:1

10
HAOAG15
852204
20
AR169:4, AR241:3, AR172:3, AR206:3, AR263:3, AR207:3, AR176:3, AR235:3, AR168:2, AR183:2, AR297:2, AR166:2,





AR163:2, AR282:2, AR171:2, AR193:2, AR178:2, AR181:2, AR162:2, AR274:2, AR182:2, AR298:2, AR217:2, AR224:2,





AR312:2, AR053:2, AR287:2, AR254:2, AR295:2, AR239:2, AR205:1, AR293:1, AR216:1, ARL7S:1, AR238:1, AR285:1,





AR316:1, AR277:1, AR033:1, AR179:1, AR267:1, AR291:1, AR288:1, AR289:1, AR089:1 L0759:3, S0314:2, L0744:2,





L0756:2, L0755:2, S0046:1, H0391:1, H0052:1, H0050:1, S0318:1, S0338:1, S0312:1, L0766:1 and H0144:1.

11
HATCD80
826098
21
AR316:50, AR055:4, AR277:3, AR060:3, AR300:3, AR282:3, AR283:2, AR218:2, AR039:1, AR104:1, AR089:1, AR240:1,





AR299:1, AR185:1 H0156:1 and H0038:1.

12
HATCI03
580805
22
AR313:42, AR039:30, AR299:20, AR096:19, AR185:19, AR277:18, AR300:18, AR089:17, AR219:15, AR240:14, AR218:13,





AR316:12, AR104:10, AR060:10, AR282:8, AR055:7, AR283:5 S6026:1, H0156:1 and S0426:1.

13
HBAGD86
838799
23
AR219:7, AR218:4, AR313:4, AR104:4, AR039:3, AR299:3, AR282:2, AR300:2, AR096:2, AR316:2, AR277:1, AR240:1,





AR089:1 L0809:4, L0766:3, L0439:3, H0624:2, H0411:2, L0794:2, L0749:2, L0756:2, L0005:1, L3649:1, S0476:1, H0599:1,





L0471:1, S0051:1, T0010:1, H0266:1, S0150:1, S0422:1, L0637:1, L0765:1, L0803:1, L0783:1, L5622:1, H0144:1, H0672:1,





S0392:1, L0748:1, L0754:1, L0779:1, L0777:1, L0731:1 and L0759:1.

14
HBHAA05
603174
24
AR313:74, AR039:45, AR299:34, AR089:33, AR185:31, AR277:29, AR096:26, AR300:26, AR240:22, AR316:20, AR218:17,





AR060:15, AR219:14, AR104:14, AR282:11, AR055:9, AR283:6 S0029:1

15
HBHAA81
846465
25
AR289:34, AR291:33, AR283:32, AR055:32, AR294:26, AR266:26, AR286:26, AR256:23, AR285:21, AR293:19, AR.259:17,





AR295:16, AR292:15, AR298:14, AR258:14, AR296:12, AR284:11, AR104:10, AR033:9, AR186:9, AR202:7, AR206:7,





AR246:7, AR204:7, AR241:6, AR194:5, AR198:4, AR244:4, AR251:4, AR060:4, AR061:4, AR282:4, AR052:4, AR053:4,





AR205:4, AR309:4, AR316:3, AR182:3, AR312:3, AR192:3, AR273:3, AR229:3, AR183:3, AR310:3, AR271:3, AR213:3,





AR248:3, AR270:3, AR277:2, AR185:2, AR275:2, AR299:2, AR269:2, AR300:2, AR247:2, AR267:2, AR175:2, AR089:2,





AR313:2, AR265:2, AR268:2, AR237:2, AR096:1, AR232:1, AR039:1, AR240:1, AR179:1, AR231:1, AR234:1 H0599:8,





S0366:7, L0485:6, H0733:5, H0734:5, L0769:5, H0735:4, H0729:3, H0728:3, H0619:2, H0706:2, L0661:2, L0756:2, L0759:2,





S0282:1, S0029:1, S0222:1, L0622:1, H0122:1, S0010:1, H0196:1, H0012:1, H0200:1, H0373:1, S6028:1, S0364:1, S0036:1,





S0294:1, L0770:1, L0638:1, L5565:1, L0657:1, L0809:1, L0789:1, L0791:1, L0438:1, L0439:1, L0750:1, L0777:1, S0260:1,





L0604:1 and S0460:1.

16
HBIAA59
806303
26
AR313:13, AR089:13, AR299:12, AR240:11, AR104:11, AR185:11, AR039:11, AR055:10, AR096:10, AR218:9, AR060:9,





AR219:7, AR316:7, AR300:7, AR282:6, AR283:5, AR277:5 L0747:13, L0757:12, L0754:8, L0749:6, L0740:5, L0731:4,





H0009:3, H0051:3, L0750:3, L0756:3, L0777:3, L0752:3, S0376:2, S0360:2, H0619:2, L3388:2, H0485:2, L3653:2, S0010:2,





H0052:2, H0251:2, S0022:2, H0090:2, H0494:2, L0662:2, L0794:2, L0806:2, L0776:2, L0665:2, H0144:2, S0390:2, L0748:2,





L0581:2, H0265:1, H0556:1, H0716:1, S0402:1, L0808:1, S0212:1, S0001:1, H0661:1, S0358:1, S0444:1, S0046:1, S6026:1,





L0717:1, H0549:1, S0222;1, H0438:1, H0592:1, H0333:1, H0632:1, H0486:1, H0013:1, H0042:1, S0049:1, H0744:1, H0545:1,





H0123:1, H0081:1, H0050:1, L0471:1, H0105:1, H0012:1, H0620:1, S00S1:1, S6028:1, H0688:1, H0553:1, L0455:1, H0598:1,





H0040:1, H0412:1, L0763:1, L0769:1, L0638:1, L0372:1, L0764:1, L0771:1, L0766:1, L0561:1, L0498:1, L0774:1, L0775:1,





L0375:1, L0378:1, L0805:1, L0657:1, L0659:1, L0809:1, L5623:1, L0789:1, L0663:1, L0438:1, H0520:1, H0518:1, H0696:1,





S3112:1, S3014:1, S0028:1, L0744:1, L0751:1, L0780:1, L0755:1, L0758:1, L0759:1, S0031:1, S0260:1, H0506:1 and H0008:1.

17
HBJAC40
841235
27
AR104:23, AR060:6, AR055:6, AR283:5, AR185:5, AR282:4, AR313:4, AR299:4, AR316:4, AR277:3, AR096:3, AR240:3,





AR219:2, AR089:2, AR300:2, AR039:2, AR218:2 L0439:18, H0052:12, L0741:8, L0438:6, S0051:5, H0556:4, L0769:4,





L0774:4, S0474:3, H0622:3, S0036:3, L3905:3, H0261:2, H0318:2, H0194:2, L0471:2, H0538:2, L0749:2, L0757:2, L0758:2,





S0436:2, L0593:2, H0624:1, H0265:1, S0342:1, H0717:1, H0650:1, H0657:1, S0212:1, S0282:1, H0730:1, S0045:1, S0476:1,





H0619:1, S0222:1, H0455:1, H0559:1, H0075:1, H0253:1, H0251:1, H0544:1, L0158:1, H0012:1, S0050:1, L0163:1, H0083:1,





H0594:1, H0615:1, T0006:1, H0708:1, H0087:1, H0056:1, S0038:1, H0494:1, S0450:1, S0144:1, L0770:1, L4747:1, L0639:1,





L0761:1, L0775:1, L0805:1, L0635:1, L5622:1, L0788:1, S0428:1, S0044:1, L0612:1, L0742:1, L0748:1, L0779:1, L0777:1,





S0011:1 and H0136:1.

18
HBJCR46
815649
28
L0794:11, L0803:10, L0779:10, H0038:9, L0777:9, L0758:9, S0358:6, L0809:5, S0408:4, H0616:4, L0748:4, L0439:4,





L0591:4, S0282:3, L0789:3, L0666:3, L0438:3, L0756:3, H0036:2, H0196:2, H0046:2, H0154:2, L0163:2, H0213:2, S0036:2,





L0804:2, L0774:2, L0655:2, L0656:2, S0374:2, S0126:2, S0328:2, S0152:2, H0521:2, S0406:2, L0731:2, L0588:2, L0485:2,





S0026:2, H0543:2, H0170:1, H0171:1, H0713:1, S6024:1, H0650:1, H0656:1, S0116:1, H0341:1, H0638:1, S0442:1, S0354:1,





S0360:1, H0580:1, S0045:1, H0619:1, H0437:1, S0222:1, H0333:1, H0574:1, H0486:1, H0575:1, H0590:1, H0618:1, H0253:1,





H0318:1, H0581:1, H0230:1, H0597:1, H0544:1, H0178:1, H0123:1, H0050:1, S0050:1, H0014:1, S6028:1, H0266:1, S0003:1,





H0033:1, H0032:1, H0673:1, H0598:1, H0163:1, H0040:1, H0551:1, H0623:1, H0100:1, T0041:1, H0561:1, S0438:1, H0641:1,





S0344:1, S0002:1, S0426:1, L0769:1, L0800:1, L0641:1, L0766:1, L0775:1, L0375:1, L0653:1, L0634:1, L0659:1, L0783:1,





L0787:1, L0663:1, L0664:1, L0665:1, H0725:1, H0670:1, H0522:1, H0436:1, H0540:1, S0027:1, L0740:1, L0751:1, L0747:1,





L0749:1, L0786:1, L0780:1, L0752:1, L0757:1, L0608:1, L0604:1, S0192:1 and S0276:1.

19
HBJDW56
520401
29
AR055:8, AR060:7, AR282:6, AR104:5, AR313:5, AR185:5, AR300:4, AR089:4, AR299:4, AR240:4, AR039:4, AR219:4,





AR316:3, AR096:3, AR283:3, AR218:3, AR277:2 H0318:1

20
HBJEL16
847030
30
H0046:2, H0009:2, H0090:2, H0494:2, L0438:2, H0547:2, H0521:2, L0439:2, L0777:2, H0543:2, H0556:1, S0342:1, S0045:1,





H0619:1, H0632:1, H0013:1, H0156:1, L0021:1, H0575:1, H0318:1, S0003:1, L0483:1, H0628:1, H0623:1, H0561:1, L0761:1





L0803:1, L0804:1, L0659:1, L0382:1, H0144:1, H0539:1, S0152:1, H0478:1, H0631:1, L0741:1, L0740:1 and L0591:1.

21
HBJIG20
866159
31
AR060:1246, AR282:1116, AR300:1098, AR055:1006, AR104:977, AR299:929, AR185:901, AR240:901, AR283:813,





AR316:761, AR277:738, AR089:695, AR039:604, AR096:571, AR313:390, AR218:357, AR219:319 H0594:11, H0596:8,





S0282:5, S0260:5, S0194:5, H0543:5, S0278:2, H0600:2, H0592:2, H0598:2, S0344:2, H0595:2, S0356:1, H0438:1, H0574:1,





H0599:1, S0346:1, H0318:1, H0597:1, S0388:1, H0316:1, S0390:1 and H0542:1.

22
HBJKD16
853358
32
AR172:63, AR171:62, AR215:61, AR274:50, AR216:48, AR213:43, AR214:41, AR272:41, AR169:41, AR224:37, AR225:37,





AR217:37, AR254:36, AR205:36, AR170:35, AR243:35, AR168:35, AR247:34, AR245:32, AR312:32, AR221:32, AR212:31,





AR161:29, AR222:28, AR162:28, AR311:27, AR308:27, AR163:26, AR275:26, AR165:25, AR164:24, AR313:23, AR053:23,





AR166:23, AR223:21, AR039:20, AR089:20, AR309:19, AR096:19, AR242:18, AR253:18, AR240:17, AR289:16, AR266:16,





AR283:16, AR263:16, AR193:16, AR316:16, AR264:16, AR204:16, AR250:15, AR282:15, AR201:15, AR277:15, AR207:14,





AR291:14, AR246:14, AR200:13, AR198:13, AR271:12, AR299:12, AR300:12, AR195:12, AR155:12, AR104:12, AR290:11,





AR192:11, AR173:11, AR255:11, AR257:11, AR060:11, AR197:11, AR252:10, AR150:1O, AR297:1O, AR179:10, AR210:10,





AR061:10, AR181:9, AR296:9, AR199:9, AR270:9, AR269:9, AR175:9, AR183:9, AR268:8, AR055:8, AR177:8, AR262:8,





AR236:8, AR288:8, AR211:8, AR188:7, AR267:7, AR293:7, AR219:7, AR285:7, AR256:7, AR294:7, AR174:7, AR176:7,





AR189:7, AR033:7, AR261:7, AR218:7, AR287:6, AR175:6, AR196:6, AR231:6, AR203:6, AR286:5, AR235:5, AR190:5,





AR230:5, AR234:5, AR191:5, AR182:5, AR260:5, AR258:4, AR295:4, AR237:4, AR233:4, AR229:4, AR238:4, AR239:3,





AR226:3, AR232:2, AR227:2, AR228:2 L0766:9, L0439:9, L0747:6, L2528:5, L0777:5, H0673:4, L0438:4, L0758:4, L0362:4,





S0116:3, L0748:3, L0752:3, H0445:3, H0156:2, T0010:2, H0615;2, H0038:2, H0616:2, H0264:2, H0646:2, L0761:2, L0776:2,





L0750:2, L0779:2, S0436:2, L0593:2, S0242:2, H0222:1, H0740:1, H0657:1, H0661:1, H0663:1, L2293:1, H0589:1, S0444:1,





H0340:1, L3646:1, H0580:1, H0749:1, H0393:1, H0549:1, S0222:1, H0574:1, H0486:1, H0013:1, H0069:1, L0021:1, S0010:1,





H0318:1, S0474:1, H0046:1, L0471:1, H0090:1, L0638:1, L0646:1, l0764:1, L0521:1, L0364:1, L0774:1, L0659:1, L0543:1,





L5622:1, L0792:1, L0666:1, L0664:1, L0665:1, S0428:1, L2657:1, L2652:1, L3663:1, L2262:1, H0435:1, L3832:1, L0741:1,





L0749:1, S0434:1, L0588:1, H0422:1, L0698:1 and L2359:1.

23
HBMTY48
637521
33
AR241:34, AR313:20, AR039:19, AR192:19, AR182:18, AR198:16, AR275:14, AR271:13, AR312:13, AR184:13, AR186:12,





AR274:12, AR290:11, AR268:11, AR185:11, AR089:10, AR204:10, AR267:10, AR096:10, AR270:9, AR299:9, AR052:9,





AR240:9, AR213:9, AR273:9, AR243:9, AR291:8, AR247:8, AR300:8, AR269:8, AR053:8, AR293:8, AR194:8, AR258:8,





AR206:8, AR316:8, AR277:7, AR104:7, AR060:7, AR292:6, AR218:6, AR246:6, AR309:6, AR183:6, AR263:6, AR244:6,





AR202:6, AR205:5, AR233:5, AR296:5, AR282:5, AR177:5, AR294:5, AR289:5, AR033:5, AR061:5, AR285:5, AR219:5,





AR175:5, AR310:4, AR295:4, AR055:4, AR298:4, AR259:4, AR249:4, AR266:4, AR229:4, AR179:3, AR265:3, AR256:3,





AR248:2, AR283:2, AR284:2, AR280:2, AR286:2, AR226:1, AR237:1 S0116:1, H0591:1, L2270:1, L0766:1, L2657:1 and





H0690:1.

24
HBMUH74
866160
34
AR218:12, AR055:8, AR060:7, AR104:7, AR219:5, AR240:5, AR299:5, AR096:4, AR316:4, AR300:4, AR039:4, AR089:3,





AR283:3, AR185:3, AR313:3, AR282:2, AR277:2 L0754:3, L0777:3, L0439:2, S0116:1, H0341:1, H0661:1, H00381,





H0412:1, L0761:1, L0667:1, L0764:1, L0788:1, H0435:1, L0749:1, L0779:1 and LO758:1.

25
HBQAB79
810542
35
AR055:7, AR218:7, AR060:6, AR039:6, AR300:5, AR185:5, AR313:5, AR240:4, AR299:4, AR089:4, AR096:3, AR316:3,





AR283:3, AR104:2, AR219:2, AR277:2, AR282:1 H0229:1

26
HBXCX15
637542
36
S0038:3, H0438:1, L0363:1 and S0053:1.

27
HCDCY76
837972
37
AR219:7, AR218:6, AR055:2, AR282:2, AR060:2, AR299:1, AR104:1, AR185:1, AR240:1, AR277:1 L1430:5, L0770:2,





L0754:2, L0747:2, L0777:2, S0360:1, S0045:1, H0486:1, H0616:1, L0803:1, L0775:1, L0783:1, L0787:1, L0789:1, L0750:1,





S0194:1 and S0276:1.

28
HCEDR26
771144
38
AR313:59, AR039:47, AR277:33, AR299:28, AR185:25, AR096:23, AR089:23, AR300:20, AR219:19, AR240:17, AR316:16,





AR104:15, AR282:13, AR218:13, AR060:13, AR283:10, AR055:10 H0052:2, H0018:1, H0264:1 and L0700:1.

29
HCEEU18
688041
39
AR313:46, AR039:35, AR299:24, AR219:21, AR277:21, AR089:20, AR096:19, AR185:19, AR218:16, AR316:14, AR300:13,





AR104:13, AR240:12, AR060:11, AR282:10, AR055:9, AR283:5 H0052:1

30
HCEGX05
827060
40
AR219:16, AR104:13, AR218:13, AR089:11, AR185:9, AR313:9, AR299:8, AR240:8, AR096:8, AR316:8, AR055:7, AR039:6,





AR060:6, AR300:6, AR283:5, AR277:4, AR282:4 L0766:11, L0748:5, L0757:4, L0662:3, H0587:2, L3816:2, L0041:2,





H0039:2, L0659:2, L0438:2, H0672:2, H0521:2, L0750:2, L0758:2, L0596:2, L0589:2, L0605:2, H0265:1, H0341:1, H0728:1,





S0222:1, H0600:1, L0623:1, H0069:1, H0052:1, H0569:1, S0388:1, T0010:1, L0055:1, L0456:1, H0560:1, H0641:1, S0426:1,





L0770:1, L0769:1, L5575:1, L0794:1, L0776:1, L0783:1, L0382:1, L0666:1, L0663:1, S0052:1, S0216:1, H0702:1, L3825:1,





L3828:1, H0670:1, H0539:1, H0522:1, S0406:1, S0390:1, L0743:1, L0744:1, L0439:1, L0740:1, L0747:1, L0779:1, L0777:1,





H0445:1, S0436:1, H0542:1, H0423:1 and H0422:1.

31
HCFLN88
610000
41
S0410:22, L0770:9, L0748:9, L0769:7, L0776:6, L0659:6, H0424:5, L0761:5, L0731:5, H0486:4, L0803:4, L0809:4, L0666:4,





H0696:4, L0754:4, L0779:4, L0758:4, H0729:3, H0618:3, H0135:3, L0637:3, L0771:3, L0766:3, L0805:3, L0665:3, L0751:3,





H0542:3, H0341:2, H0402:2, S0358:2, S0376:2, S0360:2, H0747:2, S0132:2, L3109:2, L0717:2, H0592:2, H0253:2, S0010:2,





H0052:2, H0545:2, H0050:2, H0617:2, H0087:2, H0551:2, H0100:2, H0560:2, L0763:2, L5565:2, L0646:2, L0764:2, L0655:2,





L0663:2, L2260:2, S0374:2, H0414:2, S0406:2, H0436:2, L0743:2, L0740:2, L0749:2, L0755:2, L0757:2, L0759:2, H0445:2,





H0136:2, H0543:2, H0423:2, H0352:2, H0170:1, H0171:1, H0225:1, H0713:1, S0218:1, L0785:1, H0692:1, S0212:1, H0483:1,





H0254:1, H0305:1, S0356:1, S0442:1, S0444:1, S0408:1, H0619:1, H0393:1, H0406:1, H0370:1, H0249:1, H0101:1, H0250:1,





S0280:1, H0599:1, H0575:1, H0706:1, T0048:1, H0318:1, S0474:1, H0581:1, T0115:1, H0009:1, H0572:1, H0024:1, S0051:1,





H0271:1, H0288:1, T0006:1, H0213:1, H0553:1, H0644:1, S0364:1, H0163:1, H0090:1, H0264:1, H0488:1, S0112:1, H0494:1,





H0652:1, S0344:1, S0002:1, S0426:1, L4497:1, L5575:1, L3905:1, L5566:1, L0772:1, L0641:1, L0645:1, L0773:1, L0650:1,





L0774:1, L0775:1, L0378:1, L0806:1, L0783:1, L5622:1, L0790:1, L0664:1, L3827:1, H0547:1, H0519:1, S0126:1, H0711:1,





H0672:1, S0330:1, H0521:1, S0392:1, S0037:1, L0742:1, L0439:1, L0745:1, L0747:1, L0750:1, L0777:1, S0436:1, L0485:1,





L0608:1, S0011:1, H0653:1 and H0422:1.

32
HCHAB84
834326
42
AR313:37, AR039:34, AR104:24, AR300:24, AR277:23, AR096:23, AR185:20, AR089:20, AR299:19, AR219:18, AR218:17,





AR316:16, AR240:16, AR282:13, AR283:9, AR060:8, AR055:7 S0354:9, S0358:3, H0494:3, S04762, S0474:2, 50438:2,





H0519:2, H0521:2, L0754:2, H0170:1, S0040:1, S0114:1, H0484:1, H0483:1, H0255:1, S0376:1, S0444:1, S0408:1, S0046:1,





H0619:1, H0549:1, H0042:1, H058:1, H0052:1, H0083:1, S0440:1, L0773:1, L0517:1, L0383:1, S0374:1, S0152:1, S3014:1,





L0751:1, L0759:1, S0434:1, S0436:1, H0543:1 and H0422:1.

33
HCMSX51
788643
43
L0740:16, L0745:7, L0439:6, L0438:4, H0547:4, L0750:4, L0759:4, H0619:3, H0618:3, L0770:3, L3828:3, L0749:3, L0758:3,





H0393:2, H0599:2, H0083:2, H0124:2, H0623:2, H0100:2, L3905:2, L0794:2, L0809:2, L3825:2, L3829:2, S0406:2, S0027:2,





L0743:2, L0746:2, L0777:2, L0603:2, S0040:1, L2879:1, L2906:1, S0420:1, S0442:1, S0358:1, L3311:1, L3485:1, H0261:1,





H0392:1, H0013:1, H0250:1, H0590:1, H0196:1, H0545:1, H0046:1, H0123:1, H0620:1, S0051:1, S0250:1, H0617:1, S0036:1,





H0135:1, H0634:1, H0087:1, H0269:1, H0561:1, H0509:1, H0646:1, S0426:1, L0763:1, L0769:1, L3904:1, L0662:1, L0363:1,





L0767:1, L0768:1, L0650:1, L0375:1, L0806:1, L0776:1, L0657:1, L0787:1, L4559:1, L0664:1, L2260:1, H0520:1, L3831:1,





H0670:1, H0518:1, L3834:1, H0704:1, H0436:1, L0747:1, L0780:1, L0608:1, L0595:1 and H0423:1.

34
HCNCO11
775086
44
AR055:2, AR060:2, AR277:1, AR282:1 H0597:1

35
HCNSD29
862314
45
AR252:128, AR253:67, AR245:63, AR272:55, AR308:49, AR246:47, AR263:46, AR212:40, AR053:37, AR243:35, AR312:34,





AR254:33, AR275:33, AR205:33, AR309:32, AR264:31, AR250:31, AR197:31, AR271:29, AR224:26, AR195:26, AR311:26,





AR200:26, AR223:26, AR201:25, AR198:25, AR219:23, AR274:22, AR210:22, AR172:21, AR218:21, AR222:21, AR225:20,





AR221:20, AR268:19, AR1O4:19, AR096:18, AR240:17, AR188:17, AR313:17, AR199:17, AR213:17, AR203:16, AR242:16,





AR039:15, AR316:15, AR170:15, AR193:15, AR180: 15, AR165:14, AR269:14, AR192:14, AR204:14, AR164:14, AR166: 14,





AR211:13, AR033:13, AR168:13, AR270:13, AR207:12, AR175:12, AR290:12, AR169:12, AR089:12, AR183:12, AR247:12





AR176.11, AR171:11, AR267:11, AR178:11, AR162:11, AR161:11, AR217:11, AR163:10, AR174:10, AR229:10, AR291:10,





AR173:10, AR189:9, AR214:9, AR266:9, AR255:9, AR181:8, AR196:8, AR177:8, AR297:8, AR289:8, AR299:8, AR179:8,





AR190:8, AR191:8, AR300:8, AR060:8, AR182:8, AR238:7, AR216:7, AR215:7, AR257:7, AR261:7, AR262:7, AR282:7,





AR296:7, AR185:7, AR293:7, AR235:6, AR295:6, AR283:6, AR231:6, AR055:6, AR288:6, AR285:6, AR287:6, AR234:6,





AR258:6, AR237:5, AR239:5, AR236:5, AR256:5, AR277:5 , AR286:5, AR228:5, AR230:4, AR294:4, AR226:4, AR233:4,





AR260:4, AR232:4, AR061:3, AR227:3 L0648:2, L0768:2, L0766:2, L0748:2, L0588:2, H0125:1, S0468:1, H0497:1, H0486:1,





H0744:1, H0231:1, H0266:1, H0202:1, H0641:1, S0422:1, L0638:1, L0644:1, L5572:1, L0662:1, L0650:1, L0807:1, L0657:1,





L0663:1, L0665:1, H0519:1, L0759:1, S0026:1 and L0718:1.

36
HCQBH72
637548
46
AR055:2, AR060:2, AR299:2, AR089:2, AR219:1, AR185:1, AR039:1, AR283:1, AR104:1 L0520:4, L0754:2, H0263:1,





H0272:1 and H0555:1.

37
HCQCC96
845066
47
AR252:46, AR197:44, AR204:38, AR195:35, AR253:34, AR178:31, AR230:31, AR254:31, AR233:29, AR250:28, AR18O028,





AR198:28, AR266:26, AR243:26, AR193:24, AR239:23, AR061:23, AR267:23, AR201:23, AR227:22, AR229:22, AR228:22,





AR237:21, AR162:21, AR181:21, AR163:21, AR170:21, AR161:20, AR257:20, AR192:20, AR226:20, AR176:19, AR234:19,





AR171:18, AR183:18, AR245:18, AR271:18, AR182:17, AR258:17, AR270:17, AR179:17, AR275:17, AR238:16, AR231:16,





AR296:16, AR261:16, AR033:16, AR174:15, AR185:15, AR255:15, AR164:15, AR262:15, AR207:15, AR053:15, AR165:15,





AR272:14, AR256:14, AR039:14, AR269:14, AR242:14, AR166:14, AR205:14, AR175:14, AR246:14, AR300:13, AR203:13,





AR289:12, AR236:12, AR104:12, AR316:11, AR232:11, AR293:11, AR055:11, AR287:11, AR169:11, AR260:11, AR235:11,





AR168:11, AR089:11, AR173:10, AR308:10, AR286:10, AR268:10, AR291:10, AR060:10, AR297:10, AR313:10, AR288:10,





AR212:10, AR213:10, AR299:10, AR177:10, A2R188:9, AR096:9, AR190:9, AR191:9, AR294:9, AR282:9, AR285:9, AR.283:9,





AR172:8, AR277:8, AR189:8, AR247:8, AR309:8, AR312:8, AR274:8, AR240:7, AR218:7, AR264:7, AR210:6, AR200:6,





AR295:6, AR290:6, AR219:6, AR215:6, AR199:5, AR263:5, AR196:5, AR223:5, AR311:5, AR216:5, AR214:4, AR224:4,





AR225:4, AR211:4, AR217:4, AR221:2, AR222:2 S0360:5, L0748:5, L0766:3, H0657:2, L3388:2, H0581:2, H0596:2,





H0563:2, S0003:2, H0328:2, H0670:2, L0756:2, S0436:2, S0026:2, H0170:1, H0556:1, H0344:1, H0650:1, H0656:1, H0638:1,





S0420:1, H0675:1, S0007:1, H0574:1, H0632:1, H0013:1, H0036:1, S0010:1, H0318:1, H0052:1, H0251:1, H0150:1, H0050:1,





H0090:1, H0038:1, S0440:1, H0130:1, S0142:1, S0422:1, H0529:1, L0803:1, L0659:1, L5623:1, L0666:1, S0428:1, S0126:1,





H0689:1, H0648:1, H0672:I, S0330:1, H0539:1, S0378:1, H0521:1, H0522:1, H0478:1, L0744:1, L0754:1, L07791, L0752:1,





S0260:1, H0445:1, H0343:1, H0595:1, S0434:1, H0423:1 and S0424:1.

38
HCQCJ56
832157
48
AR055:8, AR060:5, AR104:4, AR240:3, AR218:3, AR299:3, AR300:3, AR185:3, AR277:3, AR089:2, AR283:2, AR282:2,





AR039:2, AR316:2, AR096:2, AR219:2 L0779:4, L0777:3, H0050:2, H0670:2, L0748:2, L0717:1, H0596:1, L0641:1, L0794:1,





L0803:1, L0774:1, L0809:1 and L0749:1.

39
HCUDD64
835082
49
AR282:3, AR219:3 H0052:3, S3012:2, L0754:2, H0402:1, H0413:1, S0374:1, L0438:1, L0748:1 and L0740:1.

40
HCWAE64
535893
50
AR277:7, AR282:1 H0305:1

41
HDPDJ58
587265
51
AR263:8, AR249:6, AR053:5, AR270:5, AR312:5, AR039:4, AR309:4, AR096:4, AR052:4, AR198:4, AR183:4, AR253:4,





AR313:4, AR282:4, AR243:3, AR269:3, AR184:3, AR192:3, AR267:3, AR268:3, AR213:3, AR316:3, AR290:3, AR310:2,





AR240:2, AR275:2, AR273:2, AR186:2, AR238:2, AR298:2, AR206:2, AR234:2, AR277:2, AR177:2, AR292:2, AR226:1,





AR060:1, AR237:1, AR296:1, AR205:1, AR299:1, AR033:1, AR294:1, AR293:1, AR291:1, AR231;1, AR175:1, AR182:1,





AR185:1, AR284:1, AR218:1 L0766:14, H0457:10, H0486:4, H0581:4, S0406:4, H0422:4, H0171:3, L0655:3, H0521:3,





L0779:3, H0749:2, H0156:2, H0090:2, H0551:2, L0598:2, L0666:2, L0438:2, L0748:2, L0756:2, L0777:2, T0002:1, H0656:1,





S0212:1, H0662:1, H0638:1, S0442:1, S0140:1, H0747:1, H0261:1, H0587:1, L3816:1, H0574:1, L0586:1, L0022:1, H0318:1,





H0123:1, L0471:1, H0039:1, H0591:1, T0041:1, S0344:1, S0426:1, UNKWN:1, L0794:1, L0387:1, L0776:1, L0606:1, L0659:1,





L0367:1, L0792:1, L0793:1, H0690:1, H0539:1, H0436:1, L0439:1, L0780:1, L0755:1, L0759:1, H0445:1, H0423:1 and





H0506:1.

42
HDPFU43
790189
52
AR277:53, AR283:13, AR096:12, AR240:12, AR316:11, AR219:11, AR218:10, AR104:9, AR282:9, AR299:8, AR039:8,





AR313:8, AR185:8, AR300:7, AR060:7, AR055:7, AR089:7 H0585:8, L3388:8, S0474:7, H0622:4, H0141:3, H0553:3,





S0126:3, H0539:3, L0750:3, H0556:2, H0717:2, H0581:2, S0440:2, S0344:2, L0771:2, L0774:2, L0664:2, S0380:2, H0521:2,





L0751:2, L0755:2, L3643:1, H0650:1, H0306:1, S0420:1, L0617:1, S0444:1, S0360:1, H0580:1, S0046:1, H0619:1, H0549:1,





H0486:1, T0039:1, L0021:1, H0274:1, H0457:1, H0012:1, H0620:1, S0003:1, S0214:1, H0615:1, H0628:1, H0087:1, H0551:1,





S0438:1, S0422:1, H0529:1, L0770:1, L0761:1, L0767:1, L0768:1, L0804:1, L0515:1, L0809:1, H0703:1, H0711:1, H0672:1,





S0378:1, H0522:1, H0696:1, H0555:1, S3014:1, L0754:1, L0747:1, L0749:1, L0731:1, H0445:1, S0436:1, L0581:1, S0026:1,





H0543:1 and H0423:1.

43
HDPGE24
801947
53
H0555:8, S0002:7, L0748:6, H0556:5, H0179:5, L0369:5, S0222:4, S0474:4, S0045:3, H0427:3, H0599:3, H0575:3, H0271:3,





H0628:3, H0598:3, S0426:3, L0766:3, L0581:3, H0265:2, S0114:2, S0212:2, H0402:2, S0442:2, S0354:2, S0132:2, H043 1:2,





H0370:2, H0632:2, H0581:2, H0196:2, H0050:2, H0124:2, L0665:2, H0521:2, S0390:2, S0028:2, L0777:2, H0444:2, S0436:2,





H0423:2, L3643:1, S0040:1, L0002:1, H0381:1, S0116:1, H0255:1, H0662:1, S0360:1, H0676:1, H0580:1, H0729:1, H0722:1,





H0728:1, S0046:1, H0749:1, S0300:1, L0717:1, L3388:1, H0586:1, H0333:1, H0486:1, H0706:1, H0036:1, T0048:1, H0318:1,





H0251:1, H0309:1, H0121:1, H0544:1, S0050:1, H0375:1, H0266:1, S0003:1, S0214:1, H0252:1, H0031:1, H0644:1, H0708:1,





H0400:1, H0063:1, H0264:1, S0038:1, H0280:1, H0334:1, H0625:1, S0440:1, H0509:1, H0132:1, 50210:1, L0803:1, L0525:1,





L0555:1, L0529:1, L0367:1, L0532:1, S0052:1, S0428:1, S0216:1, H0547:1, H0519:1, S0126:1, H0134:1, S0406:1, H0727:1,





H0345:1, S0037:1, L0740:1, L0749:1, S0031:1, H0445:1, H0707:1, L0605:1, L0604:1, L0601:1 and H0543:1.

44
HDPIU94
813352
54
AR055:17, AR277:13, AR060:12, AR316:9, AR219:8, AR240:8, AR089:8, AR300:8, AR218:8, AR039:7, AR283:7, AR096:6,





AR282:5, AR104:5, AR185:4, AR299:4, AR313:2 L0748:6, L0666:5, L0665:5, L0768:4, L0777:4, L0595:4, H0352:4, S0045:3,





H0124:3, L0774:3, S0028:3, L0439:3, L0756:3, L0592:3, S0376:2, S0360:2, H0619:2, S0222:2, L3816:2, H0635:2, H0036:2,





H0052:2, H0046:2, L0041:2, S0312:2, H0551:2, L3815:2, L0764:2, L0663:2, H0144:2, L3825:2, L0751:2, L0754:2, L0745:2,





L0731:2, L0589:2, H0653:2, H0136:2, H0216:2, H0624:1, S6024:1, S0430:1, H0656:1, H0255:1, S0046:1, H0747:1, H0645:1,





L2759:1, H0013:1, H0156:1, H0575:1, H0050:1, S0050:1, H0373:1, H0687:1, S0314:1, S0250:1, H0031:1, H0135:1, H0634:1,





H0616:1, H0380:1, H0264:1, H0433:1, H0059:1, L0351:1, S0422:1, L0800:1, L0662:1, L0626:1, L0766:1, L0803:1, L0375:1,





L0655:1, L0659:1, L0783:1, L0809:1, L0664:1, L2263:1, L2258:1, L2259:1, H0726:1, L3826:1, L3827:1, H0648:1, S0152:1,





L3833:1, H0521:1, S0390:1, S3014:1, S0027:1, L0749:1, L0750:1, L0780:1, L0759:1, L0759:1, S0260:1 and L0366:1.

45
HDPIY31
886159
55
AR214:26, AR263:25, AR224:21, AR222:20, AR264:20, AR223:19, AR169:18, AR221:18, AR217:18, AR171:17, AR172:17,





AR195:17, AR207:16, AR170:16, AR311:16, AR235:16, AR215:16, AR225:16, AR168:15, AR216:15, AR165:14, AR197:14,





AR164:14, AR162:14, AR308:13, AR089:13, AR161:13, AR166:13, AR212:13, AR192:13, AR193:13, AR163:13, AR213:12





AR033:12, AR309:12, AR242:12, AR053:11, AR245:11, AR312:11, AR210:10, AR282:10, AR253:10, AR261:10, AR254:10





AR277:10, AR198:10, AR104:10, AR295:10, AR288:9, AR240:9, AR316:9, AR299:9, AR219:9, AR196:9, AR205:9, AR271:9,





AR252:9, AR285:9, AR250:9, AR297:9, AR272:8, AR060:8, AR096:8, AR177:8, AR269:8, AR274:8, AR246:8, AR313:8,





AR236:8, AR211:8, AR185:8, AR286:8, AR039:7, AR291:7, AR287:7, AR275:7, AR200:7, AR300:7, AR055:7, AR229:7,





AR181:7, AR283:7, AR189:7, AR218:7, AR175:7, AR174:7, AR247:7, AR238:7, AR199:6, AR289:6, AR188:6, AR293:6,





AR243:6, AR191:6, AR173:6, AR266:6. AR262:6, AR204:6, AR270:6, AR226:6, AR258:6, AR201:6, AR176:6, AR268:5,





AR296:5, AR257:5, AR183:5, AR182:5, AR234:5, AR231:5, AR180:5, AR255:5, AR230:5, AR178:5, AR256:5, AR290:5,





AR190:5, AR239:5, AR232:5, AR260:4, AR227:4, AR203:4, AR294:4, AR267:4, AR061:4, AR179:4, AR237:4, AR233:3,





AR228:3 L0439:56, L0438:20, H0556:7, H0052:7, L0776:5, S0222:4, H0438:4, S0418:3, S0278:3, L0770:3, L0771:3, L0743:3,





L0366:3, H0265:2, S0040:2, L0415:2, S0045:2, H0619:2, H0492:2, H0486:2, H0581:2, H0620:2, H0266:2, H0604:2, H0031:2,





H0100:2, L0351:2, H0144:2, L0352:2, H0672:2, H0521:2, L0756:2, L0777:2, L0731:2, H0624:1, H0140:1, H0583:1, H0255:1,





H0402:1, H0305:1, H0458:1, S0420:1, S0354:1, S0358:1, H0645:1, S6022:1, H0392:1, H0643:1, H0559:1, H0013:1, H0069:1,





H0156:1, H0590:1, S0346:1, H0085:1, H0544:1, H0545:1, H0439:1, H0150:1, H0041:1, S0388:1, S0051:1, T0010:1, H0271:1,





H0416:1, H0188:1, H0288:1, S0022:1, T0006:1, H0213:1, H0628:1, H0617:1, H0135:1, H0087:1, H0551:1, H0477:1, H0059:1,





S0038:1, L0435:1, T0042:1, H0494:1, S0344:1, S0426:1, L0640:1, L0769:1, L0638:1, L0761:1, L0642:1, L0764:1, L0768:1,





L0794:1, L0803:1, L0375:1, L0806:1, L0805:1, L0655:1, L0659:1, L0809:1, L0789:1, L0665:1, H0519:1, S0126:1, H0690:1,





H0682:1, S0330:1, H0539:1, H0522:1, S0037:1, L0751:1, L0754:1, L0746:1, L0749:1, L0786:1, L0779:1, H0445:1, L0596:1,





H0542:1 and H0543:1.

46
HDPOC24
777493
56
H0585:26, H0141:12, L0666:9, L0754:9, L0755:9, S0212:6, L0663:5, L0743:5, S0356:4, H0587:4, H0553:4, L0657:4,





L0382:4, L0740:4, L0747:4, S0045:3, S0046:3, H0024:3, L0771:3, L0648:3, L0662:3, L0659:3, L0664:3, S0126:3, H0522:3,





L0748:3, L0777:3, L0757:3, S0192:3, S0040:2, S0420:2, S0442:2, S0358:2, S0476:2, H0550:2, H0497:2, H0250:2, H0575:2,





H0052:2, H0546:2, H0266:2, H0100:2, H0646:2, S0002:2, L0763:2, L0649:2, L0803:2, L0775:2, L0653:2, L0517:2, L0809:2,





L0790:2, L0665:2, H0660:2, S0380:2, H0521:2, S3014:2, S0028:2, L0751:2, S0436:2, H0665:2, S0430:1, H0341:1, S0282:1,





H0664:1, S0418:1, S0354:1, S0444:1, H0549:1, S0222:1, H0600:1, H0333:1, H0618:1, H0253:1, S0474:1, H0581:1, H0235:1,





H0597:1, H0545:1, H0009:1, H0081:1, H0620:1, H0023:1, H0687:1, S0250:1, L0483:1, T0006:1, L0055:1, H0087:1, H0551:1,





H0379:1, H0264:1, H0494:1, H0625:1, S0352:1, H0641:1, H0529:1, L0371:1, L0769:1, L5575:1, L3905:1, L5566:1, L0772:1,





L0800:1, L0764:1, L0773:1, L0794:1, L0386:1, L0378:1, L0806:1, L0807:1, L0792:1, L0565:1, S0310:1, H0519:1, H0682:1,





H0684:1, H0670:1, H0672:1, S0328:1, S0330:1, S0332:1, H0478:1, S0432:1, S3012:1, S0390:1, S0206:1, L0742:1, L0756:1,





L0779:1, H0707:1, S0434:1, L0596:1, H0668:1, S0242:1, H0506:1 and H0008:1.

47
HDPPD93
637588
57
AR202:68, AR194:68, AR281:64, AR244:59, AR315:56, AR205:52, AR246:50, AR280:49, AR283:45, AR314:39, AR271:38,





AR232:37, AR243:37, AR241:35, AR316:34, AR282:33, AR204:33, AR263:32, AR089:32, AR192:32, AR265:31, AR277:31,





AR206:30, AR219:29, AR310:29, AR033:29, AR096:29, AR313:28, AR299:28, AR240:26, AR247:26, AR273:24, AR300:24,





AR198:24, AR295:24, AR274:24, AR218:24, AR039:23, AR275:23, AR055:23, AR213:23, AR104:22, AR251:22, AR238:20,





AR177:20, AR312:20, AR060:19, AR226:19, AR052:19, AR231:18, AR053:18, AR309:18, AR234:18, AR227:18, AR185:17,





AR292:17, AR237:17, AR229:16, AR258:16, AR183:16, AR175:15, AR294:14, AR256:13, AR259:13, AR233:13, AR293:11,





AR186:11, AR253:10, AR061:10, AR266:10, AR267:9, AR285:8, AR248:8, AR270:8, AR296:8, AR284:7, AR179:7, AR289:7,





AR249:7, AR268:6, AR269:6, AR291:6, AR184:6, AR298:5, AR286:5, AR182:5, AR290:4 L0794:6, L0748:6, H0556:5,





L0771:5, H0052:4, L0756:4, L0596:4, H0265:3, H0341:3, H0587:3, L0662:3, L0803:3, L0790:3, S0152:3, L0750:3, S0114:2,





S0360:2, H0318:2, L0471:2, L0369:2, L0763:2, L0770:2, L0764:2, L0766:2, L0774:2, L0378:2, L0789:2, L0666:2, L3825:2,





H0547:2, L0747:2, L0777:2, L0581:2, H0543:2, H0422:2, S0218:1, H0255:1, S0418:1, S0354:1, S0376:1, S0408:1, L3649:1,





S0045:1, H0747:1, H0619:1, L0717:1, S0222:1, H0431:1, H0586:1, H0013:1, H0069:1, S0049:1, H0009:1, H0071:1, H0083:1,





H0428:1, T0006:1, H0424:1, H0213:1, H0644:1, H0628:1, H0135:1, H0163:1, H0616:1, H0413:1, H0059:1, H0561:1, S0448:1,





H0647:1, L3818:1, S0002:1, L0769:1, L0500:1, L0363:1, L0767:1, L0768:1, L0649:1, L0804:1, L0806:1, L0657:1, L0512:1,





L0659:1, L0384:1, L0647:1, L5622:1, L5623:1, L0664:1, L0665:1. S0374:1, L3828:1, S0126:1, H0711:1, H0658:1, H0666:1,





H0539:1, H0753:1, H0521:1, H0522:1, S0406:1, H0555:1, H0436:1, L0439:1, L0749:1, S0031:1, L0595:1, H0136:1, H0542:1,





H0423:1, S0424:1 and H0352:1.

48
HDPPQ30
684292
58
H0542:4, S0250:3, H0521:3, H0522:3, H0485:2, H0486:1, H0494:1 and H0543:1.

49
HDQHM36
852328
59
AR313:50, AR039:48, AR277:28, AR089:26, AR096:26, AR300:25, AR185:23, AR299:22, AR240:17, AR316:17, AR104:15,





AR219:12, AR282:12, AR.218:12, AR060:10, AR055:6, AR283:5 H0521:2

50
HDTFX18
801957
60
AR313:6, AR277:5, AR039:4, AR096:4, AR055:3, AR283:3, AR300:3, AR282:3, AR316:3, AR104:2, AR299:2, AR240:2,





AR060:2, AR185:2, AR089:2, AR218:1 L0748:2, L0731:2, H0486:1, H0634:1, L0766:1, L0809:1, L0750:1 and L0777:1.

51
HE2CM39
553651
61
AR277:46, AR283:32, AR219:30, AR313:29, AR218:25, AR316:25, AR089:24, AR299:23, AR104:23, AR282:23, AR055:21,





AR300:20, AR185:18, AR039:18, AR096:17, AR240:17, AR060:13 L0759:4, L0657:3, L0789:3, L0439:3, L0752:3, L0758:3,





S0360:2, L0805:2, L0438:2, L0750:2, L0777:2, H0423:2, H0171:1, H0638:1, H0351:1, H0178:1, H0606:1, L0625:1, L0769:1,





L0771:1, L0662:1, L0794:1, L0803:1, L0804:1, L0650:1, L0774:1, L0659:1, L0809:1, L0663:1, H0436:1, L0748:1, L0740:1,





H0445:1, L0604:1 and H0422:1.

52
HE2HC60
753265
62
AR277:44, AR283:36, AR219:36, AR218:34, AR055:31, AR316:29, AR313:24, AR089:24, AR104:23, AR282:22, AR299:21,





AR039:19, AR240:19, AR096:18, AR185:18, AR300:18, AR060:14 L0439:13, L0777:9, L0717:8, L0748:6, L0659:5, L0747:4,





H0318:3, L0665:3, L0779:3, H0170:2, H0212:2, L0455:2, S0422:2, L0764:2, L0662:2, L0768:2, L0766:2, L0775:2, L0655:2,





L0809:2, H0520:2, H0672:2, L0746:2, L0755:2, L0758:2, L0759:2, L0595:2, H0624:1, H0171:1, H0685:1, H0661:1, H0402:1,





S0408:1, L3646:1, S0046:1, H0333:1, T0109:1, H0013:1, S0280:1, L0021:1, H0590:1, H0581:1, H0374:1, H0596:1, L0471:1,





H0014:1, S0051:1, S0003:1, H0328:1, H0617:1, H0040:1, H0412:1, H0494:1, H0641:1, L0761:1, L0645:1, L0773:1, L0521:1,





L0375:1, L0651:1, L0805:1, L0776:1, L0526:1, L0783:1, L0789:1, L0666:1, L0664:1, H0701:1, H0723:1, L0352:1, H0547:1,





H0658:1, H0670:1, H0648:1, H0651:1, H0436:1, L0740:1, L0754:1, L0752:1, L0757:1, S0436:1, L0591:1, L0592:1 and





H0293:1.

53
HE2PO93
771655
63
AR219:19, AR218:19, AR313:13, AR299:13, AR185:13, AR059:11, AR055:10, AR316:10, AR060:10, AR300:9, AR096:8,





AR104:7, AR039:7, AR240:6, AR282:6, AR283:5, AR277:3 L0803:5, L0731:5, S0422:4, L2903:3, S0408:2, H0040:2,





L0766:2, L0666:2, L2657:2, H0144:2, H0648:2, L0748:2, L0439:2, L0754:2, L0779:2, H0170:1, H0171:1, S0114:1, H0657:1,





L2285:1, S0354:1, S0360:1, H0580:1, H0742:1, H0741:1, H0749:1, L2777:1, L0717:1, H0411:1, H0431:1, H0586:1, H0052:1,





H0596:1, H0014:1, S0388:1, S0051:1, S0003:1, H0591:1, 50042:1, H0625:1, H0509:1, L0598:1, H0026:1, L0763:1, L0639:1,





L0372:1, L0646:1, L0641:1, L0768:1, L0649:1, L0651:1, L0805:1, L0776:1, L0635:1, L0664:1, L0665:1, L2264:1, L2262:1,





S0374:1, L0438:1, L0352:1, H0672:1, S0380:1, H0696:1, H0134:1, S0406:1, H0478:1, L0758:1, L0759:1, S0436:1, S0011:1 and





S0424:1.

54
HE6FU11
827236
64
AR089:8, AR104:6, AR039:6, AR096:5, AR060:5, AR313:5, AR185:5, AR055:4, AR284:4, AR266:4, AR316:4, AR282:4,





AR292:4, AR299:4, AR202:4, AR218:3, AR289:3, AR283:3, AR298:3, AR277:3, AR182:3, AR184:3, AR280:3, AR250:3,





AR219:3, AR315:3, AR281:3, AR300:3, AR169:3, AR294:3, AR240:3, AR291:3, AR268:2, AR172:2, AR033:2, AR270:2,





AR248:2, AR310:2, AR175:2, AR238:2, AR241:2, AR265:2, AR232:2, AR285:2, AR183:2, AR177:2, AR227:2, AR269:2,





AR263:2, AR290:2, AR288:2, AR293:2, AR229:2, AR267:2, AR061:2, AR176:2, AR257:2, AR271:2, AR296:2, AR286:2,





AR272:2, AR259:1, AR206:1, AR256:1, AR214:1, AR231:1, AR247:1, AR295:1, AR204:1, AR237:1, AR226:1, AR234:1,





AR308:1, AR253:1, AR053:1, AR311:1, AR205:1, AR243:1, AR233:1, AR173:1, AR312:1, AR251:1, AR314:1 L0759:2,





H0706:1, H0123:1, H0024:1, H0100:1, L07941 and L0789:1.

55
HE6FV29
588454
65
AR219:37, AR218:36, AR315:34, AR280:34, AR271:33, AR244:29, AR089:29, AR314:29, AR243:28,





AR281:26, AR282:25, AR273:25, AR205:24, AR192:22, AR206:22, AR198:19, AR247:19, AR316:19,





AR039:19, AR231:18, AR269:17, AR246:17, AR204:16, AR234:16, AR299:16, AR313:15, AR194:15,





AR055:14, AR186:14, AR237:14, AR060:14, AR241:13, AR270:13, AR293:12, AR240:12, AR232:11,





AR238:11, AR251:10, AR300:10, AR061:10, AR233:10, AR227:10, AR291:9, AR185:9, AR202:9,





AR266:9, AR226:9, AR229:9, AR184:8, AR179:8, AR182:8, AR175:8, AR312:8, AR268:8, AR289:7,





AR284:7, AR249:7, AR183:7, AR310:7, AR267:7, AR052:7, AR033:7, AR296:7, AR290:7, AR265:7,





AR177:7, AR309:7, AR292:6, AR298:6, AR275:6, AR277:6, AR285:6, AR294:5, AR248:5, AR053:5,





AR253:5, AR295:5, AR286:5, AR259:5, AR274:4, AR258:4, AR213:4, AR096:4, AR256:4, AR104:4,





AR283:3, AR263:2 S0440:32, S0476:22, H0494:20, L0754:17, S0372:16, S0132:13, L0666:13, S0330:13, H0046:12,





H0586:11, H0587:11, S0328:11, S0360:10, S0436:9, S0356:8, H0622:8, S0003:7, L0806:7, H0648:7, L0747:7, L0752:7,





H0674:6, L0777:6, L0362:6, L0662:5, L0659:5, L0601:5, S0430:4, S0358:4, S0408:4, H0592:4, S0214:4, H0039:4, H0031:4,





H0551:4, H0264:4, H0S60:4, L0763:4, L0653:4, L5623:4, L0663:4, S0376:3, S0444:3, S0410:3, H0370:3, H0600:3, H0644:3,





L0646:3, L0649:3, L0776:3, L0783:3, L0809:3, L0665:3, H0696:3, S0406:3, S3014:3, L0755:3, S0434:3, L0591:3, H0170:2,





S134:2, H0662:2, S0442:2, H0393:2, H0596:2, H0597:2, H0688:2, H0553:2, H0032:2, H0169:2, H0598:2, H0090:2, H0379:2,





H0380:2, L0770:2, L0372:2, L0549:2, L0376:2, L0517:2, L0518:2, L5622:2, H0658:2, H0670:2, S0380:2, S0152:2, S0350:2,





S0027:2, L0744:2, L0779:2, L0759:2, L0599:2, S0196:2, S0456:2, H0171:1, H0556:1, T0002:1, H0713:1, H0483:1, H0663:1,





L0005:1, S0354:1, T0008:1, H0742:1, H0741:1, H0411:1, H0549:1, T0039:1, H0013:1, L0021:1, H0349:1, S0010:1, H0204:1,





L0738:1, H0545:1, H0014:1, H0015:1, H0373:1, H0355:1, H0510:1, H0615:1, L0483:1, L0142:1, L0143:1, H0166:1, H0673:1,





H0708:1, H0591:1, H0038:1, H0040:1, H0634:1, T0067:1, H0272:1, H0487:1, H0412:1, H0623:1, H0059:1, H0100:1, S0352:1,





S0382:1, S0448:1, S0306:1, S0438:1, S0472:1, H0646:1, L0503:1, L0640:1, L0637:1, L0761:1, L0772:1, L0764:1, L0771:1,





L0648:1, L0794:1, L5564:1, L0551:1, L0805:1, L0382:1, L0519:1, L0789:1, L0532:1, L0664:1, H0144:1, H0520:1, H0547:1,





S0126:1, H0689:1, H0711:1, H0435:1, H06591, H06661, S03781, H07041, S0044:1, H0555:1, S0392:1, S0322:1, L0748:1,





L0740:1, L0745:1, L0749:1, L0756:1, L0757:1 and S0242:1.

56
HE8TY46
899528
66
AR226:12, AR227:11, AR238:10, AR237:10, AR175:9, AR183:9, AR232:9, AR234:8, AR182:8, AR233:7, AR291:7, AR293:7,





AR269:7, AR104:7, AR184:7, AR060:6, AR298:6, AR059:6, AR270:6, AR292:6, AR285:6, AR268:6, AR179:6, AR295:5,





AR290:5, AR284:5, AR289:5, AR294:5, AR055:5, AR316:5, AR229:5, AR231:5, AR296:5, AR185:5, AR247:5, AR096:5,





AR240:5, AR265:5, AR313:5, AR256:5, AR259:4, AR266:4, AR219:4, AR282:4, AR258:4, AR251:4, AR061:4, AR299:4,





AR177:4, AR309:4, AR286:4, AR267:4, AR033:4, AR300:3, AR039:3, AR244:3, AR218:3, AR310:3, AR283:3, AR277:3,





AR253:3, AR213:2, AR312:2, AR186:2, AR271:1, AR052:1, AR314:1 H0253:8, L0439:8, L0769:7, H0618:6, L0758:6,





H0052:5, L0749:5, H0617:4, H0135:4, L0766:4, S0406:4, S0001:3, H0255:3, S0410:3, H0619:3, L3655:3, S0422:3; L0775:3,





L0378:3, H0547:3, H0521:3, L0742:3, L0750:3, L0755:3, L0757:3, S0434:3, L0605:3, H0381:2, H0419:2, H0341:2, S0420:2,





H0733:2, H0749:2, H0550:2, H0438:2, H0599:2, H0318:2, H0046:2, H0050:2, H0012:2, H0024:2, S0050:2, T0010:2, L0455:2,





H0412:2, H0413:2, H0494:2, L0772:2, L0645:2, L0764:2, L0771:2, L0662:2, L0666:2, L0665:2, L0438:2, H0520:2, H0519:2,





H0134:2, L0741:2, L0748:2, L0751:2, L0747:2, L0777:2, L0759:2, H0445:2, L0596:2, L0603:2, L0411:1, H0556:1, S0114:1,





S0218:1, H0656:1, S0116:1, H0125:1, S0418:1, S0354:1, S0360:1, H0729:1, H0730:1, H0741:1, H0722:1, H0728:1, H0747:1,





H0771:1, L0717:1, S0278:1, H0549:1, H0370:1, H0392:1, H0613:1, H0013:1, H0427:1, H0575:1, T0082:1, H0706:1, H0036:1,





H0421:1, S0049:1, H0194:1, H0085:1, H0231:1, L0041:1, H0041:1, H0009:1, H0123:1, H0620:1, H0199:1, H0246:1, H0014:1,





L0163:1, H0594:1, S6028:1, H0266:1, H0188:1, H0687:1, H0288:1, H0033:1, H0181:1, S0364:1, S0366:1, S0036:1, H0038:1,





H0616:1, H0264:1, H0268:1, H0117:1, S0038:1, H0100:1, L0351:1, L0435:1, T0041:1, T0042:1, S0448:1, S0142:1, S0002:1,





H0529:1, L0796:1, L0639:1, L3904:1, L5575:1, L3905:1, L5566:1, L0761:1, L0374:1, L0648:1, L0768:1, L0649:1, L0803:1,





L0375:1, L0805:1, L0776:1, L0655:1, L0659:1, L0526:1, L0783:1, L5622:1, L0793:1, L0709:1, L3821:1, L2257:1, L2259:1,





L0710:1, L2261:1, L2264:1, L2262:1, L2654:1, H0144:1, H0690:1, H0660:1, S0330:1, H0539:1, S0378:1, S0152:1, H0522:1,





H0694:1, H0555:1, H0436:1, S3012:1, S0390:1, S3014:1, S0028:1, L0743:1, L0779:1, L0752:1, H0444:1, S0436:1, L0581:1,





H0543:1, H0423:1, S0458:1 and H0506:1.

57
HE9EA10
827796
67
L0794:12, H0620:3, L0756:2, L0759:2, S0408:1, S0049:1, H0544:1, H0012:1, H0615:1, H0040:1, L0764:1, L0803:1, L0806:1,





L0789:1, H0144:1, H0547:1, L0779:1, L0597:1 and L0595:1.

58
HEBCY54
600355
68
AR104:9, AR277:6, AR283:5, AR055:4, AR096:4, AR060:4, AR240:4, AR282:2, AR316:2, AR185:2, AR300:2, AR299:2,





AR089:2, AR039:2, AR313:1, AR218:1 L0438:3, L0748:3, T0010:2, L0351:2, L0769:2, H0521:2, L0439:2, L0747:2, S0116:1,





S0354:1, S0007:1, H0619:1, H0253:1, H0565:1, H0135:1, L0641:1, L0521:1, L0774:1, L0809:1, L0789:1, H0520:1, L0755:1,





L0758:1 and H0445:1.

59
HEBFR46
847064
69
AR313:58, AR039:47, AR300:30, AR096:29, AR299:29, AR277:28, AR089:27, AR185:27, AR316:22, AR219:22, AR104:21,





AR218:20, AR240:20, AR282:15, AR060:15, AR055:11, AR283:7 H0457:10, H0550:5, H0436:5, H0549:4, H0616:4, L0519:4,





H0556:3, H0580:3, S0007:3, S0046:3, L0809:3, L0747:3, L0777:3, S0436:3, H0295:2, T0040:2, H0266:2, L0761:2, L0783:2,





L0789:2, H0658:2, H0521:2, L0753:2, L0731:2, L0596:2, H0543:2, S0040:1, S0116:1, S0282:1, H0662:1, H0402:1, H0125:1,





L0534:1, L0562:1, S0356:1, S0358:1, H0749:1, L3816:1, H0559:1, H0069:1, H0599:1, H0618:1, H0253:1, H0581:1, H0546:1,





H0123:1, S0051:1, H0083:1, H0687:1, H0284:1, H0124:1, H0038:1, H0551:1, H0623:1, S0038:1, T0041:1, S0440:1, S0150:1,





L3818:1, S0002:1, L0763:1, L0769:1, L5575:1, L0627:1, L0800:1, L0662:1, L0803:1, L0793:1, L0666:1, L2264:1, L3825:1,





L3827:1, L3828:1, H0547:1, H0519:1, H0539:1, S0037:1, S0206:1, L0748:1, L0749:1, H0595:1, L0593:1, S0194:1 and S0276:1.

60
HEBGE07
798096
70
S0007:1

61
HEGAU15
834379
71
AR299:9, AR039:8, AR300:8, AR055:7, AR240:7, AR060:7, AR313;6, AR282:6, AR277:6, AR104:5, AR059:5, AR096:5,





AR185:5, AR316:4, AR283:4, AR218:4, AR219:3 H0550:2, L0749:2, H0318:1 and H0555:1.

62
HBTC116
844543
72
AR282:28, AR055:21, AR060:18, AR219:17, AR089:16, AR218:15, AR104:14, AR299:14, AR277:14, AR185:14, AR300:13,





AR240:11, AR283:10, AR316:9, AR096:9, AR039:9, AR313:4 H0046:6, L0747:6, L0756:6, L0803:5, L0740:5, L0662:4,





L0748:4, S0360:3, H0620:3, H0014:3, H0674:3, L0774:3, L5622:3, L0439:3, S0408:2, H0431:2, L0761:2, L0794:2, L0663:2,





H0659:2, L0751:2, L0779:2, L0596:2, L0588:2, T0049:1, S0442:1, S0376:1, S0444:1, S0468:1, S0045:1, S0476:1, H0645:1,





H0549:1, H0550:1, T0109:1, H0013:1, H0156:1, H0599:1, H0575:1, T0048:1, H0196:1, H0544:1, H0050:1, H0510:1, H0292:1,





H0039:1, H0135:1, H0616:1, S0016:1, L0640:1, L0770:1, L0637:1, L3905:1, L0388:1, L0805:1, L0776:1, L0659:1, L0809:1,





L0790:1, L0792:1, L0666:1, L0664:1, H0144:1, L0438:1, H0547:1, H0519:1, H0689:1, H0672:1, S0328:1, H0521:1, H0627:1,





S3014:1, S0027:1, S0028:1, L0780:1, L07571, L07581, S00261 and H0506:1.

63
HFCEI04
692438
73
AR055:7, AR060:7, AR104:6, AR240:6, AR282:6, AR218:5, AR185:5, AR283:5, AR089:4, AR300:4, AR313:3, AR299:3,





AR316:3, AR096:3, AR039:3, AR277:3, AR219:2 H0009:3

64
HFCFE20
701985
74
H0052:2, H0560:2, H0529:2, L0470:1, S0212:1, H10305:1, S0420:1, S0356:1, H0550:1, S0222:1, H0497:1, S0010:1, H0251:1,





H0009:1, H0024:1, H0083:1, H0290:1, H0379:1, H0264:1, S0150:1, S0426:1, L3905:1, H0520:1, H0547:1, S0044:1, S3014:1,





S0434:1, L0581:1, L0604:1, H0136:1, H0423:1 and S0424:1.

65
HFPCZ55
840840
75
L0756:6, L0439:4, L0777:4, L0662:3, H0672:3, S0358:2, L0659:2, L0666:2, S0031:2, S0360:1, H0411:1, H0369:1, S0222:1,





S0220:1, S0005:1, H0575:1, T0082:1, H0050:1, S6028:1, H0169:1, H0100:1, L0769:1, L0774:1, L0776:1, L0647:1, L0663:1,





H0660:1, H0651:1, S0146:1, L0743:1, L07571, L03611 and L0462:1.

66
HFPDR62
839400
76
S0222:2, S0114:1, H0305:1, H0449:1 and T0039:1.

67
HFPDS07
821646
77
AR060:37, AR104:33, AR299:19, AR039:13, AR316:12, AR313:1 1, AR185:11, AR055:10, AR096:10, AR277:9, AR218:8,





AR240:7, AR089:7, AR300:6, AR282:5, AR283:4, AR219:2 L0803:24, L0439:13, H0052:5, L0804:5, L0774:5, H0090:4,





L0659:4, H0521:4, L0751:4, S0222:3, H0486:3, H0622:3, L0766:3, H0144:3, S0126:3, H0656:2, S0360:2, H0580:2, H0575:2,





S0346:2, H0046:2, L0455:2, S0036:2, H0623:2, S0002:2, L0775:2, L0607:2, L0790:2, L0438:2, L0748:2, L0740:2, L0752:2,





L0757:2, L0759:2, H0422:2, H0222:1, L3659:1, S0418:1, S0356:1, H0437:1, H0587:1, H0590:1, S0010:1, S0665:1, S0049:1,





H0263:1, H0572:1, H0562:1, H0569:1, H0051:1, H0275:1, S6028:1, S0003:1, H0252:1, H0400:1, H0591:1, H0551:1, H0264:1,





H0488:1, H0056:1, L0351:1, L0370:1, S0438:1, S0422:1, L0637:1, L0646:1, L0662:1, L0809:1, L0647:1, L0367:1, L0666:1,





L0665:1, H0701:1, L3811:1, L3824:1, H0547:1, H0648:1, S0152:1, H0522:1, S0406:1, H0436:1, S0028:1, L0777:1, L0755:1,





L0758:1, S0260:1, S0436:1, L0366:1, S0196:1 and H0542:1.

68
HFVHW43
570948
78
H0393:1

69
HFXAV37
626595
79
AR313:13, AR039:13, AR300:8, AR299:7, AR277:6, AR096:6, AR185:5, AR059:5, AR218:4, AR104:4, AR316:4, AR282:4,





AR060:3, AR240:2, AR055:2, AR219:2, AR283:1 S0002:2, S0134:1, S0001:1 and L0589:1.

70
HFXFZ46
600361
80
AR218:9, AR219:9, AR185:2, AR039:2, AR104:1, AR055:1, AR060:1 S0001:1

71
HGBHP91
693011
81
AR313:32, AR039:24, AR299:22, AR089:21, AR185:17, AR096:16, AR060:15, AR316:13, AR300:13, AR277:13, AR240:12,





AR218:11, AR055:11, AR104:11, AR282:9, AR219:9, AR283:5 H0014:1

72
HHEAK45
765278
82
AR277:12, AR283:11, AR313:11, AR316:9, AR282:9, AR089:7, AR240:7, AR104:7, AR299:7, AR039:7, AR218:6, AR096:6,





AR055:6, AR300:6, AR185:6, AR060:4, AR219:3 L0758:9, L0748:6, L0747:6, L0779:5, L0750:4, H0556:3, S0440:3, H0658:3





H0656:2, L0770:2, L0769:2, L0804:2, L0774:2, H0144:2, H0648:2, L0439:2, L0749:2, L0596:2, H0265:1, S0444:1, H0318:1,





H0597:1, H0050:1, H0024:1, H0135:1, H0090:1, H0038:1, H0616:1, H0494:1, L0065:1, S0422:1, H0529:1, L0637:1, L0764:1,





L0768:1, L0794:1, L0387:1, L0803:1, L0805:1, L0809:1, L0788:1, L0790:1, L0664:1, L0438:1, H0555:1, L0780:1, L0731:1,





H0444:1, H0542:1, H0543:1 and H0423:1.

73
HHEOW19
886174
83
AR169:30, AR089:25, AR207:25, AR308:25, AR214:24, AR263:24, AR165:24, AR264:24, AR164:23, AR161:23, AR168:23,





AR222:23, AR171:22, AR283:22, AR166:22, AR162:22, AR311:21, AR163:21, AR096:21, AR223:21, AR213:19, AR104:19,





AR316:19, AR219:19, AR218:18, AR217:18, AR312:18, AR212:18, AR225:17, AR309:17, AR282:17, AR313:17, AR039:17,





AR272:17, AR299:16, AR216:16, AR060:15, AR172:15, AR274:15, AR083:15, AR170:15, AR240:14, AR055:14, AR185:14,





AR195:13, AR277:13, AR197:13, AR235:13, AR295:12, AR192:12, AR224:12, AR296:11, AR297:11, AR246:11, AR285:10,





AR198:10, AR245:10, AR293:10, AR252:10, AR288:10, AR205:10, AR300:10, AR221:9, AR242:9, AR287:9, AR201:9,





AR247:9, AR253:9, AR033:9, AR266:9, AR215:9, AR275:8, AR291:8, AR174:8, AR261:8, AR193:8, AR243:8, AR271:8,





AR177:8, AR270:8, AR254:8, AR289:7, AR236:7, AR286:7, AR175:6, AR204:6, AR269:6, AR189:6, AR294:6, ARI8O:6,





AR178:5, AR250:5, AR183:5, AR199:5, AR257:5, AR181:5, AR179:5, AR268:5, AR262:5, AR173:5, AR290:5, AR258:5,





AR255:4, AR061:4, AR210:4, AR191:4, AR190:4, AR229:4, AR196:4, AR176:4, AR188:3, AR226:3, AR239:3, AR182:3,





AR200:3, AR234:3, AR238:3, AR267:3, AR232:3, AR230:3, AR203:3, AR256:3, AR231:3, AR211:3, AR237:3, AR233:2,





AR227:2, AR260:2, AR228:1 L0748:4, L0745:4, L0775:3, L0776:3, L0758:3, H0458:2, H0050:2, S0003:2, H0529:2,





L0747:2, L0599:2, L0362:2, H05561, S0116:1, S0282:1, H0662:1, H0305:1, S0420:1, S0444:1, H0329:1, H0345:1, H0411:1,





S0278:1, H0438:1, T0039:1, H0635:1, H0156:1, H0235:1, H0327:1, L0471:1, H0428:1, H0031:1, H0644:1, H0032:1, S0366:1,





H0038:1, H0616:1, T0067:1, H0477:1, H0059:1, H0560:1, H0625:1, S0422:1, L0769:1, L0761:1, L0667:1, L0771:1, L0662:1,





L0806:1, L0655:1, L0809:1, L5622:1, L0789:1, L0790:1, L0665:1, S0052:1, H0144:1, H0520:1, H0547:1, H0519:1, H0435:1,





H0539:1, S0044:1, S0392:1, L0754:1, L0749:1, L0750:1, L0779:1, L0755:1, L0759:1, S0434:1, L0608:1, H0543:1 and S0452:1.

74
HHFFS40
824059
84
AR219:22, AR277:18, AR283:17, AR218:16, AR039:15, AR282:15, AR089:14, AR316:13, AR313:13, AR096:12, AR299:12,





AR104:12, AR240:10, AR055:10, AR300:10, AR185:9, AR060:8 S0422:7, L0748:6, L0591:6, L0766:5, L0754:5, H0423:5,





S0408:4, H0069:4, L0803:4, L0602:4, H0657:3, S0442:3, S0046:3, H0596:3, S0003:3, H0032:3, H0169:3, H0674:3, L0662:3,





L0794:3, L0526:3, H0670:3, L0740:3, L0759:3, S0134:2, S0212:2, H0661:2. S0444:2, H0046:2, L0471:2, H0355:2, H0038:2,





H0100:2, L0564:2, S0440:2, H0529:2, L0770:2, L0769:2, L0667:2, L0771:2, L0521:2, L0804:2, L0805:2, L0384:2, L0809:2,





L0665:2, H0659:2, L0743:2, L0750:2, L0731:2, S0436:2, L0592:2, L0599:2, L0608:2, L0362:2, H0171:1, H0556:1, H0686:1,





H0713:1, H0717:1, H0738:1, H0740:1, H0656:1, H0663:1, H0662:1, H0402:1, S0356:1, H0742:1, H0730:1, H0747:1, S0222:1,





H0574:1, H0632:1, H0486:1, H0013:1, H0581:1, S0049:1, H0052:1, H0194:1, H0309:1, H0263:1, H0123:1, H0050:1, H0373:1,





H0510:1, 56028:1, H0266:1, H0615:1, L0483:1, H0644:1, L0143:1, H0708:1, H0135:1, H0163:1, H0090:1, H0616:1, T0067:1,





H0488:1, H0412:1, H0059:1, H0494:1, S0382:1, S0306:1, S0450:1, H0509:1, H0641:1, H0647:1, H0646:1, L0520:1, L0763:1,





L0637:1, L0373:1, L0363:1, L5564:1, L0775:1, L0375:1, L0651:1, L0655:1, L0661:1, L0527:1, L0656:1, L0659:1, L0518:1,





L0532:1, L0663:1, L0664:1, S0374:1, H0682:1, H0658:1, H0660:1, H0672:1, H0539:1, H0521:1, S0044:1, S0406:1, H0478:1,





L0744:1, L0439:1, L0747:1, L0779:1, L0777:1, L0758:1, L0480:1, L0595:1, H0667:1, S0192:1, S0194:1, S0196:1, H0422:1 and





S0424: 1.

75
HHGCS78
634605
85
AR277:76, AR283:71, AR219:57, AR218:56, AR316:56, AR059:52, AR313:52, AR240:51, AR055:45, AR282:45, AR299:44,





AR104:41, AR096:41, AR185:34, AR039:33, AR060:31, AR300:30 L0770:7, H0333:3, L0783:2, L0731:2, H0445:2, S0418:1,





H0741: 1, S0002:1, L0369:1, L0643:1, L0764:1, L0794:1, L0803:1, L0775:1, L0375:1, L0378:1, L0655:1, L0509:1, L0666:1,





L0664:1, L0754:1, L0747:1, L0749:1, L0752:1 and L0591:1.

76
HHPSA85
658695
86
AR104:25, AR313:9, AR055:5, AR060:5, AR240:5, AR096:5, AR277:5, AR282:4, AR089:4, AR316:4, AR185:4, AR218:4,





AR300:4, AR299:3, AR039:3, AR283:2, AR219:2 L0756:5, H0051:4, L0438:4, L0759:4, S0031:4, S0007:3, S6028:3, L0666:3,





L0439:3, H0556:2, S6024:2, S0300:2, H0013:2, S0036:2, L0770:2, L0411:1, L0393:1, H0393:1, L3653:1, L3657:1, H0581:1,





H0235:1, H0327:1, H0046:1, H0009:1, L0157:1, H0201:1, S0051:1, H0399:1, H0064:1, H0038:1, H0040:1, H0634:1, H0100:1,





L0638:1, L0796:1, L0768:1, L0794:1, L0766:1, L0803:1, L06061, L07911, L07921, H0144:1, H06981, L38111, H05471,





H0519:1, H0659:1, L0779:1, L0752:1, S0260:1 and H0136:1.

77
HHSBI06
639097
87
AR218:14, AR060:11, AR282:11, AR055:11, AR089:10, AR219:10, AR185:10, AR277:10, AR039:9, AR104:9, AR299:8,





AR316:8, AR300:8, AR313:8, AR283:7, AR096:7, AR240:5 L0766:12, L0794:7, L0439:7, L0749:7, L0803:6, L0740:6,





L0745:6, H0052:5, L0754:5, L3181:4, L0770:4, L0666:4, L0748:4, H0553:3, L0790:3, L0589:3, H0543:3, S0114:2, S0134:2,





H0650:2, S0354:2, S0444:2, H0747:2, S0476:2, H0393:2, H0549:2, H0586:2, H0013:2, H0599:2, H0014:2, S0051:2, S0003:2,





H0032:2, H0674:2, H0135:2, S0142:2, L0372:2, L0800:2, L0764:2, L0805:2, L0655:2, L0657:2, L0659:2, L0809:2, L0789:2,





L0792:2, H0144:2, L0438:2, H0684:2, H0658:2, H0539:2, H0521:2, S0406:2, S0028:2, L0750:2, L0779:2, L0777:2, L0752:2,





L0731:2, L0758:2, S0436:2, H0653:2, H0542:2, H0556:1, H0716:1, H0381:1, S0116:1, H0661:1, S0356:1, S0442:1, S0360:1,





H0675:1, H0734:1, L2255:1, L3726:1, H0261:1, S0222:1, L3499:1, T0114:1, H0706:1, H0036:1, H0318:1, H0581:1, L0738:1,





H0123:1, H0620:1, S0050:1, H0015:1, H0051:1, H0355:1, H0416:1, H0286:1, H0328:1, H0428:1, H0622:1, T0006:1, H0030:1,





H0031:1, H0644:1, H0617:1, L0055:1, H0124:1, H0163:1, H0038:1, H0040:1, H0616:1, H0551:1, H0264:1, H0102:1, S0112:1,





L0564:1, H0280:1, H0494:1, H0561:1, S0440:1, H0633:1, L3815:1, S0002:1, L0763:1, L0769:1, L0761:1, L0646:1, L0642:1,





L0644:1, L0645:1, L0648:1, L0662:1, L0363:1, L0775:1, L0375:1, L0651:1, L0784:1, L0806:1, L0653:1, L0807:1, L0658:1,





L0540:1, L5622:1, L0368:1, L0665:1, L2655:1, L2257:1, L2263:1, L2258:1, L2262:1, S0374:1, H0723:1, L3811:1, L2670:1,





H0547:1, L3215:1, H0648:1, H0672:1, S0328:1, H0753:1, H0522:1, H0436:1, S0392:1, H06261, L0759:1, S0031:1, H0445:1,





S0434:1, L0596:1, L0588:1, S0192:1, H0423:1, S0424:1 and H0352:1.

78
HHSBI65
801910
88
AR176:10, AR216:9, AR217:8, AR168:8, AR169:8, AR182:8, AR16L:8, AR196:8, AR162:8, AR214:8, AR228:8, AR269:8,





AR231:8, AR233:7, AR171:7, AR207:7, AR229:7, AR181:7, AR223:7, AR163:7, AR198:7, AR165:7, AR172:7, AR225:7,





AR267:7, AR224:7, AR266:6, AR268:6, AR170:6, AR164:6, AR237:6, AR221:6, AR222:6, AR177:6, AR179:6, AR235:6,





AR270:6, AR183:6, AR204:6, AR288:6, AR053:6, AR239:6, AR193:5, AR236:5, AR250:5, AR191:5, AR264:5, AR293:5,





AR296:5, AR055:5, AR238:5, AR247:5, AR309:5, AR300:5, AR178:5, AR295:5, AR290:5, AR294:5, AR060:5, AR061:5,





AR287:5, AR257:5, AR201:5, AR282:5, AR291:5, AR175:5, AR311:4, AR261:4, AR234:4, AR289:4, AR275:4, AR262:4,





AR252:4, AR242:4, AR213:4, AR253:4, AR297:4, AR203:4, AR277:4, AR180:4, AR212:4, AR200:4, AR316:4, AR286:4,





AR274:4, AR255:4, AR312:4, AR240:4, AR174:4, AR215:4, AR039:4, AR192:4, AR263:4, AR205:4, AR283:3, AR232:3,





AR271:3, AR285:3, AR190:3, AR226:3, AR185:3, AR033:3, AR246:3, AR230:3, AR188:3, AR308:3, AR227:3, AR096:3,





AR173:3, AR313:3, AR089:3, AR195:3, AR272:3, AR199:3, AR189:3, AR260:3, AR197:3, AR299:3, AR104:2, AR210:2,





AR258:2, AR211:2, AR256:2, AR243:2, AR218:2, AR219:2 L0439:7, L0794:5, L0766:5, S0354:2, H0549:2, S0051:2,





S0142:2, L0372:2, L0809:2, L0438:2, H0658:2, H0650:1, H0381:1, S0116:1, S0356:1, S0360:1, H0261:1, H0586:1, H0486:1,





H0036:1, H0052:1, L0738:1, H0457:1, H0014:1, H0051:1, H0617:1, H0032:1, H0561:1, S0440:1, H0633:1, L0763:1, L0761:1,





L0800:1, L0644:1, L0645:1, L0764:1, L0648:1, L0655:1, L0657:1, L0658:1, L0368:1, L0665:l, L3811:1, S0044:1, S0406:1,





H0626:1, L0731:1, S0434:1, S0436:1, H0653:l and H0423:1.

79
HHSGL28
801912
89
L0439:8, L0438:3, S0440:2, L0666:2, H0170:1, S0442:1, H0318:1, S0049:1, H0052:1, H0050:1, H0057:1, S0388:1, S0214:1,





H0598:1, S0036:1. H0063:1, H0551:1, L0520:1, L0796:1, L0662:1, L0766:1, L0664:1, H0547:1, H0435:1, H0521:1, L0779:1,





L0777:1, L0752:1 and L0594:1.

80
HISAT67
843549
90
AR283:20, AR282:15, AR277:11, AR089:11, AR219:11, AR299:10, AR218:10, AR316:9, AR313:8, AR096:8, AR185:8,





AR104:7, AR240:7, AR055:6, ARO6O:6, AR039:6, AR300:6 L0751:8, L0754:6, L0731:6, H0556:5, L0766:5, L0439:5,





L0750:5, L0770:4, L0666:4, H0521:4, S0356:3, H0052:3, H0424:3, L0776:3, S0406:3, S0418:2, S0442:2, H0580:2, H0733:2,





H0486:2, H0575:2, S0438:2, L0769:2, L3905:2, L0659:2, L0663:2, L0665:2, L0748:2, L0749:2, S0436:2, T0002:1, H0159:1,





S6024:1, S0134:1, H0656:1, H0484:1, S0358:1, S0360:1, H0742:1, S0046:1, H0393:1, S0278:1, H0607:1, H0586:1, H0642:1,





H0632:1, H0427:1, L0021:1, H0599:1, H0318:1, H0746:1, H0194:1, L0738:1, H0178:1, H0566:1, H0051:1, T0010:1, H0408:1,





H0290:1, H0328:1, H0401:1, H0417:1, H0553:1, H0617:1, H0040:1, H0264:1, H0623:1, H0059:1, T0041:1, H0494:1, H0561:1,





H0509:1, S0144:1, L0763:1, L0638:1, L5565:1, L0772:1, L0373:1, L0764:1, L0662:1, L0626:1, L0363:1, L0649:1, L0650:1,





L0774:1, L0806:1, L0654:1, L0789:1, L0664:1, L3822:1, H0699:1, S0374:1, L3828:1, H0547:1, H0689:1, H0659:1, H0658:1,





H0670:1, H0672:1, H0539:1, L0740:1, L0746:1, L0752:1, L0755:1, L0757:1, L0584:1, L0596:1, L0608:1 and H0352:1.

81
HJBCU75
638329
91
AR162:12, AR161:12, AR163:11, AR186:10, AR244:10, AR273:8, AR222:8, AR225:8, AR221:8, AR214:8, AR216:8,





AR224:7, AR223:7, AR282:7, AR215:7, AR052:7, AR202:7, AR200:6, AR206:6, AR176:6, AR269:6, AR235:6, AR272:6,





AR217:6, AR055:6, AR182:6, AR264:6, AR275:6, AR061:6, AR183:5, AR168:5, AR171:5, AR309:5, AR270:5, AR228:5,





AR290:5, AR268:5, AR310:5, ARL8L:5, AR257:5, AR060:5, AR255:5, AR165:5, AR191:5, AR164:5, AR274:5, AR247:5,





AR246:4, AR166:4, AR178:4, AR311:4, AR291:4, AR312:4, AR236:4, AR173:4, AR249:4, AR233:4, AR240:4, AR288:4,





AR174:4, AR267:4, AR239:4, AR204:4, AR185:4, AR287:4, AR172:4, AR192:4, AR297:4, AR213:4, AR194:4, AR177:4,





AR294:4, AR261:4, AR184:4, AR190:4, AR170:3, AR284:3, AR262:3, AR210:3, AR188:3, AR089:3, AR313:3, AR196:3,





AR298:3, AR266:3, AR175:3, AR033:3, AR260:3, AR285:3, AR189:3, AR316:3, AR231:3, AR251:3, AR293:3, AR296:3,





AR237:3, AR286:3, AR265:3, AR243:3, AR277:3, AR300:3, AR039:3, AR289:3, AR315:3, AR179:3, AR271:3, AR234:3,





AR263:3, AR199:3, AR283:3, AR203:3, AR096:3, AR299:3, AR295:3, AR229:3, AR230:3, AR292:3, AR180:3, AR104:2,





AR198:2, AR308:2, AR219:2, AR053:2, AR218:2, AR211:2, AR205:2, AR238:2, AR241:2, AR258:2, AR256:2, AR226:2,





AR232:2, AR280:2, AR169:1, AR227:1, AR259:1, AR201:1 L0805:3, H0556:2, H0046:2, S0022:2, L0764:2, L0662:2,





L0748:2, H0013:1, H0050:1, H0039:1, H0040:1, H0087:1, T0042:1, L0643:1, L0794:1, L0803:1, L0804:1, L0807:1, L0809:1,





L0666:1, H0144:1, L0749:1, L0779:1 and L0758:1.

82
HJMAA03
824062
92
AR207:12, AR309:11, AR192:11, AR252:10, AR053:9, AR212:9, AR242:9, AR235:9, AR213:8, AR215:8, AR198:8, AR170:8,





AR169:8, AR161:8, AR162:8, AR253:8, AR223:8, AR165:8, AR166:8, AR263:7, AR163:7, AR164:7, AR274:7, AR224:7,





AR245:7, AR264:7, AR214:7, AR195:7, AR217:7, AR174:7, AR197:7, AR261:7, AR311:7, AR221:7, AR282:6, AR308:6,





AR222:6, AR240:6, AR312:6, AR205:6, AR171:6, AR168:6, AR193:6, AR313:6, AR246:6, AR177:6, AR173:6, AR277:6,





AR216:6, AR247:6, AR180:6, AR225:6, AR283:5, AR269:5, AR300:5, AR089:5, AR201:5, AR272:5, AR297:5, AR189:5,





AR204:5, AR183:5, AR299:5, AR175:5, AR288:5, AR176:5, AR295:5, AR271:5, AR250:5, AR096:5, AR275:5, AR270:4,





AR316:4, AR196:4, AR191:4, AR286:4, AR178:4, AR290:4, AR185:4, AR268:4, AR296:4, AR291:4, AR257:4, AR033:4,





AR199:4, AR181:4, AR039:4, AR236:4, AR229:4, AR243:4, AR285:4, AR254:4, AR289:4, AR238:3, AR172:3, AR293:3,





AR262:3, AR190:3, AR287:3, AR179:3, AR200:3, AR055:3, AR104:3, AR060:3, AR188:3, AR239:3, AR182:3, AR233:3,





AR258:3, AR294:3, AR061:3, AR237:3, AR231:3, AR234:3, AR226:3, AR203:3, AR255:3, AR232:3, AR230:2, AR211:2,





AR227:2, AR228:2, AR267:2, AR210:2, AR266:2, AR219:2, AR260:1, AR218:1, AR256:1 L0749:8, L0803:5, 1.0748:5,





L0777:5, L0794:4, L0766:4, L0804:4, H0135:3, H0551:3, L0754:3, L0599:3, H0542:3, H0556:2, H0545:2, H0674:2, L0764:2,





L0774:2, L0776:2, L0655:2, H0521:2, L0439:2, L0752:2, L0731:2, L0596:2, H0395:1, H0713:1, H0483:1, H0663:1, S0358:1,





H0580:1, H0329:1, S0045:1, H0453:1, H0427:1, H0599:1, H0706:1, H0150:1, H0123:1, L0471:1, L0163:1, H0051:1, H0275:1,





S0003:1, S0214:1, H0628:1, H0090:1, H0040:1, H0087:1, T0067:1, H0412:1, H0494:1, H0509:1, H0633:1, H0647:1, S0344:1,





L0769:1, L0637:1, L0761:1, L0772:1, L0800:1, L0374:1, L0771:1, L0363:1, L0768:1, L0806:1, L0659:1, L0382:1, L0809:1,





L0545:1, L0789:1, L0666:1, H0519:1, H0659:1, S0152:1, S0404:1, L0751:1, L0747:1, L0750:1, L0779:1, S0436:1, L0608:1,





S0276:1, H0543:1, H0506:1 and H0352:1.

83
EKABU43
838573
93
AR219:2, AR282:1, AR300:1, AR316:1 L0794:7, L0803:3, H0052:2, S0250:2, H0032:2, H0494:2, H0529:2, L0666:2, L0663:2,





L0747:2, L0759:2, H0657:1, H0664:1, H0662:1, S0442:1, H0741:1, H0735:1, H0733:1, S0046:1, H0640:1, H0331:1, H0559:1,





T0039:1, H0013:1, S0280:1, H0318:1, T0110:1, H0024:1, S0364:1, H0591:1, H0038:1, H0040:1, S0142:1, L0640:1, L0667:1,





L0764:1, L0662:1, L0804:1, L0659:1, L0517:1, L0789:1, L4559:1, L0664:1, S0126:1, H0435:1, H0539:1, S0152:1, H0521:1,





H0522:1, S0027:1, L0779:1, L0758:1, L0485:1, L0601:1, S0026:1, H0667:1, S0192:1, H0542:1 and H0506:1.

84
HKACI79
853361
94
AR313:63, AR039:48, AR300:33, AR096:31, AR089:31, AR277:26, AR185:25, AR299:24, AR316:20, AR240:19, AR218:15,





AR219:14, AR282:12, AR104:12, AR060:9, AR055:6, AR283:3 H0659:2, S0418:1, L0004:1, H0041:1, H0087:1, H0494:1,





H0646:1, S0422:1, L0373:1, L07661, L06651, S0380:1, L0748:1, L0740:1 and L0589:1.

85
HKIXC44
716213
95
AR104:28, AR055:19, AR240:15, AR219:11, AR218:11, AR185:10, AR060:9, AR299:7, AR089:7, AR283:7, AR282:6,





AR096:5, AR316:5, AR300:5, AR313:5, AR039:4, AR277:3 L0770:7, L0742:5, L0439:4, L0776:3, S0358:2, H0619:2,





S0222:2, L0769:2, L0638:2, L0796:2, L0805:2, H0593:2, L0753:2, L0485:2, L0608:2, H0329:1, H0351:1, H0441:1, H0611:1,





H0371:1, H0013:1, H0196:1, H0052:1, H02511, H00411, H00241, H06221, S0366:1, H0623:1, L0648:1, L05231, L08061,





L0788:1, L0666:1, L0663:1, H0648:1, H0539l, S0152:1, L0612:1, L0777:1, L0599:1 and S0242:1.

86
HKTAB41
695732
96
AR277:83, AR283:74, AR219:65, AR313:56, AR316:50, AR089:49, AR218:47, AR282:46, AR104:45, AR055:42, AR185:41,





AR299:40, AR096:35, AR039:31, AR240:31, AR060:26, AR300:25 L0794:5, H0574:1 and H0239:1.

87
HLDQU79
740755
97
AR253:8, AR171:7, AR245:6, AR243:5, AR183:5, AR263:5, AR264:4, AR250:4, AR269:4, AR060:4, AR180:4, AR270:4,





AR309:4, AR162:4, AR268:4, AR161:4, AR165:4, AR192:4, AR176:4, AR164:4, AR055:4, AR163:4, AR213:4, AR195:4,





AR271:4, AR166:3, AR275:3, AR240:3, AR282:3, AR312:3, AR246:3, AR178:3, AR181:3, AR311:3, AR168:3, AR289:3,





AR182:3, AR193:3, AR217:3, AR179:3, AR212:3, AR237:3, AR238:3, AR299:3, AR199:3, AR252:3, AR229:3, AR242:2,





AR185:2, AR300:2, AR277:2, AR175:2, AR293:2, AR257:2, AR308:2, AR177:2, AR198:2, ARO61:2, AR214:2, AR174:2,





AR104:2, AR231:2, AR316:2, AR201:2, AR233:2, AR230:2, AR224:2, AR236:2, AR239:2, AR228:2, AR188:2, AR223:2,





AR189:2, AR247:2, AR294:2, AR226:2, AR266:2, AR221:2, AR285:2, AR191:2, AR089:2, AR216:2, AR200:2, AR207:2,





AR272:2, AR232:2, AR190:2, AR290:2, AR283:2, AR096:2, AR222:2, AR296:2, AR039:2, AR267:2, AR205:2, AR211:1,





AR196:1, AR173:1, AR033:1, AR218:1, AR295:1, AR255:1, AR262:1, AR215:1, AR227:1, AR254:1, AR234:1, AR313:1,





AR203:1, AR256:1, AR169:1, AR225:1, AR210:1, AR170:1 L0748:9, L0731:7, L0771:6, L0759:6, H0013:5, L0764:4,





L0747:4, L0758:4, H0265:3, H0039:3, H0038:3, L0769:3, L0766:3, L0775:3, H0144:3, L0755:3, S0444:2, S0476:2, H0318:2,





H0050:2, L0471:2, H0266:2, L0374:2, L0649:2, L0805:2, L0663:2, L0664:2, H0547:2, S0126:2, H0670:2, L0740:2, L0754:2,





L0750:2, L0593:2, H0667:2, H0170:1, H0171:1, H0685:1, H0662:1, S0354:1, S0360:1, H0580:1, H0728:1, H0151:1, H0747:1,





L3388:1, H0357:1, H0586:1, H0331:1, H0574:1, H0635:1, H0575:1, H0263:1, H0596:1, H0545:1, H0012:1, H0620:1, H0350:1,





H0355:1, H0510:1, H0428:1, H0604:1, H0031:1, H0553:1, S0366:1, H0040:1, H0063:1, H0059:1, H0560:1, H0561:1, S0440:1,





S0422:1, H0529:1, L0640:1, L0637:1, L0761:1, L0772:1, L0646:1, L4556:1, L0774:1, L0375:1, L0653:1, L0382:1, L5622:1,





L0793:1, L4501:1, H0723:1, L0352:1, S0152:1, S0350:1, H0521:1, H0696:1, S0044:1, H0627:1, S0027:1, L0749:1, L0752:1,





H0595:1, S0436:1, L0591:1, L0595:1, L0361:1, S0011:1, S0194:1, S0276:1 and H0423:1.


HLDQU79
837599
191

88
HLHAP05
638476
98
L0005:3, H0024:2, H0209:1 and H0445:1.

89
HLHCS23
560663
99
AR055:5, AR060:4, AR185:3, AR218:3, AR240:3, AR300:3, AR282:3, AR299:2, AR039:2, AR283:2, AR089:2, AR219:2,





AR316:2, AR104:2, AR096:1, AR277:1 H0024:1

90
HLICE88
840321
100
AR185:21, AR240:19, AR104:13, AR039:13, AR060:13, AR089:13, AR300:12, AR282:11, AR096:11, AR055:10, AR316:10,





AR219:10, AR218:9, AR299:7, AR283:7, AR313:7, AR277:4 H0014:72, L3388:60, H0509:49, L0581:44, H0355:43,





H0574:32, H0393:30, H0632:21, H0510:18, S0438:18, H0098:15, H0144:14, H0331:13, H0015:8, L0748:8, H0722:7, L3387:7,





H0741:5, H0013:5, H0147:4, T0078:4, L0615:3, H0357:3, S0440:3, H0730:2, H0349:2, H0350:2, H0057:2, H0644:2, H0647:2,





L0605:2, L0599:2, H0170:1, L0448:1, H0149:1, L0393:1, S0444:1, L3645:1, H0749:1, L2255:1, H0351:1, H0642:1, H0427:1,





H0003:1, H0575:1, H0199:1, H0040:1, H07451, L07871, L07471 and S0436:1.

91
HLMGP50
647603
101
AR055:5, AR313:4, AR282:4, AR104:4, AR277:4, AR300:4, AR299:3, AR060:3, AR039:3, AR096:3, AR316:3, AR283:2,





AR185:2, AR218:2, AR240:2, AR089:2 H0255:2, H0385:1, L0753:1 and H0595:1.

92
HLMMX62
688051
102
AR060:7, AR055:7, AR313:7, AR299:7, AR089:7, AR218:6, AR185:6, AR240:6, AR039:5, AR300:5, AR277:5, AR096:4,





AR219:4, AR283:4, AR104:4, AR316:4, AR282:3 H0255:2, S0410:2, H0052:1 and H0673:1.

93
HLQCX36
584786
103
AR313:27, AR226:23, AR039:23, AR251:21, AR089:17, AR238:15, AR241:14, AR096:14, AR185:14, AR299:14, AR104:13,





AR258:13, AR300:13, AR316:12, AR293:12, AR253:12, AR240:12, AR198:11, AR060:11, AR219:11, AR248:11, AR249:10,





AR218:10, AR237:10, AR231:10, AR232:10, AR282:10, AR227:10, AR275:10, AR192:10, AR186:9, AR234:9, AR229:8,





AR271:8, AR312:8, AR277:8, AR204:8, AR274:7, AR292:7, AR243:7, AR294:7, AR233:7, AR244:7, AR177:7, AR183:7,





AR055:7, AR259:6, AR053:6, AR175:6, AR033:6, AR052:6, AR269:6, AR273:5, AR202:5, AR247:5, AR206:5, AR061:5,





AR309:5, AR267:5, AR213:5, AR265:5, AR256:4, AR295:4, AR184:4, AR205:4, AR179:4, AR283:3, AR268:3, AR182:3,





AR246:3, AR270:3, AR310:3, AR296:2, AR298:2, AR281:2, AR290:2, AR286:2, AR263:2, AR315:2, AR266:2, AR284:1,





AR280:1, AR289:1, AR291:1, AR285:1 L0459:1 and H0574:1.

94
HLWAF06
658701
104
AR277:1 L0664:2, L0438:2, L0439:2, L0751:2, L0755:2, H0553:1, S0002:1, L0775:1 and L0752:1.

95
HLWDB73
838453
105
L0777:13, L0803:9, L0748:9, L0731:6, H0423:5, L0794:4, L0766:4, L0740:4, L0754:4, L0779:4, H0171:3, S0408:3, H0580:3,





S0003:3, H0553:3, H0616:3, L0804:3, H0519:3, L0439:3, L0751:3, S0026:3, H0422:3, H0170:2, H0717:2, S0356:2, H0486:2,





H0318:2, H10644:2, H0068:2, H0038:2, H0551:2, H0413:2, L0598:2, L0646:2, L0662:2, L0388:2, L0784:2, L0805:2, L0776:2,





L0790:2, H0547:2, H0710:2, H0521:2, L0745:2, L0747:2, L0756:2, L0752:2, H0624:1, S0342:1, SO114:1, S0134:1, H0650:1,





L0808:1, S0354:1, S0444:1, S0360:1, S0046:1, H0411:1, H0438:1, H0600:1, H0632:1, T0039:1, H0013:1, H0427:1, H0581:1,





H0421:1, H0544:1, H0046:1, H0457:1, H0150:1, H0563:1, H0123:1, H0019:1, L0471:1, H0024:1, H0271:1, H0416:1, H0428:1,





H0039:1, L0055:1, H0361:1, H0598:1, H0090:1, H0591:1, H0268:1, H0623:1, H0560:1, S0440:1, H0647:1, H0646:1, S0344:1,





UNKWN:1, L0763:1, L0770:1, L0769:1, L0637:1, L0667:1, L0764:1, L0767:1, L0768:1, L0649:1, L0775:1, L0375:1, L0806:1,





L0807:1, L0659:1, L0783:1, L0791:1, L0792:1, L0663:1, L0664:1, H0144:1, S0374:1, L0438:1, H0520:1, S0126:1, H0658:1,





H0648:1, S0330:1, S0152:1, H0696:1, S0406:1, H0576:1, S0028:1, L0749:1, L0780:1, L0755:1, L0758:1, S0031:1, H0595:1,





L0596:1, L0581:1, L0604:1, H0665:1 and S0194:1.

96
HLYDF73
566869
106
AR277:12, AR283:9, AR282:6, AR316:6, AR300:5, AR055:5, AR089:5, AR104:4, AR299:4, AR185:4, AR096:4, AR218:4,





AR313:4, AR240:4, AR039:3, AR219:3, AR060:2 H0445:1

97
HLYGB19
838083
107
AR240:33, AR096:27, AR313:22, AR055:15, AR282:15, AR316:13, AR060:10, AR089:9, AR039:9, AR300:7, AR277:7,





AR299:7, AR104:5, AR219:4, AR185:4, AR283:4, AR218:1 L0752:10, L0471:9, L0731:8, H0422:8, H0040:5, L0641:5,





L0662:4, L0439:4, L0755:4, S0114:3, S0360:3, L0766:3, L0747:3, L0749:3, L0757:3, H0445:3, H0543:3, H0265:2, H0556:2,





S0116:2, H0013:2, H0244:2, H0135:2, H0264:2, L0769:2, L0639:2, L0761:2, L0774:2, L0775:2, L0776:2, L0384:2, L0663:2,





L0665:2, L0565:2, H0658:2, H0539:2, L3832:2, L0744:2, L0748:2, L0750:2, L0779:2, L0758:2, L0759:2, S0134:1, H0657:1,





S0212:1, S0400:1, S0420:1, L3645:1, S0046:1, S0476:1, L0717:1, S0220:1, L2491:1, H0599:1, H0706:1, L0563:1, H0545:1,





H0150:1, H0009:1, H0024:1, T0010:1, H0354:1, H0028:1, H0553:1, L0456:1, H0616:1, H0413:1, L0351:1, S0438:1, H0646:1,





L3818:1, S0208:1, L0796:1, L3904:1, L0667:1, L0644:1, L0764:1, L0768:1, L0649:1, L0655:1, L0606:1, L0634:1, L0659:1,





L0809:1, L0367:1, L0793:1, L0666:1, H0144:1, L0438:1, L2670:1, H0689:1, H0666:1, H0672:1, S0378:1, H0436:1, L0756:1,





L0599:1, L0595:1, S0242:1, H0542:1, H0423:1 and L3796:1.

98
HLYGY91
658703
108
AR313:6, AR316:5, AR218:3, AR300:3, AR299:3, AR055:3, AR185:2, AR039:2, AR096:2, A2R277:2, AR219:1, AR089:1





H0692:10, L0777:10, L0805:5, L0803:3, L2497:2, H0328:2, L0662:2, L0794:2, L0809:2, L3832:2, L0748:2, L0752:2, L0599:2,





H0170:1, H0402:1, S0444:1, S0360:1, H0747:1, L2486:1, L3503:1, H0427:1, H0644:1, H0038:1, L0800:1, L0648:1, L0804:1,





H0670:1, H0478:1, L0731:1, L0758:1, H0445:1, S0434:1, L0591:1 and L0362:1.

99
HMDAB29
584789
109
AR313:127, AR039:86, AR299:64, AR089:56, AR185:51, AR096:50, AR277:50, AR300:42, AR316:37, AR240:33, AR218:27,





AR219:25, AR104:22, AR060:22, AR282:20, AR055:16, AR283:9 H0346:1, H0598:1 and S0330:1.

100
HMDAD44
566854
110
AR277:44, AR283:35, AR219:28, AR316:26, AR089:24, AR218:23, AR313:22, AR282:22, AR055:22, AR104:21, AR299:20,





AR185:19, AR240:19, AR096:17, AR039:16, AR060:14, AR300:14 L0749:3, H0346:1, H0370:1, H0427:1 and L0439:1.

101
HMEDI90
840077
111
AR104:7, AR316:6, AR055:5, AR060:5, AR300:4, AR185:4, AR218:4, AR282:3, AR283:3, AR240:3, AR089:3, AR219:2,





AR299:2, AR039:1, AR313:1, AR096:1, AR277:1 L0439:8, S6028:2, H0266:2, L0438:2, L0745:2, L0717:1, S0222:1, H0052:1,





H0194:1, H0009:1, T0010:1, S0036:1, L0776:1, L0789:1, S0028:1, L0756:1 and L0779:1.

102
HMIBF07
603528
112
AR055:5, AR060:4, AR240:4, AR300:3, AR299:3, AR104:3, AR283:3, AR219:2, AR218:2, AR185:2, AR039:2, AR089:2,





AR277:2, AR096:2, AR316:2, AR282:1, AR313:1 S6028:1

103
HMICP65
847403
113
AR313:25, AR039:24, AR277:13, AR104:13, AR300:11, AR096:11, AR299:10, AR185:10, AR089:9, AR219:9, AR316:8,





AR218:8, AR240:6, AR060:6, AR282:5, AR055:3, AR283:2 S0474:12, H0156:5, H0650:3, L0666:3, H0341:2, H0393:2,





H0486:2, H0052:2, H0039:2, H0135:2, S0330:2, L0748:2, L0439:2, L0757:2, L0601:2, H0224:1, H0225:1, S0134:1, H0583:1,





H0657:1, S0212:1, S0282:1, H0735:1, S0046:1, H0550:1, H0431:1, L3653:1, H0013:1, H0042:1, H0590:1, S0010:1, H0318:1,





H0046:1, H0009:1, H0050:1, H0242:1, S0388:1, S6028:1, H0271:1, H0031:1, H0644:1, L0455:1, L0370:1, T0042:1, H0560:1,





H0538:1, L3904:1, L0804:1, L0805:1, L0653:1, L0776:1, L0659:1, L0787:1, L2264:1, H0547:1, H0648:1, H0539:1, L0745:1,





S0436:1 and S0242:1.

104
HMSHC86
840402
114
S0002:4 and H0695:1.

105
HMUAN45
833072
115
AR236:442, AR228:429, AR211:336, AR230:331, AR287:325, AR191:311, AR239:297, AR174:289, AR233:252, AR232:252,





AR190:238, AR288:237, AR176:228, AR203:222, AR262:218, AR260:210, AR199:209, AR181:193, AR173:191, AR163:186,





AR178:181, AR200:175, AR162:165, AR189:164, AR297:164, AR166:154, AR161:154, AR164:149, AR188:148, AR227:147,





AR234:145, AR261:144, AR311:142, AR257:141, AR210:140, AR179:139, AR165:137, AR272:136, AR295:134, AR226:134,





AR255:130, AR231:129, AR180:129, AR285:127, AR196:127, AR308:126, AR275:123, AR286:123, AR177:118, AR238:117,





AR258:116, AR235:114, AR237:104, AR294:100, AR175:99, AR264:98, AR182:96, AR212:96, AR293:93, AR185:92,





AR291:90, AR267:79, AR229:78, AR061:75, AR240:74, AR060:74, AR256:73, AR269:65, AR104:64, AR247:63, AR274:60,





AR201:60, AR300:59, AR033:56, AR263:53, AR289:51, AR183:49, AR193:48, AR290:43, AR195:41, AR316:40, AR270:40,





AR296:37, AR282:36, AR312:34, AR197:33, AR218:33, AR277:29, AR299:29, AR055:28, AR089:28, AR250;27, AR268:27,





AR207:26, AR309:25, AR053:25, AR252:24, AR266:21, AR213:20, AR242:19, AR224:19, AR219:19, AR313:18, AR223:18,





AR169:18, AR222:17, AR171:17, AR168:16, AR172:16, AR205:15, AR217:14, AR096:14, AR245:14, AR214:14, AR204:12,





AR225:12, AR170:11, AR246:11, AR254:11, AR198:11, AR283:10, AR192:10, AR216:10, AR271:9, AR253:9, AR215:8,





AR221:7, AR039:5, AR243:4, AR184:1, AR310:1 H0271:5, H0083:3, L0794:3, H0656:2, H0457:2, H0179:2, L0791:2,





H0521:2, L0744:2, H0707:2, H0265:1, H0556:1, H0657:1, H0449:1, H0580:1, S0046:1, H0411:1, H0437:1, H0333:1, H0486:1,





H0250:1, S6028:1, H0615:1, H0628:1, L0055:1, H0040:1, H0634:1, S0144:1, H0529:1, L0769:1, L0768:1, L0766:1, L0803:1,





L0653:1, L0793:1, L0666:1, S0052:1, H0689:1, H0522:1, H0436:1, L0743:1, L0749:1, L0779:1, H0445:1 and H0542:1.

106
HMVBC31
825598
116
AR055:5, AR316:4, AR218:3, AR282:3, AR104:2, AR283:2, AR185:2, AR096:2, AR313:2, AR240:2, AR060:2, AR300:2,





AR299:1, AR277:1, AR089:1, AR039:1 L0748:10, H0556:5, S0442:5, L0438:4, L0439:4, L0754:4, H0050:3, H0040:3,





L0769:3, L0806:3, L0757:3, L0759:3, L0601:3, T0002:2, S0418:2, S0358:2, S0360:2, H0580:2, S0476:2, H0549:2, H0644:2,





H0529:2, L0773:2, L0768:2, L0766:2, L0805:2, L0776:2, L0663:2, L0740:2, L0747:2, L0749:2, S0436:2, H0717:1, S0212:1,





H0484:1, H0661:1, S0376:1, H0729:1, H0733:1, S0007:1, H0643:1, L0622:1, L3653:1, H0013:1, H0042:1, H0052:1, L0157:1,





L0471:1, H0373:1, H0083:1, H0266:1, T0006:1, H0090:1, H0268:1, H0494:1, H0509:1, H0633:1, H0646:1, S0422:1, S0002:1,





L0761:1, L0772:1, L0643:1, L0644:1, L0794:1, L0803:1, L0555:1, L0659:1, L0783:1, L0809:1, L5622:1, H0690:1, H0658:1,





S0328:1, S0330:1, S0152:1, H0521:1, H0696:1, S0044:1, S0027:1, L0780:1, L0752:1, L0753:1, L0755:1, S0434:1, L0485:1,





H0667:1, S0276:1 and S0456:1.

107
HMWBL03
822861
117
AR313:27, AR299:16, AR219:16, AR218:14, AR316:12, AR300:6, AR039:5, AR089:5, AR055:5, AR060:4, AR240:4,





AR096:4, AR282:4, AR283:3, AR277:3, AR185:3, AR104:2 S0422:18, L0766:7, H0341:5, S0356:5, H0543:5, H0591:4,





H0656:3, S0354:3, H0013:3, T0042:3, H0659:3, L0748:3, L0750:3, L0777:3, S0418:2, S0442:2, S0444:2, S0410:2, L0471:2,





H0040:2, H0063:2, H0494:2, L0646:2, L0626:2, L0806:2, L0655:2, L0663:2, S0374:2, H0547:2, S0206:2, L0756:2, S0436:2,





L0588:2, H0624:1, H0171:1, S0342:1, H0650:1, S0360:1, T0008:1, H0733:1, S0046:1, H0257:1, H0263:1, L0735:1, H0046:1,





L0157:1, H0039:1, H0068:1, H0135:1, H0090:1, T0041:1, H0560:1, S0440:1, H0529:1, L0640:1, L0771:1, L0768:1, L0634:1,





L0529:1, L5623:1, L0666:1, L0665:1, H0520:1, H0519:1, S0328:1, S0152:1, S0406:1, L0751:1, L0747:1, L0759:1, L0591:1,





L0608:1, H0542:1, H0423:1 and H0721:1.

108
HNFGR08
825417
118
AR055:5, AR060:4, AR185:3, AR240:3, AR300:2, AR104:2, AR282:2, AR089:2, AR283:2, AR219:2, AR218:2, AR316:1,





AR039:1, AR096:1 H0271:1

109
HNGAK51
603910
119
AR313:60, AR039:47, AR299:29, AR277:29, AR089:26, AR185:24, AR096:23, AR240:20, AR300:19, AR316:17, AR218:14,





AR060:14, AR219:14, AR104:14, AR055:11, AR282:10, AR283:6 S0052:1

110
HNGDX18
1145071
120
AR228:8, AR176:7, AR161:6, AR162:6, AR163:6, AR251:5, AR223:5, AR151:5, AR171:5, AR225:4, AR060:4, AR267:4,





AR055:4, AR216:4, AR261:4, AR235:4, AR236:4, AR268:4, AR230:4, AR269:4, AR288:4, AR191:4, AR052:4, AR182:4,





AR221:4, AR239:4, AR254:3, AR242:3, AR255:3, AR312:3, AR233:3, AR287:3, AR272:3, AR262:3, AR165:3, AR271:3,





AR244:3, AR178:3, AR229:3, AR164:3, AR173:3, AR257:3, AR290:3, AR274:3, AR266:3, AR061:3, AR297:3, AR166:3,





AR282:3, AR198:3, AR053:3, AR231:3, AR199:3, AR291:3, AR177:3, AR201:3, AR214:3, AR264:3, AR247:3, AR196:3,





AR224:3, AR190:3, AR174:3, AR270:3, AR296:2, AR286:2, AR300:2, AR309:2, AR203:2, AR089:2, AR200:2, AR294:2,





AR289:2, AR249:2, AR311:2, AR240:2, AR168:2, AR293:2, AR238:2, AR188:2, AR217:2, AR175:2, AR285:2, AR179:2,





AR234:2, AR310:2, AR185:2, AR033:2, AR298:2, AR260:2, AR226:2, AR316:2, AR227:2, AR222:2, AR313:2, AR265:2,





AR197:2, AR277:2, AR237:2, AR189:2, AR295:2, AR299:2, AR193:2, AR283:2, AR172:2, AR183:2, AR275:2, AR232:2,





AR211:2, AR253:2, AR210:2, AR104:2, AR096:2, AR213:1, AR258:1, AR292:1, AR308:1, AR273:1, AR194:1, AR180:1,





AR184:1, AR284:1, AR252:1, AR205:1 H0457:4, S0052:4, H0271:3, L0766:3, H0543:3, H0255:2, H0402:2, H0253:2,





L0805:2, L0754:2, H0422:2, H0583:1, H0650:1, H0656:1, H0484:1, H0483:1, H0254:1, L3659:1, S0442:1, S0360:1, H0580:1,





S0140:1, H0747:1, H0393:1, H0486:1, H0250:1, H0618:1, H0050:1, H0630:1, H0719:1, H0182:1, H0063:1, H0087:1, H0264:1,





H0488:1, H0487:1, L0351:1, 50041:1, S0448:1, S0002:1, L0761:1, L0378:1, L0655:1, L4501:1, H0539:1, S0188:1, S0146:1,





H0707:1, L0599:1, H0136:1, H0423:1 and H0677:1.


HNGDX18
866177
192

111
HNGGP65
597449
121
AR313:17, AR039:13, AR219:10, AR299:10, AR300:10, AR185:9, AR096:9, AR104:8, AR089:8, AR218:8, AR060:8,





AR055:7, AR277:7, AR282:7, AR316:6, AR240:6, AR283:4 S0052:1

112
HNGIV64
561572
122
AR185:8, AR039:8, AR060:8, AR313:7, AR055:7, AR096:6, AR300:6, AR089:6, AR240:6, AR218:6, AR299:6, AR277:6,





AR316:5, AR104:5, AR283:4, AR282:3, AR219:1 S0052:1

113
HNGMW45
838613
123
AR316:3 S0428:1

114
HNGPJ25
834942
124
AR060:7, AR055:7, AR218:6, AR240:6, AR282:5, AR185:5, AR277:5, AR300:5, AR299:4, AR283:3, AR089:3, AR104:3,





AR316:3, AR096:3, AR039:2, AR313:2, AR219:2 H0251:8, H0624:4, L0752:4, H0286:1, L0598:1, S0428:1 and H0144:1.

115
HNHEN82
836157
125
AR055:5, AR060:4, AR300:4, AR104:3, AR282:2, AR283:2, AR219:2, AR089:2, AR039:2, AR185:2, AR299:2, AR218:2,





AR313:2, AR316:2, AR277:1, AR240:1

116
HNHFE71
834487
126
AR055:9, AR060:8, AR218:6, AR300:5, AR185:5, AR240:5, AR277:5, AR299:5, AR282:5, AR104:5, AR283:4, AR089:4,





AR039:4, AR316:4, AR096:3, AR313:3, AR219:2 S0053:1

117
HNHKV56
800877
127
S0216:1 and L0746:1.

118
HOACG07
792928
128
AR202:138, AR194:120, AR315:99, AR281:99, AR198:98, AR244:88, AR246:86, AR243:85, AR205:85, AR192:82, AR241:80,





AR280:79, AR204:73, AR265:70, AR283:66, AR206:63, AR310:59, AR263:57, AR271:57, AR314:56, AR273:54, AR053:50,





AR052:49, AR282:49, AR033:48, AR277:47, AR275:46, AR213:45, AR274:44, AR312:43, AR316:43, AR309:42, AR104:40,





AR240:40, AR300:40, AR289:39, AR096:39, AR266:38, AR251:37, AR247:36, AR089:36, AR299:36, AR284:35, AR313:35,





AR186:34, AR039:33, AR055:33, AR175:32. AR219:31, AR295:31, AR291:29, AR177:28, AR218:28, AR185:27, AR268:26,





AR285:26, AR270:25, AR286:25, AR253:24, AR183:24, AR060:24, AR232:23, AR061:23, AR292:23, AR298:23, AR258:22,





AR256:20, AR248:20, AR296:19, AR182:19, AR238:18, AR269:18, AR290:18, AR294:18, AR267:17, AR231:17, AR226:17,





AR259:17, AR229:16, AR227:16, AR234:16, AR293:15, AR233:15, AR237:14, AR249:13, AR184:13, AR179:11 L0748:4,





L0749:4, H0265:3, S0442:2, H0587:2, H0427:2, S0142:2, L0769:2, L0761:2, L0800:2, L0794:2, L0657:2, L0659:2, L0663:2,





L0665:2, H0689:2, L0731:2, H0677:2, H0713:1, H0716:1, H0657:1, H0661:1, H0638:1, S0360:1, H0722:1, H0122:1, H0545:1,





H0009:1, H0123:1, S0388:1, H0252:1, T0023:1, T0006:1, H0135:1, H0163:1, H0412:1, L0351:1, S0440:1, H0529:1, L0372:1,





L0646:1, L0645:1, L0764:1, L0662:1, L0767:1, L0766:1, L0649:1, L0803:1, L0776:1, L0655:1, L0382:1, L0809:1, L0789:1,





H0672:1, H0627:1, L0751:1, L0750:1, L0753:1, L0757:1, L0758:1 and L0759:1.

119
HODBB70
520196
129
AR055:7, AR218:6, AR060:5, AR104:5, AR240:4, AR300:4, AR299:4, AR096:4, AR219:4, AR283:4, AR039:3, AR185:3,





AR089:3, AR316:3, AR282:3, AR277:2 H0328:1, L0789:1, L0742:1 and L0439:1.

120
HOEBK60
789396
130
AR219:21, AR215:19, AR316:12, AR313:10, AR039:9, AR096:8, AR089:8, AR299:7, AR240:7, AR055:7, AR300:7, AR060:7,





AR282:6, AR185:5, AR283:5, AR104:4, AR277:4 L0439:10, L0731:7, L0605:6, L0766:5, L0803:5, L0471:4, L0655:4,





L0659:4, L0756:4, S0408:3, S0440:3, H0648:3, L0777:3, H0170:2, S0420:2, L0021:2, H0014:2, T0010:2, L0143:2, H0090:2,





H0591:2, H0641:2, L0638:2, L0662:2, L0794:2, L0776:2, L0657:2, L0809:2, L0666:2, L0663:2, H0547:2, S0126:2, H0518:2,





L0751:2, L0755:2, L0758:2, L0588:2, H0543:2, H0624:1, H0740:1, S0430:1, H0657:1, H0656:1, S0212:1, S0110:1, H0661:1,





H0663:1, S0442:1, S0358:1, S0376:1, S0360:1, S0476:1, L0717:1, L0586:1, T0114:1, H0427:1, L0105:1, H0318:1, S0474:1,





H0581:1, H0052:1, H0309:1, L0157:1, H0024:1, H0071:1, H0275:1, H0354:1, S6028:1, H0266:1, S0003:1, H0328:1, H0615:1,





H0428:1, H0031:1, H0169:1, H0163:1, H0038:1, H0040:1, H0551:1, H0272:1, T0041:1, S0014:1, S0438:1, H0646:1, S0142:1,





S0210:1, S0426:1, H0529:1, L0372:1, L0645:1, L0764:1, L0651:1, L0656:1, L5623:1, L0789:1, L0665:1, H0520:1, H0689:1,





H0682:1, H0435:1, H0659:1, S0380:1, H0696:1, L0740:1, L0754:1, L0746:1, L0750:1, L0779:1, L0752:1, S0260:1, S0434:1,





S0436:1, L0485:1 and H0423:1.

121
HOFAA78
836646
131
AR316:60

122
HOFNB74
762821
132
AR277:18, AR283:18, AR282:14, AR313:12, AR316:11, AR055:9, AR089:9, AR240:8, AR104:8, AR218:8, AR299:8,





AR096:8, AR219:8, AR300:7, AR185:7, AR039:6, AR060:5 H0415:1

123
HORBS82
638293
133
H0706:2, L0809:2, S0360:1, L0623:1, H0122:1, H0041:1, H0095:1, H0292:1, H0424:1, S0364:1, L0794:1, L0787:1, L0663:1,





H0780:1, H0435:1, L0743:1, L0747:1 and L0731:1.

124
HOSDO75
862049
134
AR060:6, AR055:6, AR218:6, AR240:5, AR277:5, AR300:4, AR185:4, AR299:4, AR283:4, AR089:4, AR282:3, AR104:3,





AR316:3, AR096:3, AR313:2, AR039:2, AR219:2 L0766:2, L0362:2, 50358:1, H0580:1, S0046:1, H0266:1, S0003:1, H0553:1





S0344:1, L0761:1, L0794:1, S0152:1, L0777:1 and L0755:1.

125
HOUDR07
745404
135
AR177:34, AR197:30, AR195:28, AR207:25, AR171:23, AR182:23, AR192:23, AR275:22, AR199:22, AR169:21, AR170:21,





AR226:21, AR183:20, AR189:19, AR168:18, AR263:18, AR179:18, AR174:18, AR175:18, AR176:18, AR224:18, AR269:17,





AR235:17, AR311:17, AR270:16, AR231:16, AR214:16, AR222:16, AR181:16, AR225:16, AR229:16, AR190:15, AR196:15,





AR271:15, AR237:15, AR309:15, AR261:15, AR061:15, AR295:15, AR245:15, AR210:14, AR201:14, AR230:14, AR173:14,





AR221:13, AR191:13, AR283:13, AR161:13, AR223:13, AR291:13, AR165:13, AR308:13, AR213:13, AR268:13, AR162:13,





AR164:13, AR266:13, AR166:13, AR240:13, AR163:13, AR195:13, AR277:13, AR217:12, AR264:12, AR289:12, AR247:12,





AR211:12, AR239:12, AR236:12, AR212:12, AR216:12, AR288:12, AR172:12, AR286:12, AR205:11, AR178:11, AR218:11,





AR228:11, AR215:11, AR312:11, AR238:11, AR243:11, AR258:11, AR246:11, AR316:11, AR297:11, AR287:10, AR053:10,





AR285:10, AR227:10, AR204:10, AR193:10, AR282:10, AR180:10, AR242:9, AR089:9, AR039:9, AR267:9, AR296:9,





AR290:9, AR234:9, AR188:9, AR272:9, AR033:9, AR233:8, AR299:8, AR293:8, AR300:8, AR255:8, AR262:8, AR254:8,





AR256:7, AR200:7, AR203:7, AR252:7, AR253:7, AR219:7, AR313:7, AR257:7, AR274:7, AR260:6, AR104:6, AR055:6,





AR250:6, AR096:6, AR294:6, AR060:6, AR185:6, AR232:6 S0212:10, L0659:10, L0755:6, L0731:6, H0599:5, L0757:5,





L0775:4, L0603:4, H0713:3, S0132:3, H0427:3, S0312:3, H0622:3, H0553:3, H0628:3, L5565:3, L0439:3, L0740:3, S0040:2,





H0717:2, H0587:2, H0635:2, T0010:2, H0266:2, S0314:2, H0163:2, L0770:2, L3904:2, L0804:2, L5622:2, L0751:2, L0747:2,





L0750:2, H0668:2, S0194:2, H0716:1, S0116:1, H0661:1, S0358:1, S0360:1, H0730:1, H0728:1, S0045:1, S0046:1, S0476:1,





H0456:1, H0549:1, H0431:1, H0370:1, H0592:1, H0632:1, L0623:1, H0486:1, L0021:1, H0706:1, H0590:1, H0618:1, H0705:1,





T0071:1, S0049:1, H0545:1, H0086:1, H0024:1, N0007:1, L3647:1, H0510:1, H0594:1, H0124:1, H0551:1, S0352:1, S0438:1,





H0509:1, H0647:1, L0769:1, L0637:1, L0764:1, L0773:1, L0378:1, L0661:1, L4558:1, L0783:1, L0788:1, H0144:1, H0519:1,





H0555:1, S0390:1, S0028:1, L0753:1, L0758:1, S0434:1 and S0276:1.

126
HPEAD23
773409
136
AR277:68, AR283:65, AR219:61, AR316:49, AR218:41, AR059:41, AR104:39, AR299:38, AR055:36, AR185:36, AR039:34,





AR282:33, AR240:31, AR096:31, AR313:31, AR060:30, AR300:25 H0585:18, L0779:3, L0775:2, S0374:2, H0341:1, S0358:1,





S0360:1, S0408:1, H0559:1, T0039:1, H0156:1, H0253:1, S0182:1, H0318:1, H0545:1, H0083:1, H0165:1, L0768:1, L0774:1,





L0750:1, L0752:1 and S0031:1.

127
HPFCI36
855966
137
AR218:18, AR219:16, AR313:14, AR089:9, AR055:7, AR282:6, AR060:6, AR316:6, AR185:6, AR299:5, AR240:5, AR039:5,





AR300:4, AR104:4, AR283:4, AR096:4, AR277:2 L0591:4, L0754:3, H0450:2, H0486:2, H0046:2, S0003:2, H0494:2,





S0422:2, L0659:2, S0126:2, H0659:2, L0750:2, L0601:2, H0170:1, H0556:1, H0657:1, S0420:1, S0354:1, H0734:1, H0749:1,





H0455:1, H0403:1, H0600:1, H0013:1, H0156:1, H0599:1, H0744:1, H0082:1, S0214:1, H0622:1, H0031:1, H0673:1, H0169:1,





H0090:1, H0038:1, H0022:1, H0560:1, L0643:1, L0771:1, L0773:1, L0655:1, L0807:1, L3872:1, L0792:1, L0665:1, L3811:1,





S0378:1, H0518:1, S0152:1, H0521:1, L0748:1, L0749:1, L0757:1, L0759:1, S0434:1, L0596:1, L0605:1 and H0653:1.

128
HPFDI37
862056
138
AR055:5, AR218:5, AR060:4, AR299:3, AR300:3, AR283:3, AR104:3, AR282:3, AR240:3, AR039:2, AR219:2, AR277:2,





AR185:2, AR316:2, AR089:2, AR313:2, AR096:1 L0771:13, L0752:12, L0748:9, L0731:7, S0360:6, L0769:6, S0358:5,





H0318:5, L0770:5, L0747:5, L0758:5, L0599:5, H0140:4, H0545:4, H0673:4, L0774:4, L0655:4, L0659:4, L0664:4, L0665:4,





H0659:4, H0648:4, L0740:4, L0754:4, L0588:4, H0662:3, H0169:3, H0413:3, L0638:3, L0775:3, L0783:3, L0666:3, L0663:3,





H0660:3, H0521:3, L0749:3, L0750:3, L0757:3, H0543:3, H0170:2, S0110:2, H0574:2, H0581:2, H0535:2, L0065:2, H0647:2,





S0344:2, L0763:2, L0764:2, L0662:2, L0767:2, L0803:2, L0806:2, L0776:2, S0374:2, S0328:2, S0380:2, H0576:2, S0028:2,





L0591:2, H0352:2, H0265:1, T0002:1, H0686:1, H0685:1, H0295:1, H0657:1, L0760:1, S0212:1, H0484:1, H0177:1, H0638:1,





L0617:1, L0005:1, S0442:1, S0376:1, H0637:1, H0411:1, H0370:1, H0587:1, H0632:1, T0109:1, H0156:1, H0085:1, H0327:1,





H0530:1, H0046:1, H0041:1, S0388:1, H0630:1, H0271:1, H0644:1, H0628:1, H0181:1, H0617:1, H0674:1, H0068:1, H0040:1,





H0488:1, L0564:1, T0041:1, H0494:1, H0633:1, S0144:1, S0210:1, S0422:1, L0369:1, L0762:1, L0372:1, L0646:1, L0765:1,





L0363:1, L0768:1, L0651:1, L0653:1, L0629:1, L0657:1, L0526:1, L0532:1, S0053:1, S0216:1, H0144:1, H0519:1, S0126:1.





H0689:1, H0690:1, H0684:1, S0027:1, L0742:1. L0756:1, L0780:1, L0755:1, H0444:1, H0445:1, L0596:1, L0605:1, L0592:1,





L0593:1, L0362:1, L0603:1 and H0136:1.

129
HPIAA80
829972
139
AR218:13, AR219:11, AR282:9, AR089:8, AR055:8, AR240:7, AR104:6, AR060:6, AR283:6, AR277:6, AR039:6, AR316:5,





AR299:5, AR096:4, AR185:4, AR300:4, AR313:3 L0750:3, H0672:2, L0744:2, H0587:1, L0021:1, S0010:1, H0024:1,





H0266:1, S0364:1, H0068:1, H0038:1, T0004:1, H0625:1, S0150:1, L0769:1, L0667:1, L0649:1, L0784:1, L0526:1, L0790:1,





L0792:1, L0793:1, L0663:1, H0696:1, L0747:1, L0608:1 and S0276:1.

130
HPJCW58
612866
140
AR055:8, AR060:6, AR282:6, AR218:5, AR104:4, AR300:4, AR240:4. AR283:4, AR219:4, AR299:3, AR089:3, AR185:3,





AR316:3, AR096:3, AR039:2, AR277:2, AR313:1 S0152:1

131
HPRBH85
695752
141
AR284:14, AR295:10, AR271:8, AR293:7, AR246:7, AR243:7, AR291:7, AR244:6, AR286:6, AR290:6, AR241:6, AR285:6,





AR206:6, AR310:5, AR249:5, AR273:5, AR198:5, AR280:5, AR312:5, AR186:5. AR270:5, AR294:5, AR204:5, AR269:5,





AR251:5, AR202:5, AR292:5, AR275:4, AR033:4, AR253:4, AR053:4, AR182:4, AR314:4, AR259:4, AR315:4, AR298:4,





AR265:4, AR309:4, AR282:4, AR274:4, AR183:4, AR061:4, AR205:4, AR296:4, AR289:4, AR052:4, AR313:4, AR175:3,





AR213:3, AR238:3, AR268:3, AR248:3, AR055:3, AR177:3, AR247:3, AR231:3, AR104:3, AR256:3, AR226:2, AR185:2,





AR283:2, AR277:2, AR267:2, AR227:2, AR300:2, AR096:2, AR089:2, AR258:2, AR219:2, AR263:2, AR299:2, AR237:2,





AR240:2, AR060:2, AR316:2, AR218:2, AR281:2, AR039:2, AR233:1, AR232:1, AR184:1, AR234:1, AR266:1, AR192:1





L0439:5, L0740:4, L0777:4, L0755:4, L0794:2, L0803:2, L0438:2, L0602:2, L0752:2, L0599:2, H0713:1, H0583:1, 50360:1,





L3653:1, L0471:1, H0510:1, H0032:1, H0488:1, H0413:1, L0662:1, L0804:1, L0775:1, L0805:1, L0655:1, L0809:1, L0519:1,





S0148:1, H0547:1, L0747:1, L0686:1 and H0665:1.

132
HPRCA64
824074
142
AR295:13, AR285:10, AR297:8, AR191:8, AR291:7, AR261:7, AR288:7, AR189:6, AR296:6, AR188:6, AR178:6, AR271:6,





AR170:5, AR181:5, AR165:5, AR175:5, AR174:5, AR270:5, AR235:5, AR223:5, AR164:5, AR190:5, AR166:5, AR272:5,





AR172:5, AR210:5, AR255:5, AR236:5, AR274:5, AR196:5, AR217:5, AR287:5, AR246:4, AR096:4, AR089:4, AR286:4,





AR176:4, AR168:4, AR195:4, AR269:4, AR183:4, AR173:4, AR060:4, AR219:4, AR222:4, AR214:3, AR199:3, AR268:3,





AR033:3, AR218:3, AR257:3, AR104:3, AR161:3, AR163:3, AR275:3, AR171:3, AR240:3, AR299:3, AR182:3, AR289:3,





AR290:3, AR177:3, AR316:3, AR207:3, AR245:3, AR180:3, AR162:3, AR300:3, AR216:3, AR262:3, AR293:3, AR197:3,





AR282:3, AR258:3, AR179:3, AR294:3, AR200:2, AR185:2, AR238:2, AR201:2, AR203:2, AR225:2, AR267:2, AR193:2,





AR232:2, AR260:2, AR266:2, AR231:2, AR211:2, AR205:2, AR283:2, AR053:2, AR264:2, AR256:2, AR247:2, AR192:2,





AR237:2, AR226:2, AR243:2, AR204:1, AR277:1, AR239:1, AR229:1, AR169:1, AR313:1 L0005:7, L0662:7, L0665:7,





L0666:6, L0740:6, S0222:5, L0659:5, L0439:5, L0483:4, H0547:4, L0754:4, L0756:4, L0779:4, S0194:4, S0049:3, L0646:3,





L0521:3, L0663:3, L0664:3, L0438:3, H0696:3, L0777:3, H0171:2, S0356:2, S0442:2, S0360:2, S0408:2, H0052:2, H0563:2,





L0471:2, S0388:2, S0051:2, S6028:2, H0266:2, H0623:2, S0440:2, L0598:2, L0520:2, L0641:2, L0771:2, L0768:2, L0774:2,





L0805:2, L0776:2, L0518:2, H0670:2, H0648:2, H0672:2, S0028:2, L0751:2, L0731:2, L0758:2, S0031:2, L0596:2, S0026:2,





S0196:2, H0624:1, H0170:1, H0686:1, H0685:1, H0717:1, H0381:1, S0212:1, H0662:1, S0354:1, S0376:1, S0045:1, S0046:1,





H0411:1, H0369:1, H0602:1, T0040:1, H0013:1, H0427:1, H0390:1, S0474:1, H0545:1, H0046:1, H0178:1, H0562:1, H0123:1,





H0201:1, S0003:1, H0428:1, T0006:1, H0031:1, H0553:1, H0032:1, H0163:1, H0551:1, L0564:1, S0370:1, S0450:1, L0769:1,





L0637:1, L5565:1, L0372:1, L0773:1, L0650:1, L0806:1, L0527:1, L0526:1, L0783:1, L0809:1, S0374:1, L0565:1, H0519:1,





H0682:1, H0435:1, H0659:1, H0660:1, S0328:1, S0330:1, S0380:1, L0602:1, H0555:1, L0753:1, L0755:1, L0759:1, S0260:1,





S0434:1, S0436:1, L0595:1, H0667:1 and S0242:1.

133
HPRCD35
853551
143
AR104:14, AR089:11, AR055:11, AR185:10, AR219:10, AR299:10, AR218:10, AR313:10, AR282:10, AR240:9, AR316:9,





AR283:9, AR096:8, AR060:7, AR039:6, AR300:5, AR277:5 L0748:5, L0754:5, L0803:4, L0805:4, L0777:4, L0662:3,





L0766:3, H0556:2, H0013:2, H0551:2, H0264:2, L0800:2, L0806:2, L0664:2, L0439:2, L0756:2, L0758:2, S0192:2, H0657:1,





S0442:1, S0358:1, S0045:1, S0046:1, H0747:1, H0550:1, H0392:1, S0280:1, H0575:1, H0545:1, H0046:1, H0050:1, H0644:1,





H0617:1, L0055:1, H0032:1, H0212:1, H0038:1, H0560:1, S0438:1, S0210:1, L0769:1, L0796:1, L5575:1, L0768:1, L0794:1,





L0649:1, L0776:1, L0659:1, L0542:1, L0526:1, L0663:1, H0144:1, L0565:1, L3811:1, H0683:1, H0659:1, S0152:1, H0521:1,





H0522:1, H0540:1, S0118:1, S0032:1, L073L1, L07591 and H0668:1.

134
HPTRM02
812879
144
H0617:7, H0087:6, H0657:5, S0410:3, L0754:3, S0356:2, L0717:2, H0150:2, H0687:2, H0424:2, H0551:2, L0769:2, L0774:2,





L0743:2, L0758:2, L0592:2, H0556:1, T0002:1, H0686:1, H0685:1, T0049:1, H0663:1, S0442:1, S0444:1, S0360:1, S0476:1,





H0550:1, H0486:1, H0250:1, L0021:1, T0048:1, S0474:1, S0049:1, H0052:1, H0309:1, H0597:1, H0544:1, H0014:1, H0107:1,





S6028:1, H0622:1, H0644:1, H0102:1, S0038:1, L0351:1, S0450:1, S0344:1, S0002:1, L0764:1, L0766:1, L0805:1, L0776:1,





L0655:1, L0661:1, L0657:1, L0809:1, L0666:1, L0665:1, L2652:1, L2260:1, L2261:1, H0689:1, H0435:1, H0521:1, H0696:1,





H0555:1, L0744:1, L0439:1, L0749:1, L0777:1, L0755:1, L0759:1, S0436:1, L0597:1, L0599:1, L0366:1 and S0196:1.

135
HRAAD30
866187
145
AR282:5, AR277:5, AR060:5, AR055:4, AR185:4, AR300:4, AR299:3, AR104:3, AR218:3, AR283:3, AR316:3, AR089:3,





AR039:2, AR096:2, AR313:2, AR240:1 L0731:6, L2800:4, H0617:4, H0547:4, L0758:4, S0420:3, H0013:3, L0748:3, L0747:3,





S0358:2, L3278:2, L0770:2, S0126:2, L0439:2, L0751:2, L0777:2, L0757:2, H0543:2, S0040:1, L3012:1, H0341:1, S0046:1,





H0550:1, H0497:1, H0333:1, H0427:1, H0618:1, H0253:1, S0474:1, H0052:1, H0546:1, H0571:1, L0471:1, H0024:1, H0051:1,





S6028:1, H0286:1, H0622:1, H0644:1, L0455:1, T0067:1, H0561:1, S0440:1, H0130:1, H0529:1, L0763:1, L0769:1, L0772:1,





L0372:1, L0662:1, L0806:1, L0807:1, L0659:1, L5622:1, L4501:1, L0666:1, L0664:1, L0709:1, L2261:1, H0144:1, L2402:1,





S0374:1, H0520:1, L3831:1, H0555:1, S0027:1, L0740:1, L0750:1, L0752:1, L0759:1, S0436:1, L0592:1, L0604:1, H0542:1,





S0398:1, L3837:1 and H0677:1.

136
HRADF49
866481
146
AR244:12, AR296:6, AR205:6, AR183:6, AR292:6, AR104:5, AR249:5, AR291:5, AR285:5, AR298:5, AR206:5, AR289:4,





AR240:4, AR293:4, AR275:4, AR270:4, AR295:4, AR294:4, AR284:3, AR213:3, AR186:3, AR060:3, AR286:3, AR234:3,





AR229:3, AR282:3, AR267:3, AR184:3, AR096:3, AR283:3, AR033:3, AR251:3, AR300:2, AR313:2, AR316:2, AR185:2,





AR039:2, AR299:2, AR218:2, AR256:2, AR089:2, AR219:2, AR061:2, AR243:2, AR055:2, AR269:2, AR277:2, AR233:2,





AR238:2, AR182:2, AR268:2, AR175:2, AR259:2, AR266:2, AR232:2, AR258:2, AR227:2, AR315:2, AR263:1, AR226:1,





AR309:1, AR314:1, AR053:1, AR290:1, AR052:1, AR231:1 H0618:9, L0751:7, L0754:6, L0758:6, H0253:5, L0748:5,





L0439:5, H0580:3, L3816:3, H0052:3, L0770:3, L0663:3, H0556:2, H0733:2, H0351:2, H0706:2, H0567:2, H0625:2, S0142:2,





L0639:2, L3905:2, L0659:2, L0543:2, L5623:2, L0749:2, S0436:2, H0423:2, L3643:1, H0381:1, S0212:1, H0254:1, H0663:1,





H0638:1, S0418:1, H0741:1, H0735:1, S0045:1, S0046:1, S0476:1, S6022:1, H0549:1, H0550:1, S0222:1, H0370:1, H0497:1,





H0574:1, L0622:1, L0623:1, L3655:1, H0101:1, H0427:1, S0280:1, H0122:1, H0194:1, H0596:1, H0570:1, H0081:1, H0620:1,





H0014:1, H0083:1, H0355:1, H0510:1, H0424:1, H0030:1, H0553:1, H0628:1, S0364:1, S0366:1, H0038:1, H0551:1, H0100:1,





L0351:1, H0494:1, S0438:1, H0633:1, S0144:1, S0422:1, L0371:1, L0769:1, L3904:1, L0772:1, L0648:1, L0497:1, L0375:1,





L0511:1, L0666:1, L0709:1, L0710:1, H0144:1, L3811:1, L3824:1, H0520:1, H0593:1, H0682:1, H0670:1, H0672:1, H0539:1,





L3833:1, S0044:1, H0626:1, H0732:1, S3012:1, S3014:1, S0027:1, S0028:1, L0779:1, L0584:1, L0608:1, L0593:1, H0667:1 and





H0542:1.

137
HRDDQ39
840405
147
AR313:36, AR039:33, AR185:27, AR299:20, AR089:18, AR300:17, AR096:17, AR240:16, AR218:15, AR277:14, AR316:13,





AR060:11, AR219:10, AR104:9, AR055:8, AR282:7, AR283:7 S0001:2, H0436:2, S0134:1, H0657:1, H0441:1, H0009:1,





H0123:1, H0050:1, H0428:1, H0124:1, H0529:1, H0521:1 and H0352:1.

138
HRDEX93
816046
148
AR104:30, AR218:25, AR219:24, AR240:22, AR096:21, AR185:17, AR039:16, AR316:16, AR313:16, AR055:14, AR060:14,





AR299:13, AR089:13, AR282:9, AR277:9, AR300:9, AR283:6 H0694:12, L0748:10, L0731:7, L0754:6, H0556:5, L0758:5,





H0265:4, S0420:4, S0408:4, L0517:4, H0657:3, H0618:3, H0052:3, H0083:3, H0553:3, H0494:3, L0763:3, L0666:3, L0663:3,





S0126:3, L0747:3, H0295:2, S0134:2, S0418:2, H0637:2, S0046:2, H0431:2, H0545:2, H0014:2, H0271:2, H0039:2, H0424:2,





H0124:2, H0641:2, L0764:2, L0766:2, L0774:2, L0775:2, L0776:2, L0655:2, L0783:2, L0665:2, H0519:2, H0522:2, S0044:2,





L0755:2, S0436:2, L0595:2, L0362:2, H0543:2, S0040:1, H0740:1, H0656:1, S0212:1, H0484:1, H0661:1, H0662:1, S0360:1,





H0733:1, H0619:1, S0222:1, H0486:1, H0156:1, H0575:1, H0706:1, H0253:1, S0010:1, S0346:1, H0318:1, H0596:1, H0231:1,





H0046:1, H0150:1, H0081:1, H0050:1, H0012:1, H0620:1, L0163:1, S0051:1, T0010:1, S6028:1, H0266:1, H0179:1, H0292:1,





H0031:1, H0644:1, H0182:1, H0617:1, H0606:1, H0673:1, L0455:1, L0456:1, H0598:1, H0038:1, H0040:1, H0616:1, H0087:1,





T0067:1, H0264:1, T0041:1, H0131:1, H0647:1, S0002:1, L0772:1, L0642:1, L0662:1, L0767:1, L0657:1, L0659:1, L0382:1,





L5623:1, L0664:1, S0374:1, H0593:1, H0690:1, H0682:1, H0659:1, H0658:1, H0666:1, H0651:1, H0539:1, H0521:1, S0406:1,





H0576:1, L0743:1, L0740:1, L0750:1, L0779:1 and H0445:1.

139
HROEA08
866190
149
S0474:57, H0521:14, L0766:12, S0422:10, L0809:8, H0069:7, H0591:7, H0556:6, H0650:5, H0749:5, H0486:5, H0090:5,





L0655:5, S0114:4, S0134:4, H10747:4, S0003:4, H0268:4, H0641:4, L0770:4, H0518:4, L0777:4, H0656:3, H0638:3, H0271:3,





H0039:3, L0598:3, L0763:3, L0659:3, H0436:3, L0754:3, L0756:3, L0731:3, H0423:3, H0422:3, H0265:2, H0657:2, S0354:2,





H0013:2, L0105:2, H0581:2, H0622:2, H0598:2, H0100:2, S0144:2, S0002:2, H0529:2, L0648:2, L5564:2, L0803:2, L0776:2,





L0789:2, L0663:2, H0696:2, H0445:2, H0542:2, H0506:2, H0624:1, H0583:1, H0305:1, H0125:1, S0360:1, H0549:1, H0586:1,





L3657:1, T0060:1, H0075:1, H0002:1, H0599:1, H0004:1, H0318:1, H0421:1, H0251:1, H0457:1, H0178:1, L0471:1, T0003:1,





H0024:1, S0388:1, S0051:1, S0024:1, H0266:1, H0687:1, H0644:1, H0040:1, H0264:1, T0069:1, H0625:1, S0440:1, H0646:1,





S0344:1, S0426:1, L0761:1, L0641:1, L0662:1, L0794:1, L0650:1, L0774:1, L0375:1, L0784:1, L0805:1, L0515:1, H0144:1,





H0702:1, S0374:1, H0520:1, S0126:1, H0689:1, H0659:1, H0658:1, S0378:1, H0S22:1, H0555:1, H0727:1, S3014:1, L0747:1,





L0779:1, L0752:1, L0753:1, L0757:1, S0260:1, S0242:1, H0543:1 and S0412:1.

140
HRTAP63
780698
150
AR219:53, AR218:46, AR313:33, AR104:28, AR096:26, AR089:25, AR316:24, AR039:20, AR299:19, AR185:18, AR300:17,





AR060:17, AR282:16, AR055:15, AR240:13, AR277:10, AR283:9 S0474:28, H0521:15, L0758:14, L0752:13, L0731:13,





L0755:10, H0641:9, L0766:8, H0179:6, L0748:6, L0439:6, L07S9:6, H0638:5, S0222:5, H0581:5, L0662:5, L0655:5, H0436:5,





L0740:S, L0777:5, S0436:5, H0457:4, L077S:4, L0809:4, H0S22:4, L0742:4, L0754:4, L0747:4, L0749:4, L0750:4, L0757:4,





H0580:3, H0619:3, H0052:3, S0003:3, H0038:3, H0623:3, L0666:3, L0663:3, S0053:3, S0126:3, S0434:3, S0026:3, S0212:2,





S0442:2, S0408:2, H0747:2, S0476:2, H0156:2, L0021:2, H0599:2, S0010:2, H0014:2, S0214:2, H0031:2, H0644:2, H0628:2,





S0036:2, H0090:2, H0616:2, S0144:2, S0002:2, L0598:2, L0764:2, L0768:2, L0774:2, L0806:2, L0653:2, L0657:2, L0659:2,





H0520:2, H0547:2, H0539:2, H0710:2, S0027:2, L0779:2, L0588:2, L0592:2, L0594:2, L0366:2, H0665:2, H0739:1, H0170:1,





T0002:1, S0342:1, H0717:1, H0740:1, S0114:1, S0218:1, H0650:1, H0657:1, S0282:1, L3659:1, S0418:1, S0420:1, L0005:1,





T0008:1, H0742:1, H0722:1, H0735:1, S0007:1, S0046:1, H0749:1, L3388:1, H0351:1, H0406:1, S0278:1, H0461:1, H0601:1,





H0586:1, H0497:1, H0574:1, T0039:1, L3655:1, H0013:1, H0427:1, H0575:1, H0590:1, H0421:1, S0049:1, H0327:1, H0545:1,





H0046:1, H0572:1, H0570:1, H0050:1, L0471:1, H0373:1, H0510:1, H0375:1, S6028:1, H0266:1, H0271:1, H0719:1, H0416:1,





S0340:1, S0312:1, H0615:1, L0483:1, T0006:1, H0169:1, H0674:1, H0163:1, H0591:1, H0551:1, H0264:1, H0412:1, H0059:1,





H0494:1, S0015:1, S0344:1, UNKWN:1, H0529:1, L0520:1, L0637:1, L0761:1, L0667:1, L0772:1, L0641:1, L0648:1, L0521:1,





L0794:1, L0649:1, L0803:1, L0805:1, L0783:1, L0384:1, L5622:1, L0793:1, L0665:1, L0665:1, S0052:1, S0428:1, S0216:1,





H0144:1, L3811:1, H0658:1, H0670:1, H0666:1, H0672:1, H0651:1, L0355:1, S0328:1, S0152:1, H0696:1, S0406:1, H0555:1,





S0028:1, S0032:1, L0744:1, L0745:1, L0780:1, L0362:1, H0422:1 and H0721:1.

141
HSAVW42
637660
151
AR277:28, AR283:24, AR219:20, AR055:17, AR218:16, AR316:16, AR282:16, AR313:15, AR089:15, AR104:13, AR299:12,





AR185:11, AR240:11, AR096:11, AR039:10, AR300:9, AR060:8 H0412:2, S0114:1, S0222:1, H0169:1, L0520:1, L0805:1,





L0776:1, L0750:1 and L0777:1.

142
HSAYC41
688057
152
S0114:1, H0411:1, H0179:1, L0665:1 and H0435:1.

143
HSLHX15
777861
153
AR060:8, AR055:7, AR218:6, AR240:5, AR300:5, AR282:4, AR185:4, AR089:4, AR283:4, AR104:4, AR299:4, AR277:3,





AR316:3, AR219:3, AR096:3, AR039:2, AR3131 T0040:1, L0564:1, S0028:1 and L0480:1.

144
HSNBM34
635131
154
AR185:18, AR039:18, AR299:15, AR104:11, AR055:9, AR277:9, AR060:8, AR096:8, AR282:8, AR300:7, AR218:7, AR240:7,





AR313:6, AR316:6, AR219:5, AR283:4, AR089:4 H0599:9, H0144:8, H0457:7, H0266:7, H0494:6, H0046:5, H0031:5,





H0553:5, L5622:5, H0593:5, H0521:5, H0734:4, H0013:4, H0135:4, S0436:4, S0212:3, H0069:3, H0036:3, H0052:3, S0022:3,





H0708:3, H0551:3, H0696:3, S0434:3, H0713:2, H0717:2, S0418:2, S0354:2, H0580:2, H0728:2, H0733:2, H0550:2, H0587:2,





H0559:2, H0706:2, H0253:2, H0355:2, H0039:2, S0364:2, H0038:2, H0634:2, H0433:2, H0560:2, S0440:2, H0646:2, L3818:2,





S0002:2, L0506:2, L5623:2, H0547:2, S0126:2, H0518:2, H0436:2, H0478:2, S3014:2, L0601:2, H0506:2, H0265:1, H0556:1,





T0002:1, S0114:1, H0583:1, S0116:1, S0356:1, S0442:1, S0358:1, S0376:1, S0360:1, S0408:1, H0340:1, H0742:1, H0735:1,





S0132:1, S0476:1, H0619:1, S0278:1, S0222:1, H0409:1, H0602:1, H0592:1, H0586:1, H0486:1, H0270:1, S0280:1, H0042:1,





H0575:1, H0122:1, H0590:1, S0010:1, T0048:1, H0318:1, S0474:1, S0049:1, H0173:1, H0085:1, H0597:1, H0231:1, H0327:1,





H0544:1, H0123:1, H0050:1, H0095:1, H0373:1, L0163:1, H0051:1, T0010:1, T0023:1, H0213:1, L0142:1, H0383:1, S0366:1,





H0163:1, H0040:1, H0616:1, H0057:1, H0379:1, S0038:1, T0042:1, S0150:1, S0144:1, H0529:1, L0369:1, L3905:1, L0766:1,





L0775:1, L0532:1, H0693:1, H0689:1, H0670:1, L0602:1, H0522:1, S0044:1, L0611:1, S0027:1, S0028:1, L0741:1, L0743:1,





L0748:1, L0439:1, L0592:1, L0485:1, L0608:1, L0366:1, H0668:1, H0542:1, H0543:1 and H0423:1.

145
HSSEF77
658725
155
H0617:7, L0750:7, H0556:5, L0769:5, L0783:5, L0758:5, L0759:5, L0665:4, L0741:4, S0132:3, L0761:3, L0742:3, L0439:3,





L0755:3, L0592:3, H0618:2, H0620:2, H0038:2, L0771:2, L0662:2, L0659:2, L0666:2, S0126:2, H0670:2, S0328:2, S0380:2,





L0747:2, L0753:2, L0731:2, H0395:1, H0295:1, H0294:1, H0657:1, H0656:1, H0341:1, H0484:1, H0663:1, H0638:1, S0356:1,





S0444:1, H0741:1, L3271:1, H0549:1, H0550:1, H0370:1, H0455:1, H0632:1, H0486:1, T0039:1, T0112:1, H0156:1, H0581:1,





H0052:1, H0545:1, H0046:1, H0150:1, H0081:1, S0051:1, H0107:1, H0061:1, H0188:1, H0288:1, S0250:1, H0428:1, H0135:1,





L0766:1, L5574:1, L0774:1, L0775:1, L0375:1, L0806:1, L0776:1, L0807:1, L0657:1, L0658:1, L0540:1, L0384:1, L0809:1,





L0663:1, L0438:1, H0672:1, H0754:1, S0188:1, S0406:1, H0436:1, H0576:1, S3014:1, L0748:1, L0779:1, L0757:1 and





H0506:1.

146
HT4FV41
853400
156
AR244:10, AR185:10, AR204:9, AR275:9, AR202:9, AR194:9, AR052:9, AR271:8, AR246:8, AR289:8, AR219:7, AR316:7,





AR104:7, AR198:7, AR089:7, AR277:7, AR310:7, AR060:7, AR309:7, AR206:7, AR039:7, AR229:7, AR282:7, AR053:7,





AR283:7, AR269:6, AR205:6, AR270:6, AR312:6, AR184:6, AR186:6, AR299:6, AR096:6, AR240:6, AR251:6, AR274:6,





AR182:6, AR055:6, AR033:6, AR291:6, AR218:6, AR243:6, AR266:6, AR290:6, AR192:5, AR268:5, AR280:5, AR247:5,





AR213:5, AR298:5, AR231:5, AR273:5, AR313:5, AR234:5, AR284:5, AR296:5, AR061:5, AR248:5, AR238:5, AR285:5,





AR183:5, AR253:5, AR300:5, AR267:4, AR286:4, AR315:4, AR175:4, AR293:4, AR294:4, AR177:4, AR249:4, AR281:3,





AR227:3, AR233:3, AR292:3, AR263:3, AR265:3, AR232:3, AR295:3, AR226:3, AR237:3, AR258:2, AR314:2, AR256:1,





AR259:1, AR179:1, AR241:1 L0794:10, L0800:7, L0769:6, L0751:6, L0761:4, L0809:4, H0521:4, L0439:4, H0585:3,





H0617:3, H0494:3, L0659:3, L0665:3, L0777:3, H0265:2, H0255:2, S0354:2, S0376:2, H0370:2, H0069:2, H0083:2, H0040:2,





L0770:2, L0764:2, L0662:2, L5622:2, L0666:2, L0663:2, L0438:2, L0743:2, L0780:2, S0436:2, H0667:2, H0423:2, S0040:1,





H0713:1, H0295:1, H0254:1, H0638:1, S0418:1, S0420:1, S0360:1, H0734:1, S0046:1, S0476:1, H0587:1, H0635:1, H0575:1,





H0004:1, H0618:1, H0052:1, H0194:1, H0544:1, H0545:1, H0373:1, T0010:1, H0267:1, H0179:1, H0622:1, L0194:1, H0181:1,





H0124:1, H0087:1, H0412:1, H0413:1, H0646:1, S0144:1, L0369:1. L0640:1, L0763:1, L0772:1, L0646:1, L0643:1, L0644:1,





L0768:1, L0803:1, L0805:1, L0655:1, L0518:1, L0783:1, L0384:1, L0789:1, S0052:1, L2263:1, L0710:1, H0547:1, H0682:1,





S0152:1, H0187:1, H0727:1, S0390:1, L0752:1, L0757:1, L0758:1, H0665:1, H0543:1, H0422:1 and S0424:1.

147
HT5FX79
794169
157
AR313:23, AR055:11, AR089:11, AR316:10, AR060:10, AR039:9, AR277:9, AR096:8, AR240:8, AR283:8, AR299:7,





AR300:7, AR282:7, AR185:7, AR104:6, AR219:5, AR218:4 H0584:45, L0748:8, H0167:6, L0766:6, L0779:5, L0758:5,





H0445:5, L0777:4, H0581:3, H0529:3, L0805:3, L0789:3, L0750:3, H0333:2, L0471:2, H0024:2, L0483:2, H0090:2, H0494:2,





L0769:2, L0768:2, L0774:2, L0783:2, L5622:2, H0593:2, L0747:2, L0755:2, L0731:2, L0589:2, H0556:1, S0114:1, S0134:1,





H0255:1, L0481:1, S0360:1, H0675:1, H0645:1, H0619:1, H0453:1, H0574:1, H0575:1, H0309:1, L0157:1, H0014:1, H0083:1,





H0615:1, H0622:1, H0553:1, H0708:1, H0598:1, H0038:1, H0616:1, H0551:1, H0477:1, H0056:1, S0016:1, H0561:1, S0002:1,





L0763:1, L0637:1, L0761:1, L0646:1, L0771:1, L0794:1, L0803:1, L0375:1, L0806:1, L0653:1, L0776:1, L0655:1, L0527:1,





L0790:1, L0792:1, L4501:1, L4508:1, H0547:1, H0689:1, H0690:1, H0659:1, H0539:1, S0380:1, H0518:1, H0521:1, H0696:1,





S0406:1, L0754:1, L0749:1, S0394:1, S0106:1, S0026:1, S0276:1, H0542:1, H0543:1, H0423:1, S0424:1 and H0506:1.

148
HT5GR59
801930
158
AR240:19, AR096:15, AR316:10, AR300:9, AR055:9, AR039:8, AR313:8, AR282:8, AR277:7, AR185:7, AR060:7, AR219:7,





AR218:6, AR299:6, AR104:6, AR283:6, AR089:5 H0584:36, H0585:22, H0141:11, H0167:9, H0457:7, H0521:6, S0474:4,





H0575:3, L0731:3, H0265:2, H0556:2, H0581:2, L0761:2, H0543:2, H0140:1, H0638:1, S0358:1, S0140:1, H0747:1, H0619:1,





H0497:1, H0559:1, H0069:1, H0635:1, H0427:1, S0280:1, H0252:1, H0477:1, L0667:1, L0768:1, L0775:1, L0659:1, L0791:1,





L0792:1, S0053:1, L0777:1, L0758:1, H0445:1 and H0506:1.

149
HTAE178
637684
159
AR249:6, AR060:5, AR248:5, AR241:4, AR202:4, AR055:4, AR273:4, AR244:4, AR053:4, AR184:3, AR052:3, AR253:3,





AR186:3, AR182:3, AR213:3, AR206:3, AR175:3, AR251:3, AR270:3, AR282:3, AR312:3, AR274:2, ARO61:2, AR286:2,





AR300:2, AR269:2, AR243:2, AR309:2, AR298:2, AR192:2, AR246:2, AR089:2, AR185:2, AR275:2, AR316:2, AR204:2,





AR299:2, AR268:2, AR233:2, AR240:2, AR283:2, AR310:2, AR238:2, AR198:2, AR294:2, AR277:2, AR033:2, AR267:2,





AR247:2, AR104:2, AR292:2, AR284:2, AR295:2, AR285:1, AR096:1, AR313:1, AR226:1, AR177:1, AR290:1, AR218:1,





AR259:1, AR231:1, AR281:1, AR039:1, AR291:1, AR237:1, AR293:1, AR229:1, AR205:1, AR296:1 H0069:1, S0474:1 and





L0766:1.

150
HTEAG62
812332
160
AR310:2, AR282:2, AR206:2, AR273:2, AR186:1, AR295:1, AR294:1, AR175:1 L0766:6, H0038:5, L0758:4, H0616:3,





S0422:2, L0779:2, L0752:2, H0638:1, S0376:1, S0132:1, L3388:1, H0250:1, L0564:1, L0794:1, L0803:1, L0666:1, L0777:1,





L0755:1, H0595:1, S0434:1 and H0542:1.

151
HTEDJ28
762845
161
AR219:24, AR218:21, AR089:19, AR055:18, AR313:16, AR299:15, AR096:13, AR316:13, AR104:13, AR060:11, AR185:11,





AR283:10, AR039:9, AR277:9, AR282:9, AR300:8, AR240:7 L0747:9, L0439:8, L0809:6, L0766:5, L0750:5, L0758:5,





L0740:4, L0752:4, L0731:4, L0662:3, H0547:3, L0779:3, L0777:3, L0757:3, H0375:2, L0646:2, L0774:2, L0783:2, H0144:2,





L0759:2, S0442:1, H0333:1, T0060:1, H0327:1, H0399:1, L0483:1, H0038:1, L0564:1, S0382:1, H0538:1, H0743:1, L0763:1,





L0638:1, L0765:1, L0771:1, L0649:1, L0522:1, L0775:1, L0655:1, L0659:1, L0792:1, L0663:1, L0438:1, H0648:1, L0756:1,





L0753:1, L0596:1, L0590:1, L0592:1, L0608:1, H0423:1 and S0460:1.

152
HTEDS12
838621
162
H0253:4, L0779:2, H0618:1, H0050:1, H0038:1, L0151:1, L0758:1 and H0445:1.

153
HTBHA56
806461
163
AR104:41, AR089:32, AR299:24, AR313:21, AR055:19, AR096:18, A2R240:18, AR060:17, AR185:17, AR316:16, AR039:15,





AR282:12, AR300:11, AR219:11, AR277:10, AR218:9, AR283:8 L0754:8, L0770:5, L0794:5, L0805:5, H0553:4, L0803:4,





H0615:3, L0769:3, L0439:3, L0777:3, L0752:3, H0052:2, H0038:2, L0637:2, L3905:2, L0768:2, L0659:2, L5623:2, L0666:2,





L0664:2, L3828:2, H0547:2, H0593:2, H0682:2, H0539:2, H0521:2, L0744:2, L0757:2, L0604:2, L0601:2, H0624:1, H0657:1,





H0656:1, S0212:1, H0254:1, H0662:1, L0005:1, L2323:1, S0045:1, H0619:1, H0550:1, H0592:1, L3655:1, H0013:1, H0036:1,





H0618:1, H0620:1, H0023:1, S0051:1, H0188:1, H0028:1, H0428:1, T0006:1, H0213:1, L0455:1, H0135:1, H0616:1, H0264:1,





H0413:1, T0069:1, S0038:1, H0100:1, L3180:1, L0763:1, L0638:1, L5565:1, L0761:1, L0667:1, L0804:1, L0650:1, L0375:1,





L0776:1, L0807:1, L0809:1, L0519:1, L0663:1, L0665:1, L0710:1, L3811:1, L3825:1, L3827:1, H0520:1, S0126:1, H0684:1,





H0435:1, H0659:1, H0648:1, S0190:1, S0404:1, H0555:1, L0741:1, L0751:1, L0747:1, L0753:1 and L0758:1.

154
HTEJD29
695798
164
H0038:2

155
HTENR63
877952
165
AR277:32, AR283:29, AR218:25, AR219:22, AR282:21, AR316:21, AR089:20, AR313:19, AR104:18, AR055:17, AR299:16,





AR096:16, AR240:16, AR185:15, AR300:14, AR039:14, AR060:11 L0748:9, L0777:6, L0439:5, L0749:5, L0766:4, L0438:4,





L0755:4, L0752:3, L0594:3, L3814:2, S0212:2, H0014:2, H0032:2, H0598:2, H0038:2, H0100:2, L0775:2, S0330:2, L0754:2,





L0750:2, L0731:2, L0758:2, L0759:2, L0485:2, S0192:2, S0040:1, H0583:1, S0356:1, H0733:1, S0046:1, H0747:1, L3652:1,





H0613:1, H0024:1, H0373:1, H0375:1, H0179:1, H0166:1, H0673:1, H0591:1, H0616:1, H0551:1, H0412:1, H0129:1, H0529:1,





L0761:1, L0771:1, L0804:1, L0784:1, L0806:1, L0655:1, L0783:1, L0666:1, H0144:1, L3811:1, S0126:1, S0328:1, H0539:1,





S0152:1, L0740:1, L0756:1, L0779:1, L0757:1, H0445:1, L0599:1 and S0026:1.

156
HTGGM44
842856
166
AR246:5, AR244:5, AR184:5, AR253:5, AR309:4, AR313:4, AR186:4, AR052:3, AR206:3, AR312:3, AR310:3, AR204:3,





AR274:3, AR291:3, AR060:3, AR055:3, AR229:3, AR053:3, AR292:3, AR269:3, ARO6I:3, AR243:3, AR205:3, AR247:3,





AR213:2, AR299:2, AR294:2, AR089:2, AR273:2, AR270:2, AR266:2, AR263:2, AR039:2, AR265:2, AR293:2, AR316:2,





AR275:2, AR282:2, AR267:2, AR251:2, AR277:2, AR231:2, AR283:2, AR238:2, AR284:2, AR185:2, AR271:2, AR300:2,





AR182:2, AR226:2, AR096:1, AR237:1, AR033:1, AR234:1, AR290:1, AR240:1, AR241:1, AR259:1, AR194:1, AR227:1,





AR104:1, AR298:1 L0748:8, L0805:2, L0599:2, S0218:1, T0040:1, H0635:1, S0250:1, H0212:1, H0634:1, H0063:1, S0002:1,





L0766:1, H0144:1, S0126:1 and H0518:1.

157
HTHBZ06
832477
167
AR218:86, AR219:83, AR089:54, AR104:50, AR282:48, AR313:44, AR283:40, AR055:34, AR316:31, AR240:29, AR185:27,





AR096:23, AR060:22, AR299:22, AR300:16, AR039:15, AR277:11 S0414:9, L0005:7, L0065:7, S0360:6, S0422:5, H0545:4,





AR0648:4, L0777:4, L0758:4, H0716:3, H0657:3, S0474:3, L0770:3, L0666:3, L0665:3, L0600:3, H0674:2, H0494:2, L0769:2,





L0638:2. L0637:2, L0768:2, L0803:2, L0774:2, L0805:2, L0664:2, L0438:2, H0520:2, H0696:2. L0751:2, L0745:2, L0749:2,





L0756:2, L0779:2, L0757:2, L3643:1, H0484:1, H0671:1, S0358:1, L3649:1, H0742:1, H0741:1, S0132:1, L0623:1, H0581:1,





S0214:1, H0063:1, H0412:1, H0413:1, S0002:1, L0369:1, L0796:1, L0662:1, L0766:1, L0375:1, L0656:1, L0659:1, L0647:1,





L3872:1, L0663:1, H0684:1, S0328:1, S0350:1, H0436:1, L0743:1, L0754:1, L0755:l, L0731:1, S0031:1, S0436:1, L0485:1,





L0608:1, L0362:1, H0506:1 and H0352:1.

158
HTLBT80
840045
168
AR251:22, AR273:18, AR053:18, AR309:16, AR310:16, AR183:15, AR313:15, AR274:15, AR263:15, AR247:15, AR312:14,





AR314:14, AR266:14, AR265:14, AR219:14, AR175:13, AR218:13, AR285:12, AR280:12, AR182:12, AR268:12, AR293:12,





AR213:12, AR052:12, AR292:11, AR290:11, AR286:11, AR267:11, AR277:11, AR289:11, AR315:11, AR296:11, AR256:11,





AR295:11, AR177:10, AR291:10, AR269:10, AR271:10, AR284:10, AR096:9, AR243:9, AR270:9, AR299:9, AR283:9,





AR249:9, AR300:9, AR033:9, AR253:9, AR238:9, AR184:8, AR179:8, AR248:8, AR231:8, AR298:8, AR234:8, AR061:8,





AR226:8, AR282:8, AR232:8, AR229:8, AR316:8, AR258:8, AR259:7, AR233:7, AR240:7, AR186:7, AR294:7, AR185:7,





AR198:7, AR237:7, AR275:6, AR281:6, AR039:6, AR192:6, AR227:6, AR089:6, AR104:6, AR246:6, AR055:6, AR244:6,





AR202:5, AR204:5, AR060:5, AR206:4, AR205:4, AR241:4, AR194:1 L0659:6, H0556:4, H0521:4, L0439:4, L0745:4,





L0759:4, H0657:3, S0360:3, L0761:3, L0662:3, L0766:3, L0809:3, H0549:2, H0392:2, H0253:2, H0581:2, H0620:2, H0051:2,





H0551:2, H0494:2, L0770:2, L0794:2, L0649:2, L0665:2, H0520:2, S0032:2, L0741:2, L0743:2, L0748:2, L0747:2, L0779:2,





L0758:2, L0605:2, H0650:1, H0484:1, H0254:1, H0402:1, S0358:1, H0580:1, H0741:1, S0007:1, S0132:1, S0476:1, H0393:1,





H0369:1, H0550:1, H0409:1, H0256:1, H0250:1, H0042:1, H0036:1, H0318:1, S0049:1, H0050:1, H0014:1, H0375:1, S6028:1,





H0266:1, H0292:1, H0428:1, H0622:1, H0031:1, H0617:1, L0456:1, H0135:1, H0040:1, H0379:1, H0264:1, H0056:1, H0623:1,





H0100:1, H0633:1, S0002:1, H0529:1, L0762:1, L5575:1, L0772:1, L0646:1, L0771:1, L0773:1, L0767:1, L0768:1, L0803:1,





L0805:1, L0653:1, L5622:1, L4501:1, L0666:1, H0689:1, H0690:1, H0682:1, H0670:1, H0522:1, S0044:1, H0436:1, S0027:1,





L0754:1, L0749:1, L0753:1, L0731:1, S0436:1, H0653:1, S0192:1, H0542:1, H0543:1, H0423:1 and S0424:1.

159
HTLDU78
637702
169
L0758:3, H0253:1 and L0779:1.

160
HTLFA13
535937
170
AR313:11, AR089:10, AR039:9, AR096:8, AR299:8, AR282:7, AR277:7, AR283:7, AR104:7, AR219:7, AR060:7, AR270:7,





AR316:6, AR218:6, AR310:6, AR300:6, AR183:5, AR233:5, AR294:5, AR185:5, AR237:5, AR226:5, AR055:5, AR238:5,





AR231:4, AR227:4, AR296:4, AR251:4, AR312:4, AR240:4, AR033:4, AR269:4, AR290:3, AR285:3, AR052:3, AR184:3,





AR295:3, AR258:3, AR186:3, AR292:3, AR274:2, AR205:2, AR179:2, AR053:2, AR061:2, AR206:2, AR309:2, AR293:2,





AR266:2, AR273:2, AR259:1, AR194:1, AR234:1, AR182:1, AR232:1, AR241:1, AR284:1 H0253:2 and S0011:1.

161
HTLGI89
835069
171
AR283:67, AR277:61, AR219:56, AR282:51, AR104:48, AR240:47, AR316:46, AR218:45, AR313:44, AR089:42, AR096:40,





AR185:37, AR299:36, AR055:34, AR300:32, AR039:32, AR060:31 L0758:16, L0748:10, H0620:7, L0731:6, H0246:5,





S0007:4, H0253:4, L0769:4, L0754:4, H0052:3, H0100:3, L0638:3, L5575:3, L0766:3, L0650:3, L0774:3, S0152:3, S3014:3,





L0439:3, H0265:2, H0556:2, T0002:2, S6024:2, H0656:2, H0341:2, S0212:2, S0376:2, H0619:2, H0261:2, S0222:2, H0318:2,





H0196:2, H0012:2, T0010:2, H0068:2, H0551:2, H0413:2, H04942, L0770:2, L5565:2, L3905:2, L0768:2, L0776:2, S3012:2,





L0741:2, L0749:2, L0750:2, L0759:2, S0434:2, L0608:2, L0595:2, H0352:2, S0040:1, L0760:1, S0116:1, S0282:1, H0638:1,





S0418:1, S0356:1, S0444:1, H0730:1, H0747:1, S0476:1, H0393;1, H0549:1, H0550:1, H0592:1, H0333:1, H0486:1, T0114:1,





H0250:1, H0069:1, S0280:1. H0156:1, F10599:1, H0575:1, H0036:1, H0618:1, H0597:1, H0178:1, N0006:1, H0563:1, H0197:1,





H0199:1, H0051:1, H0083:1, H0060:1, H0188:1, H0290:1, H0284:1, H0428:1, H0622:1, L0483:1, H0124:1, H0135:1, H0163:1,





H0040:1, H0264:1, H0412:1, L0564:1, H0130:1, H0641:1, S0144:1, S0002:1, L0763:1, L0761:1, L0372:1, L0643:1, L0764:1,





L0771:1, L0648:1, L0767:1, L0803:1, L0804:1, L0375:1, L0378:1, L0659:1, L0544:1, L0665:1, H0703:1, L0352:1, H0670:1,





S0328:1, S0330:1, H0753:1, H0522:1, H0134:1, S0027:1, L0747:1, L0756:1, L0777:1, L0753:1, S0260:1, H0445:1, S0436:1,





L0597:1, H0653:1 and S0194:1.

162
HTNBK13
831967
172
L0779:5, L0731:4, L0593:4, H0046:3, L0776:3, L0666:3, H0031:2, L0772:2, L0774:2, L0805:2, H0670:2, L0439:2, L0754:2,





L0777:2, L0758:2, L0590:2, T0002:1, L0717:1, H0632:1, L0622:1, T0082:1, H0581:1, H0263:1, T0115:1, H0597:1, L0471:1,





H0012:1, H0620:1, H0163:1, T0067:1, L0770:1, L0637:1, L0388:1, L0657:1, L0382:1, L0664:1, S0126:1, H0660:1, S0378:1,





H0521:1, L0747:1, L0750:1, L0756:1, L0752:1, L0755:1, L0759:1, S0031:1, L0599:1 and L0603:1.

163
HTOAM11
664508
173
AR313:30, AR039:27, AR185:18, AR299:16, AR300:13, AR277:13, AR096:13, AR089:12, AR218:11, AR219:11, AR316:9,





AR240:9, AR104:8, AR060:7, AR055:6, AR282:6, AR283:3 S0010:1 and H0264:1.

164
HTOHO21
732808
174
H0556:3 and H0264:1.

165
HTPCO75
853645
175
AR104:12, AR219:11, AR089:9, AR039:9, AR282:8, AR218:8, AR313:8, AR060:8, AR300:8, AR055:7, AR316:7, AR277:7,





AR299:7, AR096:7, AR185:6, AR240:5, AR283:4 H0039:5, L0756:5, S0448:3, L0805:3, L0759:3, H0265:2, S0354:2, L0471:2,





H0674:2, S0422:2, L0794:2, L0517:2, L0666:2, L0779:2, L0777:2, L0758:2, H0170:1, H0556:1, S0342:1, S0134:1, H0637:1,





H0599:1, H0318:1, H0263:1, H0596:1, H0252:1, H0428:1, H0673:1, H0040:1, H0264:1, H0268:1, H0773:1, H0538:1, L0764:1,





L0768:1, L0766:1, L0804:1, L0774:1, L0652:1, L0527:1, L0809:1, L0519:1, L0791:1, H0144:1, H0520:1, H0519:1, H0689:1,





H0648:1, S0028:1, L0439:1, L0731:1, H0444:1, H0445:1, S0434:1, S0026:1, S0242:1 and H0543:1.

166
HTSFJ32
637720
176
AR104:9, AR039:7, AR277:5, AR282:4, AR313:4, AR299:4, AR240:3, AR089:3, AR283:3, AR300:3, AR096:3, AR185:3,





AR055:2, AR316:2, AR219:2, AR218:2, AR060:2 H0556:1, S0114:1, H0087:1, H0538:1, H0695:1 and L0774:1.

167
HTTCB60
853401
177
L0794:11, L0809:11, L0750:9, L0805:8, L0791:7, L0747:7, L0800:6, L0758:6, L0759:6, H0620:5, L0749:5, S0358:4, H0135:4,





L0769:4, L0659:4, L0780:4, H0556:3, L0471:3, H0040:3, L0804:3, S0360:2, H0393:2, H0550:2, H0592:2, H0333:2, S0049:2,





H0124:2, S0438:2, L0771:2, L0662:2, L0803:2, L4501:2, H0547:2, L3832:2, L0779:2, L0755:2, L0731:2, S0434:2, L0603:2,





H0506:2, H0713:1, H0717:1, H0294:1, H0662:1, H0729:1, H0734:1, S0045:1, H0607:1, H0586:1, H0587:1, L3816:1, T0040:1,





L3653:1, S0280:1, H0590:1, S0010:1, H0581:1, H0251:1, H0041:1, H0565:1, H0570:1, H0123:1, H0081:1, H0050:1, H0188:1,





H0039:1, H0622:1, H0038:1, H0063:1, H0412:1, H0413:1, S0440:1, S0210:1, S0002:1, L0763:1, L0770:1, L3905:1, L0761:1,





L0641:1, L0768:1, L0766:1, L0375:1, L0806:1, L0776:1, L0789:1, L0790:1, L0666:1, L0663:1, L0665:1, L2258:1, L2654:1,





H0520:1, H0660:1, H0672:1, H0539:1, S0380:1, H0521:1, H0696:1, H0555:1, L0744:1, L0748:1, L0757:1, H0445:1, L0584:1,





L0589:1, S0242:1, S0194:1, H0008:1 and H0352:1.

168
HTTEE41
840950
178
AR219:84, AR218:59, AR316:43, AR313:32, AR104:24, AR089:24, AR185:24, AR039:23, AR096:23, AR299:21, AR055:20,





AR060:17, AR282:14, AR300:14, AR283:11, AR240:11, AR277:10 H0040:17, H0251:14, L0758:10, L0748:8, L0731:8,





H0494:7, L0666:7, H0144:7, H0659:7, L0747:7, L0749:7, L0757:7, H0038:6, H0529:6, L0770:6, L0662:6, L0659:6, H0013:5,





H0318:5, H0616:5, S0440:5, L0775:5, L0776:5, H0519:5, L0588:5, L0592:5, H0341:4, S0360:4, H0412:4, L0663:4, H0547:4,





L0754:4, L0595:4, H0542:4, H0543:4, H0423:4, H0171:3, H0657:3, H0656:3, S0045:3, L3388:3, H0581:3, S0049:3, T0110:3,





H0046:3, H0090:3, H0591:3, H0551:3, H0100:3, H0022:3, H0625:3, H0633:3, S0422:3. L0375:3, L0664:3, H0682:3, S0406:3,





L0740:3, H0556:2, H0241:2, H0638:2, S0418:2, L0005:2, S0442:2, S0376:2, H0722:2, H0393:2. L0717:2, S0222:2, H0574:2,





H0486:2, T0040:2, L0471:2, S0051:2, S0003:2, H0252:2, L0483:2, T0006:2, H0031:2, H0032:2, H0124:2, H0634:2, H0264:2,





T0042:2, S0150:2, H0646:2, L0763:2, L0637:2, L0646:2, L0374:2, L0764:2, L0768:2, L0653:2, L0665:2, H0593:2, H0435:2,





H0658:2, H0536:2, S0152:2, L3832:2, H0521:2, S3014:2, S0027:2, S0028:2, L0439:2, L0750:2, L0777:2, S0436:2, L0596:2,





L0608:2, L0604:2, L0594:2, L0362:2, 50026:2, H0667:2, S0452:2, H0506:2, L0411:1, H0624:1, H0170:1, H0395:1, H0265:1,





T0002:1, H0220:1, H0140:1, H0159:1, H0686:1, H0583:1, H0650:1, S0212:1, H0484:1, H0664:1, L0481:1, S0356:1, S0354:1,





S0358:1, S0444:1, S0408:1, L3649:1, H0580:1, H0747:1, H0437:1, H0431:1, T0104:1, H0600:1, H0592:1, H0586:1, L3817:1,





H0642:1, H0632:1, L2482:1, T0114:1, H0244:1, H0250:1, H0069:1, H0156:1, L0021:1, H0599:1, H0036:1, S0346:1, H0596:1,





H0544:1, H0009:1, N0006:1, L0157:1, H0569:1, H0123:1, H0242:1, H0024:1, H0083:1, H0375:1, H0328:1, H0615:1, H0428:1,





H0039:1, H0622:1, H0213:1, H0553:1, L0142:1, H0628:1, H0674:1, H0388:1, L0456:1, H0708:1, H0068:1, H0598:1, S0036:1,





H0135:1, H0087:1, H0380:1, H0413:1, H0056:1, L0351:1, T0041:1, H0334:1, H0561:1, H0366:1, S0448:1, S0294:1, H0130:1,





H0641:1, H0649:1, S0208:1, S0002:1, S0426:1, L0520:1, L0631:1, L0769:1, L0638:1, L5565:1, L0667:1, L0772:1, L0372:1,





L0641:1, L0626:1, L0794:1, L0766:1, L0381:1, L0650:1, L0651:1, L0806:1, L0655:1, L0807:1, L0657:1, L0636:1, L0518:1,





L0782:1, L0382:1, L0809:1, L3391:1, L2263:1, L2259:1, L2262:1, L0565:1, H0693:1, L3827:1, H0520:1, S0126:1, H0689:1,





H0670:1, H0660:1, H0666:1, H0648:1, L0602:1, H0710:1, H0518:1, S0176:1, H0134:1, H0555:1, H0436:1, H0478:1, H0631:1,





L0779:1, L0752:1, S0434:1, L0605:1, L0591:1, L0599:1, H0665:1, S0196:1, L2368:1, H0008:1 and H0352:1.

169
HTWEH94
561680
179
AR313:9, AR039:7, AR096:5, AR300:3, AR185:3, AR089:3, AR299:3, AR277:2, AR316:2, AR240:2, AR060:2, AR104:2,





AR218:1, AR282:1, AR055:1 L0766:1 and H0436:1.

170
HTXFA72
853410
180
AR313:47, AR039:45, AR299:26, AR089:23, AR096:23, AR185:22, AR300:20, AR277:19, AR219:17, AR316:16, AR240:14,





AR104:14, AR060:12, AR218:11, AR282:10, AR055:8, AR283:5 H0265:1

171
HTXKF95
834438
181
AR266:6, AR309:6, AR178:6, AR176:5, AR162:5, AR161:5, AR163:5, AR282:5, AR096:5, AR312:5, AR104:5, AR053:5,





AR271:4, AR185:4, AR060:4, AR055:4, AR183:4, AR308:4, AR246:4, AR168:4, AR181:4, AR269:4, AR229:4, AR192:4,





AR263:4, AR175:4, AR274:4, AR193:4, AR225:4, AR165:4, AR267:4, AR182:4, AR164:4, AR316:4, AR213:4, AR228:4,





AR166:4, AR242:3, AR240:3, AR270:3, AR272:3, AR212:3, AR264:3, AR283:3, AR243:3, AR275:3, AR313:3, AR179:3,





AR235:3, AR268:3, AR239:3, AR293:3, AR237:3, AR171:3, AR180:3, AR289:3, AR173:3, AR215:3, AR172:3, AR089:3,





AR231:3, AR210:2, AR061:2, AR290:2, AR230:2, AR201:2, AR233:2, AR204:2, AR196:2, AR277:2, AR223:2, AR226:2,





AR299:2, AR177:2, AR190:2, AR247:2, AR300:2, AR296:2, AR257:2, AR285:2, AR222:2, AR033:2, AR200:2, AR039:2,





AR191:2, AR311:2, AR199:2, AR297:2, AR291:2, AR288:2, AR203:2, AR227:2, AR232:2, AR188:2, AR294:2, AR236:2,





AR255:2, AR189:2, AR286:2, AR260:2, AR238:2, AR211:2, AR174:2, AR287:1, AR234:1, AR261:1, AR256:1, AR216:1,





AR262:1, AR252:1 L0754:41, L0747:8, L0755:5, L0659:4, H0265:2, H0556:2, H0586:2, L0471:2, H0553:2, L0764:2, L0662:2,





L0794:2, L0748:2, L0751:2, L0749:2, L0750:2, H0305:1, S0358:1, S0046:1, H0441:1, H0599:1, H0569:1, H0050:1, H0051:1,





H0030:1, H0124:1, H0616:1, L0770:1, L0769:1, L0800:1, L0644:1, L0363:1, L0803:1, L0804:1, L0775:1, L0806:1, L0783:1,





L0666:1, L0665:1, HOl44:1, H0555:1, S3012:1, L0779:1, L0731:1, L0605:1, L0599:1, L0603:1, H0543:1, H0422:1 and





H0506:1.

172
HUKDF20
566823
182
AR055:7, AR218:6, AR060:6, AR300:5, AR282:4, AR104:4, AR313:4, AR283:4, AR185:4, AR299:4, AR277:3, AR219:3,





AR089:3, AR316:3, AR039:3, AR240:3, AR096:2 H0261:1, H0266:1 and H0059:1.

173
HUSCJ14
894699
183
AR239:10, AR228:10, AR227:9, AR237:9, AR230:8, AR233:8, AR287:8, AR203:7, AR288:7, AR176:6, AR184:6, AR199:6,





AR229:6, AR215:6, AR190:6, AR200:5, AR245:5, AR174:5, AR234:5, AR191:5, AR180:4, AR297:4, AR232:4, AR226:4,





AR289:4, AR298:4, AR194:4, AR170:4, AR257:4, AR061:3, AR292:3, AR173:3, AR231:3, AR262:3, AR242:3, AR284:3,





AR286:3, AR251:3, AR179:3, AR236:3, AR238:3, AR255:3, AR161:3, AR189:3, AR162:3, AR235:3, AR293:3, AR282:3,





AR188:3, AR294:3, AR165:3, AR163:3, AR164:3, AR166:3, AR285:2, AR201:2, AR181:2, AR295:2, AR177:2, AR247:2,





AR290:2, AR205:2, AR300:2, AR225:2, AR260:2, AR198:2, AR261:2, AR193:2, AR291:2, AR268:2, AR175:2, AR270:2,





AR183:2, AR211:2, AR296:2, AR196:2, AR185:2, AR258:2, AR250:2, AR240:2, AR178:2, AR204:2, AR195:2, AR060:2,





AR312:1, AR311:1, AR210:1, AR224:1, AR243:1, AR299:1, AR269:1, AR316:1, AR275:1, AR186:1, AR172:1, AR039:1,





AR267:1, AR256:1, AR263:1, AR055:1, AR089:1, AR217:1 L2654:6, L0741:4, S0192:4, H0677:4, H0556:3, H0013:3,





H0052:3, L0766:3, L0744:3, L0439:3, L0757:3, H0265:2, S0040:2, S0410:2, H0599:2, H0545:2, H0266:2, H0030:2, H0135:2,





L3905:2, L5622:2, H0520:2, H0547:2, H0519:2, L0748:2, L0756:2, L0777:2, L0780:2, L0758:2, L0485:2, L0604:2, H0739:1,





H0713:1, S0134:1, S0218:1, H0656:1, L2909:1, S0212:1, H0663:1, S0420:1, L1562:1, S0360:1, S0408:1, H0742:1, S0132:1,





S0476:1, H0393:1, H0587:1, T0040:1, H0575:1, H0309:1, H0009:1, L0471:1, H0620:1, H0510:1, H0290:1, S0250:1, S0022:1,





T0023:1, H0488:1, H0268:1, T0041:1, T0042:1, H0538:1, S0210:1, L0763:1, L0800:1, L0771:1, L0794:1, L0804:1, L0774:1,





L0775:1, L5623:1, L0793:1, L2652:1, L2257:1, L2260:1, L0710:1, L2262:1, H0144:1, H0593:1, H0435:1, H0521:1, H0555:1,





L0743:1, L0754:1, L0779:1, L0752:1, S0031:1, S0436:1, L0596:1, L0605:1, L0601:1, S0106:1, H0667:1, S0276:1 and L3576:1.

174
HUVDJ48
564853
184
AR055:6, AR060:5, AR283:5, AR039:5, AR185:4, AR096:4, AR240:4, AR104:4, AR299:4, AR300:3, AR089:3, AR316:3,





AR313:3, AR282:3, AR218:2, AR277:2, AR219:2 H0393:1, H0056:1 and L0662:1.

175
HBDAB91
864374
185
AR282:3, AR219:1 H0551:2, L0803:2, L0439:2, L0750:2, S0308:2, L0644:1, L0655:1, L0809:1, L0780:1 and L0752:1.


HBDAB91
789532
193

176
HELGG84
851137
186
AR218:39, AR219:33, AR299:7, AR104:6, AR055:5, AR313:4, AR300:4, AR185:4, AR060:4, AR240:3, AR282:3, AR089:3,





AR316:2, AR283:2, AR039:2, AR277:2, AR096:1 L0750:5, S0045:2, S0212:1, H0742:1, S0300:1, S0010:1, H0505:1, S0049:1,





H0266:1, L0598:1, L0662:1, L0809:1, S0374:1, H0696:1, L0758:1 and S0434:1.


HELGG84
674456
194

177
HILCA24
869856
187
AR316:4, AR282:2, AR096:1, AR299:1, AR039:1 L0748:4, H0090:2, L0659:2, H0521:2, L0777:2, L0608:2, H0543:2,





T0002:1, S0114:1, L3658:1, S0358:1, S0408:1, L3649:1, T0109:1, H0581:1, H0622:1, H0031:1, H0644:1, S0002:1, L0657:1,





L0526:1, L0789:1, L0664:1, S0380:1, H0522:1, L0749:1 and L0779:1.


HILCA24
782450
195

178
HYABC84
865064
188
AR313:19, AR219:16, AR218:13, AR104:13, AR096:11, AR316:11, AR240:10, AR299:10, AR089:10, AR282:9, AR185:9,





AR039:9, AR283:8, AR277:7, AR060:6, AR055:5, AR300:5


HYABC84
789854
196

179
HMCAZ04
668249
189
AR219:29, AR218:27, AR240:25, AR039:21, AR316:20, AR096:16, AR089: 15, AR299:13, AR283:13, AR282:12, AR060:9,





AR313:9, AR055:8, AR300:8, AR277:7, AR185:6, AR104:4 S0410:22, S0408:18, S0476:15, S0132:14, H0584:9, S0358:9,





S0002:8, H0521:8, L3388:7, L0748:7, S0442:6, H0494:6, L0599:6, S0142:5, L0777:5, S0278:4, L0483:4, L0775:4, L0659:4,





H0543:4, S0444:3, H0733:3, H0046:3, H0284:3, H0039:3, H0674:3, H0591:3, H0641:3, S0144:3, L0771:3, L0773:3, S0374:3,





L0439:3, H0422:3, H0677:3, H0556:2, T0002:2, H0657:2, S0212:2, S0360:2, H0574:2, H0486:2, H0635:2, H0575:2, H0231:2,





H0024:2, H0286:2, H0622:2, H0644:2, H0673:2, S0440:2, H0633:2, S0426:2, L0764:2, L0766:2, L0774:2, L0651:2, L0655:2,





L0664:2, H0593:2, H0658:2, H0710:2, S0044:2, S0404:2, L0745:2, L0747:2, L0731:2, S0434:2, L0581:2, S0011:2, S0276:2,





H0423:2, H0506:2, H0171:1, H0167:1, H0713:1, H0716:1, H0656:1, S0298:1, H0662:1, H0459:1, H0638:1, S0348:1, S0354:1,





S0376:1, H0580:1, H0729:1, H0742:1, H0722:1, H0734:1, H0208:1, S0045:1, H0632:1, H0075:1, H0156:1, H0042:1, H0036:1,





H0318:1, H0581:1, H0251:1, H0309:1, H0545:1, H0107:1, H0083:1, H0179:1, H0687:1, H0292:1, S0214:1, H0031:1, H0628:1,





H0617:1, L0055:1, H0032:1, H0316:1, H0090:1, H0038:1, H0040:1, H0063:1, T0067:1, H0264:1, L0564:1, H0202:1, S0014:1,





H0560:1, S0372:1, H0649:1, S0344:1, L0640:1, L0371:1, L0770:1, L0667:1, L0765:1, L0803:1, L0376:1, L0805:1, L0653:1,





L0542:1, L0783:1, L0809:1, L0663:1, H0701:1, S0126:1, H0689:1, H0672:1, S0328:1, H0539:1, L0602:1, H0522:1, S0406:1,





H0187:1, H0727:1, S0028:1, S0206:1, L0743:1, L0756:1, L0779:1, L0752:1, L0759:1, S0308:1, H0343:1, S0436:1, L0485:1,





L0601:1, H0653:1, S0196:1 and H0542:1.


HMCAZ04
887445
197


HMCAZ04
867910
198


HMCAZ04
858210
199


HMCAZ04
839783
200

180
HE8FD92
901142
190
AR055:6, AR299:6, AR060:6, AR240:5, AR218:5, AR219:5, AR300:5, AR185:5, AR089:4, AR316:3, AR096:3, AR104:3,





AR039:3, AR277:3, AR283:2, AR282:2, AR313:2


HE8FD92
888274
201


HE8FD92
869847
202


HE8FD92
856544
203


HE8FD92
843825
204

























































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
TABLE 1C cDNA Clone SEQ ID CONTIG BAC ID: SEQ ID EXON ID NO: X ID: A NO: B From-To H6EEU40 12 757048 AC026285 399 1-414 440-1918 H6EEU40 12 757048 AC023920 400 1-416 442-1920 H6EEU40 12 757048 AC026285 401 1-1458 1729-1836 H6EEU40 12 757048 AC023920 402 1-1459 1730-1837 HACAB68 13 584773 AL160283 403 1-2811 HACAB68 13 584773 AL354793 404 1-3734 3843-4723 HACAB68 13 584773 AL356058 405 1-3055 3165-4045 HACBJ56 14 847112 AC069497 406 1-117 2470-3367 4908-5262 5641-5756 7886-8200 9815-11138 HACBJ56 14 847112 AC007104 407 1-802 2342-2695 3074-3189 5319-5633 7248-8571 HACBJ56 14 847112 AC069497 408 1-453 HACBJ56 14 847112 AC007104 409 1-453 HADMB15 15 847116 AC026666 410 1-385 406-780 HADMB15 15 847116 AC026281 411 1-114 430-875 896-1262 HAGDW20 16 637489 AC006453 412 1-1568 HAGDW20 16 637489 AC005629 413 1-1569 HAGDW20 16 637489 AC010098 414 1-1569 HAGDW20 16 637489 AC006453 415 1-438 HAGDW20 16 637489 AC006453 416 1-375 HAGDW20 16 637489 AC005629 417 1-438 HAGDW20 16 637489 AC005629 418 1-375 HAJCH70 19 827275 AL159987 419 1-2168 HATCD80 21 826098 AL158801 420 1-1974 HATCD80 21 826098 AL158801 421 1-90 HATCI03 22 580805 AL137119 422 1-81 824-941 972-1185 2432-2705 3880-4812 4880-5011 5828-6591 8231-8398 8618-8767 9466-9728 HATCI03 22 580805 AL138688 423 1-81 825-942 973-1186 2433-2706 3881-4795 4870-5001 5818-6581 8221-8388 8608-8757 9456-9718 HATCI03 22 580805 AL137119 424 1-542 HATCI03 22 580805 AL138688 425 1-542 HBAGD86 23 838799 AC016755 426 1-41 1648-1993 2035-3552 3554-6713 HBAGD86 23 838799 AC016755 427 1-161 696-809 2256-2753 6910-6991 7733-7857 9267-9458 10650-10734 11114-11562 11678-11801 12524-12817 14494-15914 HBAGD86 23 838799 AC016755 428 1-217 HBHAA05 24 603174 AL353743 429 1-677 HBHAA05 24 603174 AL161453 430 1-677 HBHAA05 24 603174 AL161453 431 1-339 HBHAA81 25 846465 AC006059 432 1-230 1619-1699 1953-2090 2986-3054 3665-3786 3902-4406 4457-4674 5129-5531 5660-5811 5934-5969 7563-7959 8086-9195 9591-9735 ‘9788-10149 HBHAA81 25 846465 AC018471 433 1-230 1619-1699 1965-2090 2986-3054 3665-3786 3902-4405 4456-4673 5128-5530 5659-5810 5933-5968 7561-7957 8084-9193 9589-9733 9786-10146 HBHAA81 25 846465 AC006059 434 1-340 501-802 HBHAA81 25 846465 AC006059 435 1-661 1538-1684 3489-3680 3832-3933 4241-4410 5782-5872 5998-6150 HBHAA81 25 846465 AC018471 436 1-661 1539-1672 HBHAA81 25 846465 AC018471 437 1-340 501-802 HBIAA59 26 806303 AL121929 438 1-1114 1186-3742 HBIAA59 26 806303 AL121929 439 1-226 HBIAA59 26 806303 AL121929 440 1-243 HBJAC40 27 841235 AC007606 441 1-89 520-616 811-972 1604-1874 1982-5038 5125-5260 6459-6956 7370-7473 7507-7774 8952-9321 HBJAC40 27 841235 AC007606 442 1-403 428-1236 HBJDW56 29 520401 AC005532 443 1-626 HBJDW56 29 520401 AC005532 444 1-516 HBJDW56 29 520401 AC005532 445 1-176 HCDCY76 37 837972 AP001528 446 1-3072 HCDCY76 37 837972 AP001528 447 1-380 HCEEU18 39 688041 AC008469 448 1-169 HCEEU18 39 688041 AC026400 449 1-170 HCEEU18 39 688041 AC008469 450 1-304 420-602 1427-2108 2323-2645 3613-3987 4129-4442 4600-4731 4868-5039 5408-5538 5624-5776 6317-7734 HCEEU18 39 688041 AC008469 451 1-294 HCEEU18 39 688041 AC026400 452 1-98 HCEEU18 39 688041 AC026400 453 1-407 HCEGX05 40 827060 AL133227 454 1-32 712-1071 3453-3870 4197-4326 4639-4751 5131-5202 5588-5638 7454-8108 8670-8767 9511-9692 9754-10134 11109-11226 12456-12607 15237-15316 18143-18311 18429-18478 20682-20982 20988-21295 22686-23061 23358-23495 24076-24612 25196-25334 26760-26926 27041-27152 27271-27379 27697-28289 29024-29340 29761-29840 31168-32681 HCEGX05 40 827060 AL161661 455 1-130 443-555 935-1006 1392-1442 3258-3912 4474-4571 5315-5496 5558-5938 6915-7032 8262-8413 11042-11121 13948-14116 14234-14283 16487-16787 16793-17100 18494-18869 19166-19303 19884-20420 21002-21140 22566-22732 22847-22958 23077-23185 23503-24095 24826-25142 25563-25642 26969-28482 HCEGX05 40 827060 AL133227 456 1-51 476-521 842-1226 1375-1490 3745-4016 4046-4229 4430-4855 5300-6053 6598-6883 7406-7446 7461-8437 8550-8681 8888-8919 8943-9353 9458-9544 9834-10607 11550-11629 12196-12374 13532-14886 HCEGX05 40 827060 AL161661 457 1-418 HCEGX05 40 827060 AL161661 458 1-50 475-520 841-1225 3744-4014 4044-4227 4428-4853 4925-5089 5298-6051 6649-6801 HCFLN88 41 610000 AC005089 459 1-594 1779-2065 2224-2411 3295-3588 3962-4463 5317-5561 5835-6210 6750-7793 HCFLN88 41 610000 AC005089 460 1-141 HCFLN88 41 610000 AC005089 461 1-215 HCMSX51 43 788643 U96629 462 1-3014 HCMSX51 43 788643 AC040975 463 1-3014 HCNCO11 44 775086 AC011319 464 1-700 HCNCO11 44 775086 AC069204 465 1-700 HCNCO11 44 775086 AC011319 466 1-354 HCNCO11 44 775086 AC011319 467 1-338 HCNCO11 44 775086 AC069204 468 1-354 HCQBH72 46 637548 AC073530 469 1-1790 HCQBH72 46 637548 AC073530 470 1-106 HCQBH72 46 637548 AC073530 471 1-410 HCWAE64 50 535893 AL157935 472 1-1319 2024-2316 2937-2984 3126-3281 5595-5703 5788-6574 6667-6733 6788-6880 6962-7303 8111-11869 12019-12418 12420-12679 13140-13191 HCWAE64 50 535893 AL157935 473 1-1316 HCWAE64 50 535893 AL157935 474 1-309 HDPIY31 55 886159 AL356790 475 1-114 949-1189 2041-2318 2541-2711 2797-2871 3152-7720 HDP1Y31 55 886159 AL118506 476 1-241 1067-1317 1567-1737 1823-1897 2178-6746 HDP1Y31 55 886159 AL356790 477 1-104 116-297 HDPIY31 55 886159 AL356790 478 1-481 HDP1Y31 55 886159 AL118506 479 1-89 HDPPQ30 58 684292 AL022315 480 1-968 HDPPQ30 58 684292 AL022315 481 1-255 HDTFX18 60 801957 AC013303 482 1-832 1218-1410 2344-2463 3663-3955 4307-4617 5925-6011 10329-10419 11011-11162 12512-12600 13752-13894 14068-14375 14493-15033 15161-15932 HE2CM39 61 553651 AC018391 483 1-3570 3779-3904 4646-5979 6339-6701 6710-8473 HE2CM39 61 553651 AC018391 484 1-438 HE2CM39 61 553651 AC018391 485 1-1402 1586-1871 2685-2797 3088-3503 4900-5170 5789-5882 6089-6195 HE2P093 63 771655 AC020894 486 1-353 749-1198 2724-2986 4932-5578 7481-7617 8108-8257 8515-8849 9840-9968 10287-10827 11376-14474 14652-15073 15510-17083 17304-20501 HE2P093 63 771655 AC008590 487 1-648 2551-2687 3178-3327 3585-3919 4910-5038 5357-5897 6446-10147 10584-12159 12380-15574 HE2P093 63 771655 AC021468 488 1-353 749-1198 2724-2986 4934-5579 7482-7618 8109-8258 8516-8850 9841-9969 10288-10828 11377-13627 13631-13748 13762-15078 15515-17088 17309-20507 HB2P093 63 771655 AC020894 489 1-372 HE2P093 63 771655 AC020894 490 1-315 893-1242 HE2P093 63 771655 AC021468 491 1-350 HE2P093 63 771655 AC021468 492 1-372 HE6FU11 64 827236 AL021578 493 1-116 2674-2776 3489-4063 7279-7402 9706-10120 10217-10368 12042-12219 12315-12924 14271-14380 14463-14842 16153-16301 HE6FV29 65 588454 AL162401 494 1-1425 HEBFR46 69 847064 AC006483 495 1-70 282-644 789-4243 HEBFR46 69 847064 AC073481 496 1-2167 2174-3461 HEBFR46 69 847064 AC006483 497 1-344 HEBFR46 69 847064 AC006483 498 1-195 HEBGE07 70 798096 AC021918 499 1-1899 HEBGE07 70 798096 AC021918 500 1-225 HEGAU15 71 834379 AC009404 501 1-1121 HEGAU15 71 834379 AC011638 502 1-1119 HEGAU15 71 834379 AC009404 503 1-363 HEGAU15 71 834379 AC009404 504 1-446 HEGAU15 71 834379 AC011638 505 1-446 HEGAU15 71 834379 AC011638 506 1-363 HFCEI04 73 692438 AC068996 507 1-865 HFCEI04 73 692438 AC068303 508 1-865 HFCFE20 74 701985 AC044815 509 1-746 1575-2050 2508-3293 4818-5412 6081-6547 6748-6935 7843-8192 8425-8581 9095-9217 9266-9407 11036-11432 12081-12179 13701-13787 13976-16347 HFCFE20 74 701985 AC026587 510 1-2259 HFCFE20 74 701985 AL355175 511 1-2260 HFPDR62 76 839400 AC024938 512 1-2651 HFPDS07 77 821646 AC067945 513 1-3965 HFPDS07 77 821646 AC067945 514 1-814 HFPDS07 77 821646 AC067945 515 1-743 HFVHW43 78 570948 AL132795 516 1-253 1142-1455 1576-2150 2529-2966 4374-4471 4991-5361 6514-7738 7936-8053 9858-9979 11930-12101 12401-12525 12531-12712 16593-16786 17053-17214 18919-19396 21174-21327 21724-22296 22515-23071 HFVHW43 78 570948 AL132795 517 1-6181 HFVHW43 78 570948 AL132795 518 1-287 622-861 HGBHP91 81 693011 AL356056 519 1-1048 HGBHP91 81 693011 AL136982 520 1-1048 HGBHP91 81 693011 AL356056 521 1-238 HGBHP91 81 693011 AL356056 522 1-135 HGBHP91 81 693011 AL136982 523 1-238 HGBHP91 81 693011 AL136982 524 1-136 HHEAK45 82 765278 AL035690 525 1-2148 4277-4419 5252-5365 5452-6322 6863-7710 HHEAK45 82 765278 AC010388 526 1-2149 HHEAK45 82 765278 AL035690 527 1-732 HHEAK45 82 765278 AL035690 528 1-86 175-566 HHFFS40 84 824059 AC022423 529 1-2017 HHFFS40 84 824059 AC025178 530 1-2017 HHFFS40 84 824059 AC022444 531 1-2017 HHPSA85 86 658695 AL354831 532 1-291 2324-3412 HHPSA85 86 658695 AC018674 533 1-291 2324-3412 HHSBI06 87 639097 AF285442 534 1-1170 1250-1439 1565-1850 2214-2632 HHSBI06 87 639097 AF271897 535 1-1174 1253-1444 1568-1853 2217-2659 4394-4876 5269-6156 7228-8366 8574-8852 HHSBI06 87 639097 AC025857 536 1-1172 1254-1442 1566-1851 2215-2618 4423-4905 5298-6179 7253-8391 8599-8877 HHSBI06 87 639097 AF285442 537 1-483 HHSBI06 87 639097 AF285442 538 1-323 420-676 774-935 1372-1637 HHSBI06 87 639097 AF271897 539 1-522 HHSBI06 87 639097 AF271897 540 1-323 420-676 774-935 1372-1637 HHSBI06 87 639097 AC025857 541 1-522 HHSBI06 87 639097 AC025857 542 1-323 420-676 774-935 1372-1637 HHSBI65 88 801910 AF205589 543 1-1703 1798-2217 2302-3089 HHSBI65 88 801910 AF205589 544 1-531 571-1759 1862-2104 2219-2722 HHSGL28 89 801912 AC024242 545 1-2154 HHSGL28 89 801912 AC020584 546 1-215 233-1205 HHSGL28 89 801912 AC024242 547 1-216 952-1969 HHSGL28 89 801912 AC020584 548 1-635 HISAT67 90 843549 AC013403 549 1-753 852-1545 1734-1816 1930-2061 2259-2428 2573-2648 2685-2987 3135-4126 4242-4543 4732-4905 5033-5145 5298-5341 5530-5715 6059-6126 HISAT67 90 843549 AC013403 550 1-102 HKACI79 94 853361 AC006512 551 1-658 3090-3543 4479-5105 5885-6846 7103-9707 9914-10293 11523-12034 12067-12181 13769-14031 14199-14291 14584-14790 15123-15154 17039-17482 17539-17987 18697-19052 19112-19380 20023-20268 21158-21598 21817-22221 23565-23665 23906-24076 24981-25506 25510-25861 25981-26645 26661-27449 27717-27812 27991-28024 28437-28888 29651-33442 33621-34089 34245-34808 34819-35284 35854-35960 38525-38771 HKACI79 94 853361 AC011841 552 1-710 902-1864 1997-2121 2334-3824 4232-5905 HKACI79 94 853361 AC011043 553 1-712 904-1867 1874-1906 2000-2124 2337-3891 HKACI79 94 853361 AC078939 554 1-646 837-1797 1804-1836 1930-3820 4161-5834 HKACI79 94 853361 AC006512 555 1-315 439-531 707-1080 1144-1227 1491-1845 2113-2321 2700-3556 3818-4307 4336-4813 4958-5775 HKACI79 94 853361 AC006512 556 1-738 HKACI79 94 853361 AC011841 557 1-541 HKACI79 94 853361 AC011841 558 1-105 HKACI79 94 853361 AC011043 559 1-105 HKACI79 94 853361 AC078939 560 1-564 HKAC179 94 853361 AC078939 561 1-105 HKIXC44 95 716213 AC016240 562 1-195 476-552 679-763 1040-1119 1998-3764 HKIXC44 95 716213 AF261720 563 1-195 475-551 678-762 1039-1118 1998-3764 HKIXC44 95 716213 AC016240 564 1-423 HKIXC44 95 716213 AF261720 565 1-423 HKIXC44 95 716213 AF261720 566 1-206 HKTAB41 96 695732 AC006451 567 1-737 HLHAP05 98 638476 AC009097 568 1-101 HLHCS23 99 560663 AL356385 569 1-1419 HLHCS23 99 560663 AC016501 570 1-1419 HLHCS23 99 560663 AL356385 571 1-560 HLHCS23 99 560663 AC016501 572 1-560 HLMGP50 101 647603 AC019101 573 1-1039 HLMGP50 101 647603 AC01910I 574 1-100 HLMMX62 102 688051 AL356320 575 1-275 HLMMX62 102 688051 AL356320 576 1-122 HLMMX62 102 688051 AL356320 577 1-377 HLQCX36 103 584786 AC013758 578 1-316 HLQCX36 103 584786 AC024953 579 1-303 HLQCX36 103 584786 AC018740 580 1-280 942-1052 HLQCX36 103 584786 AC016539 581 1-293 HLQCX36 103 584786 AC012278 582 1-272 HLQCX36 103 584786 AC068854 583 1-183 HLQCX36 103 584786 AC019205 584 1-149 HLQCX36 103 584786 AC011175 585 1-318 HLQCX36 103 584786 AL157366 586 1-140 HLQCX36 103 584786 AC067890 587 1-223 HLQCX36 103 584786 AC032044 588 1-281 HLQCX36 103 584786 AC016615 589 1-300 HLQCX36 103 584786 AC018753 590 1-205 HLQCX36 103 584786 AC015900 591 1-167 HLQCX36 103 584786 AL162592 592 1-300 HLQCX36 103 584786 AC073568 593 1-226 HLQCX36 103 584786 AC026967 594 1-305 HLQCX36 103 584786 AC026107 595 1-275 HLQCX36 103 584786 AC011127 596 1-1365 HLQCX36 103 584786 AC034243 597 1-312 2334-2364 HLQCX36 103 584786 AL356441 598 1-289 HLQCX36 103 584786 AC027531 599 1-298 HLQCX36 103 584786 AC022950 600 1-311 HLQCX36 103 584786 AC010958 601 1-131 HLQCX36 103 584786 AC010073 602 1-175 HLQCX36 103 584786 AP001397 603 1-1365 HLQCX36 103 584786 AC055119 604 1-320 HLQCX36 103 584786 AC027105 605 1-308 HLQCX36 103 584786 AC022795 606 1-300 HLQCX36 103 584786 AC018445 607 1-140 HLQCX36 103 584786 AL358115 608 1-142 HLQCX36 103 584786 AL355975 609 1-322 HLQCX36 103 584786 AL096841 610 1-281 HLQCX36 103 584786 AC027414 611 1-270 HLQCX36 103 584786 AC023008 612 1-296 HLQCX36 103 584786 AL357125 613 1-278 HLQCX36 103 584786 AC073446 614 1-299 HLQCX36 103 584786 AC015989 615 1-1365 HLQCX36 103 584786 AC020873 616 1-306 HLQCX36 103 584786 AC068854 617 1-1087 HLQCX36 103 584786 AC018753 618 1-523 HLQCX36 103 584786 AC011127 619 1-686 HLQCX36 103 584786 AP001397 620 1-98 HLQCX36 103 584786 AL358115 621 1-2327 HLQCX36 103 584786 AC015989 622 1-685 HLQCX36 103 584786 AC015989 623 1-98 HLQCX36 103 584786 AC020873 624 1-126 HLYDF73 106 566869 AL122127 625 1-583 HMDAB29 109 584789 AC027264 626 1-147 HMDAB29 109 584789 AC068682 627 1-153 HMDAB29 109 584789 AL354887 628 1-1433 HMDAB29 109 584789 AL157408 629 1-1434 HMDAB29 109 584789 AL354887 630 1-577 HMDAB29 109 584789 AL354887 631 1-196 HMDAB29 109 584789 AL157408 632 1-577 HMDAB29 109 584789 AL157408 633 1-196 HMDAD44 110 566854 AC012370 634 1-145 2813-4454 HMDAD44 110 566854 AC034121 635 1-1569 HMDAD44 110 566854 AC012370 636 1-787 HMDAD44 110 566854 AC012370 637 1-622 HMIBF07 112 603528 AC022833 638 1-1721 HMICP65 113 847403 AL162741 639 1-45 HMICP65 113 847403 AL162741 640 1-102 HMWBL03 117 822861 AC012052 641 1-130 548-784 4520-4887 5112-6285 6741-6888 7577-7727 7951-8582 8927-10292 HMWBL03 117 822861 AC011667 642 1-138 1281-1681 2270-2632 3070-3372 3865-3990 4407-4644 8378-8745 8970-10143 10599-10746 11435-11585 11809-12440 12785-14150 HMWBL03 117 822861 AC012052 643 1-303 HNFGRO8 118 825417 AC006369 644 1-1423 HNGAK51 119 603910 AC013443 645 1-913 HNGAK51 119 603910 AC013443 646 1-406 HNGAK51 119 603910 AC013443 647 1-297 HNGDX18 120 1145071 AL391069 648 1-1403 HNGDX18 120 1145071 AL158846 649 1-193 208-577 894-1167 1401-1629 1918-3320 4039-4082 9400-10337 HNGDX18 120 1145071 AL391069 650 1-274 HNGDX18 120 1145071 AL158846 651 1-117 HNGPJ2S 124 834942 AP002781 652 1-1472 HNHKV56 127 800877 AC009396 653 1-1605 HODBB70 129 520196 AC006322 654 1-561 HODBB70 129 520196 AC073110 655 1-561 HODBB70 129 520196 AC025553 656 1-561 HODBB70 129 520196 AC006322 657 1-1741 HODBB70 129 520196 AC006322 658 1-354 HODBB70 129 520196 AC073110 659 1-1741 HODBB70 129 520196 AC073110 660 1-354 HORBS82 133 638293 AL034419 661 1-1798 HORBS82 133 638293 AL034419 662 1-1186 HPFCI36 137 855966 AL161652 663 1-174 313-4710 HPFD137 138 862056 AC000090 664 1-29 566-712 1355-1425 3075-3241 3725-3806 4295-4357 5382-5571 6510-7016 7981-8321 HPJCW58 140 612866 AC024735 665 1-1160 HRADF49 146 866481 AC068946 666 1-142 359-1108 1191-1345 1445-2140 2314-2935 3040-3156 3395-4126 4311-4460 4749-5820 HRADF49 146 866481 AC060820 667 1-142 359-1109 1193-1348 1448-2142 2318-2944 3056-3166 3405-4136 4321-4472 4762-5836 HRADF49 146 866481 AC068946 668 1-812 1124-1263 1281-2283 2470-2572 2752-2935 3851-3974 4153-4548 4602-4810 4980-5111 5262-5346 5434-5498 5609-5695 5871-5930 6448-6487 HRADF49 146 866481 AC060820 669 1-686 HRDDQ39 147 840405 AC009152 670 1-755 HROEA08 149 866190 AC010894 671 1-3018 HROEA08 149 866190 AC010894 672 1-138 HROEA08 149 866190 AC010894 673 1-299 HSAVW42 151 637660 AC021117 674 1-865 HSAVW42 151 637660 AC021117 675 1-336 397-651 HSAVW42 151 637660 AC021117 676 1-185 HSLHX15 153 777861 AC072032 677 1-364 HSLHX15 153 777861 AC002518 678 1-247 HSLHX15 153 777861 AC022305 679 1-686 HSLHX15 153 777861 AC078916 680 1-364 HSLHX15 153 777861 AC072032 681 1-288 HSLHX15 153 777861 AC078916 682 1-288 HSSEF77 155 658725 AC005041 683 1-68 87-493 711-838 997-1167 2227-2960 3326-4641 4768-5786 HSSEF77 155 658725 AC005041 684 1-2920 3439-3667 3839-4332 HSSEF77 155 658725 AC005041 685 1-143 HT4FV41 156 853400 AC011547 686 1-170 793-936 2771-3041 3691-3788 5141-5252 5755-6030 6325-6407 7214-7551 8653-8940 9033-9136 9428-9907 11266-11659 12082-12263 13451-13544 13664-13699 13769-13936 14571-14761 14897-14997 15135-17127 HT4FV4I 156 853400 AC005331 687 1-88 224-324 462-2454 HT4FV41 156 853400 AC023470 688 1-80 204-242 313-477 1213-1300 1436-1536 1673-3658 HT4FV41 156 853400 AC005331 689 1-607 HT4FV41 156 853400 AC023470 690 1-606 HT5FX79 157 794169 AC020978 691 1-4351 4423-4590 4875-5061 5211-5413 5519-5726 5755-6138 6281-6319 6402-7114 7359-7460 7715-7918 8030-8144 8612-9037 9280-9760 HT5FX79 157 794169 AC020978 692 1-431 HT5FX79 157 794169 AC020978 693 1-38 115-354 HTEDJ28 161 762845 AC025974 694 1-2357 HTEDJ28 161 762845 AC013370 695 1-2357 HTEDS12 162 838621 AC021491 696 1-124 290-781 1447-1562 1650-1767 2309-2417 3273-3466 3935-4120 5213-5358 6216-6605 7621-7744 8491-8761 9044-9175 9353-9523 9966-10843 11395-11839 HTEDS12 162 838621 AC021491 697 1-228 3662-4225 HTEHA56 163 806461 AC008751 698 1-469 1023-1372 1542-1694 1724-3063 3371-3477 3651-3905 4073-4931 4999-6547 HTEHA56 163 806461A C009763 699 1-1487 HTEHA56 163 806461A C008749 700 1-1487 HTEHA56 163 806461A C008751 701 1-575 HTEHA56 163 806461A C009763 702 1-577 HTEHA56 163 806461A C009763 703 1-859 HTEHA56 163 806461A C008749 704 1-575 HTEHA56 163 806461A C008749 705 1-582 HTEJD29 164 695798A L354733 706 1-1292 HTEJD29 164 695798A C007943 707 1-1292 HTEJD29 164 695798A L354733 708 1-184 HTEJD29 164 695798A L354733 709 1-59 1212-1284 1905-1956 2351-2840 4126-5105 5892-6298 6726-7122 7204-7713 7747-7932 HTHBZ06 167 832477 AC068768 710 1-835 HTLBT80 168 840045 AL133227 711 1-51 476-521 842-1226 1375-1490 3745-4016 4046-4229 4430-4855 5300-6053 6598-6883 7406-7446 7461-8437 8550-8681 8888-8919 8943-9353 9458-9544 9834-10607 11550-11629 12196-12374 13532-14886 HTLBT80 168 840045 AL133227 712 1-32 712-1071 3453-3870 4197-4326 4639-4751 5131-5202 5588-5638 7454-8108 8670-8767 9511-9692 9754-10134 11109-11226 12456-12607 15237-15316 18143-18311 18429-18478 20682-20982 20988-21295 22686-23061 23358-23495 24076-24612 25196-25334 26760-26926 27041-27152 27271-27379 27697-28289 29024-29340 29761-29840 31168-32681 HTLDU78 169 637702 AC011444 713 1-1305 HTLDU78 169 637702 AC011444 714 1-285 HTLDU78 169 637702 AC011444 715 1-274 HTLFA13 170 535937 AC022007 716 1-1127 HTLFA13 170 535937 AC021995 717 1-1115 HTLFA13 170 535937 AC007783 718 1-1144 HTLFA13 170 535937 AC022007 719 1-1729 HTLFA13 170 535937 AC022007 720 1-179 184-696 HTLFA13 170 535937 AC021995 721 1-106 132-190 674-831 1456-1588 4811-4933 5118-5304 HTLFA13 170 535937 AC021995 722 1-179 184-696 894-945 HTLFA13 170 535937 AC007783 723 1-169 180-258 681-859 864-1376 3240-3503 HTLFA13 170 535937 AC007783 724 1-1729 HTLGI89 171 835069 AC048342 725 1-130 HTLGI89 171 835069 AC009453 726 1-143 HTLGI89 171 835069 AC022231 727 1-151 HTLGI89 171 835069 AC009524 728 1-151 HTLGI89 171 835069 AC048342 729 1-118 HTOAM11 173 664508 AC002369 730 1-586 2559-2651 3329-3426 3756-5088 HTOAM11 173 664508 AP001486 731 1-1191 HTOAM11 173 664508 AP000875 732 1-1192 HTOAM11 173 664508 AC002369 733 1-228 HTOAM11 173 664508 AP001486 734 1-711 HTOAM11 173 664508 AP001486 735 1-374 HTOAM11 173 664508 AP000875 736 1-710 HTOHO21 174 732808 AC022221 737 1-85 394-740 781-1562 1622-2429 3831-4082 4239-6053 7230-7365 8195-8379 11677-11990 12508-12710 HTOHO21 174 732808 AC007897 738 1-1586 2763-2898 3728-3912 7210-7523 8041-8243 HTOHO21 174 732808 AC022221 739 1-184 HTOHO21 174 732808 AC007897 740 1-184 HTSFJ32 176 637720 AC015734 741 1-80 562-915 925-4400 HTSFJ32 176 637720 AC015734 742 1-463 HTSFJ32 176 637720 AC015734 743 1-359 HTTEE41 178 840950 AC018921 744 1-92 318-578 837-912 1091-1249 1321-1387 1862-2192 2485-2579 2708-2831 3685-4257 4547-5127 5811-6037 6562-7076 7541-7678 8069-8191 10100-10207 11102-11688 11721-11847 12201-12335 12532-12641 12888-12991 13027-13546 13637-16146 HTTEE41 178 840950 AC018921 745 1-100 HTWEH94 179 561680 AC004858 746 1-1349 1370-1744 HTWEH94 179 561680 AC004858 747 1-94 HTWEH94 179 561680 AC004858 748 1-199 HTXFA72 180 853410 AP001812 749 1-1015 HTXFA72 180 853410 AP000822 750 1-1015 HTXFA72 180 853410 AP001812 751 1-130 HTXFA72 180 853410 AP000822 752 1-527 HTXKF95 181 834438 AC004242 753 1-981 HTXKF95 181 834438 AC008083 754 1-981 HTXKF95 181 834438 AC004242 755 1-984 HTXKF95 181 834438 AC004242 756 1-118 HTXKF95 181 834438 AC008083 757 1-984 HTXKF95 181 834438 AC008083 758 1-173 HUSCJ14 183 894699 AC007040 759 1-149 394-889 1061-1139 2097-2249 2852-3007 5021-5089 5217-5919 6119-8896 HUSCJ14 183 894699 AC007040 760 1-854 HUSCJ14 183 894699 AC007040 761 1-397 HELGG84 186 851137 AC025019 762 1-2121 1-2121 HELGG84 186 851137 AC012202 763 1-2122 1-2122 HELGG84 186 851137 AC025019 764 1-573 1-573 HELGG84 186 851137 AC025019 765 1-40 1158-2410 1-40 1158-2410 HELGG84 186 851137 AC012202 766 1-573 1-573 HYABC84 188 865064 AL132825 767 1-2512 2604-2740 2974-3241 1-2512 2604-2740 2974-3241 HYABC84 188 865064 AL132825 768 1-553 1-553 1059-1263 1059-1263 3121-3476 3121-3476 5284-5734 5284-5734 6284-6513 6284-6513 6786-7426 6786-7426 8674-8733 8674-8733 10656-10933 10656-10933 11453-11555 11453-11555 12991-13079 12991-13079 13839-14281 13839-14281 14527-14827 14527-14827 15156-15685 15156-15685 15835-16046 15835-16046 16166-16604 16166-16604 16736-19566 16736-19566 19658-19794 19658-19794 20028-20295 20028-20295 HYABC84 188 865064 AL132825 769 1-188 1-188 HE8FD92 190 901142 AL359176 770 1-2410 1-2410 1-2410 2420-4226 2420-4226 2420-4226 HE8FD92 190 901142 AL139152 771 1-826 863-2063 2125-4935 4945-6753 1-826 863-2063 2125-4935 4945-6753 1-826 863-2063 2125-4935 4945-6753 1-826 863-2063 2125-4935 4945-6753 HE8FD92 190 901142 AL109937 772 1-168 233-930 1572-1748 2463-3391 HE8FD92 190 901142 AC027209 773 1-1201 1-1201 1-1201 1263-1531 1263-1531 1263-1531 1648-5860 1648-5860 1648-5860 HE8FD92 190 901142 AL356004 774 1-1052 1062-2871 HE8FD92 190 901142 AL139152 775 1-560 760-1714 1740-3644 3736-4319 4872-4998 1-560 760-1714 1740-3644 3736-4319 4872-4998 1-560 760-1714 1740-3644 3736-4319 4872-4998 1-560 760-1714 1740-3644 3736-4319 4872-4998 HE8FD92 190 901142 AL109937 776 1-437 HE8FD92 190 901142 AC027209 777 1-560 1-560 1-560 747-1721 747-1721 747-1721 1875-3650 1875-3650 1875-3650 3698-4325 3698-4325 3698-4325 4657-4693 4657-4693 4657-4693 4879-5008 4879-5008 4879-5008 HE8FD92 190 901142 AC027209 778 1-423 1-423 1-423 HE8FD92 190 901142 AL356004 779 1-560

Table 1D: The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating diabetes mellitus comprising administering to a patient in which such detection, treatment, prevention, and/or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diabetes mellitus.
In another embodiment, the present invention also encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus;
comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.
Table 1D provides information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1D also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Table 1A through Table 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, Table 1B, and Table 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity.
Table 1D describes the use of, inter alia, FMAT technology for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound flurophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for high throughput screening using flourometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to up regulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
Table 1D also describes the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).
TABLE 1D AA SEQ Gene cDNA ID Biological No. Clone ID NO:Y Activity Exemplary Activity Assay 1 H6EDM64 205 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 2 H6EEU40 206 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art viability and and may be used or routinely modified to assess the ability of polypeptides of the invention proliferation of (including antibodies and agonists or antagonists of the invention) to regulate viability and pancreatic beta proliferation of pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability cells. assay measures the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Ex Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 3 HACAB68 207 Activation of Assays for the activation of transcription through the cAMP response element are well-known in transcription the art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP and regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell in immune cells functions. Exemplary assays for transcription through the cAMP response element that may be used (such as T-cells). or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 3 HACAB68 207 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 3 HACAB68 207 Activation of Kinase assay. JNK and p38 kinase assays for signal transduction that regulate cell proliferation, Endothelial Cell activation, or apoptosis are well known in the art and may be used or routinely modified to assess p38 or JNK the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the Signaling invention) to promote or inhibit cell proliferation, activation, and apoptosis. Exemplary assays for Pathway. JNK and p38 kinase activity that may be used or routinely modified to test JNK and p38 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Endothelial cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are endothelial cells which line venous blood vessels, and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. 3 HACAB68 207 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 4 HACBJ56 208 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art viability and and may be used or routinely modified to assess the ability of polypeptides of the invention proliferation of (including antibodies and agonists or antagonists of the invention) to regulate viability and pancreatic beta proliferation of pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability cells. viability assay measures the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR. et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 5 HADMB15 209 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 5 HADMB15 209 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis of routinely modified to assess the ability of polypeptides of the invention (including antibodies and immune cells agonists or antagonists of the invention) to regulate caspase protease-mediated apoptosis in immune (such as mast cells (such as, for example, in mast cells). Mast cells are found in connective and mucosal tissues cells). throughout the body, and their activation via immunoglobulin E-antigen, promoted by T helper cell type 2 cytokines, is an important component of allergic disease. Dysregulation of mast cell apoptosis may play a role in allergic disease and mast cell tumor survival. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity induced by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Masuda A, et al., J Biol Chem, 276(28): 26107-26113 (2001); Yeatman CF 2nd, et al., J Exp Med. 192(8): 1093-1103 (2000); Lee et al., FEBS Lett 485(2-3): 122- 126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary immune cells that may be used according to these assays include mast cells such as the HMC human mast cell line. 5 HADMB15 209 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Natural Killer regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signaling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary natural killer cells that may be used according to these assays include the human natural killer cell lines (for example, NK-YT cells which have cytolytic and cytotoxic activity) or primary NK cells. 6 HAGDW20 210 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 7 HAGEQ79 211 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 7 HAGEQ79 211 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be reactivated to enhance responsiveness to immunomodulatory factors. 8 HAJBV67 212 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 9 HAJCH70 213 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 10 HAOAG15 214 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 10 HAOAG15 214 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Natural Killer regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signaling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary natural killer cells that may be used according to these assays include the human natural killer cell lines (for example, NK-YT cells which have cytolytic and cytotoxic activity) or primary NK cells. 11 HATCD80 215 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 12 HATCI03 216 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 12 HATCI03 216 Upregulation of CD69 FMAT. CD69 is an activation marker that is expressed on activated T cells, B cells, and NK CD69 and cells. CD69 is not expressed on resting T cells, B cells, or NK cells. CD69 has been found to be activation of associated with inflammation. Assays for immunomodulatory proteins expressed in T cells, B cells, T cells and leukocytes are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD69, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Ferenczi et al., J Autoimmun 14(1): 63-78 (200); Werfel et al., Allergy 52(4): 465-469 (1997); Taylor-Fishwick and Siegel, Eur J Immunol 25(12): 3215-3221 (1995); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 13 HBAGD86 217 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 13 HBAGD86 217 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may be used to identify factors that activate adipocytes. the cAMP signaling pathway. CREB plays a major role in adipogenesis, and is involved in differentiation into adipocytes. CRE contains the binding sequence for the transcription factor CREB (CRE binding protein). Exemplary assays for transcription through the cAMP response element that may be used or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of each of which are herein incorporated by reference in its entirety. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 13 HBAGD86 217 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in pre-adipocytes. transcription through the SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. Pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 13 HBAGD86 217 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorpor- ated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used acc- ording to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 13 HBAGD86 217 Activation of This reporter assay measures activation of the NFAT signaling pathway in HMC-1 human mast cell transcription line. Activation of NFAT in mast cells has been linked to cytokine and chemokine production. through NFAT Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) response element response element are well-known in the art and may be used or routinely modified to assess the in immune cells ability of polypeptides of the invention (including antibodies and agonists or antagonists of the (such as mast invention) to regulate NFAT transcription factors and modulate expression of genes involved in cells). immunomodulatory functions. Exemplary assays for transcription through the NFAT response element that may be used or routinely modified to test NFAT-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali et al., J Immunol 165(12): 7215- 7223 (2000); Hutchinson and McCloskey, J Biol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188: 527—537 (1998), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 13 HBAGD86 217 Activation of This reporter assay measures activation of the NFkB signaling pathway in HMC-1 human mast cell transcription line. Activation of NFkB in mast cells has been linked to production of certain cytokines, such as through NFKB IL-6 and IL-9. Assays for the activation of transcription through the NFKB response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate NFKB (such as mast transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for cells). transcription through the NFKB response element that may be used or rountinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Stassen et al, J Immunol 166(7): 4391-8 (2001); and Marquardt and Walker, J Allergy Clin Immunol 105(3): 500-5 (2000), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 13 HBAGD86 217 Activation of This reporter assay measures activation of the NFkB signaling pathway in Ku812 human basophil transcription cell line. Assays for the activation of transcription through the NFKB response element are well- through NFKB known in the art and may be used or routinely modified to assess the ability of polypeptides of the response element invention (including antibodies and agonists or antagonists of the invention) to regulate NFKB in immune cells transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for (such as transcription through the NFKB response element that may be used or rountinely modified to test basophils). NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), the contents of each of which are herein incorporated by reference in its entirety. Basophils that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human basophil cell lines that may be used according to these assays include Ku812, originally established from a patient with chronic myelogenous leukemia. It is an immature prebasophilic cell line that can be induced to differentiate into mature basophils. 13 HBAGD86 217 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to bind the serum response element response factor and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as T-cells). SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells, such as the MOLT4, that may be used according to these assays are publicly available (e.g., through the ATCC). 13 HBAGD86 217 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression of in immune cells multiple genes. Exemplary assays for transcription through the STAT6 response element that may (such as natural be used or routinely modified to test STAT6 response element activity of the polypeptides of the killer cells). invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). 13 HBAGD86 217 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary human T cells, such as the SUPT cell line, that may be used according to these assays are publicly available (e.g., through the ATCC). 13 HBAGD86 217 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as natural element that may be used or routinely modified to test NFAT-response element activity of killer cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. NK cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human NK cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 14 HBHAA05 218 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 15 HBHAA81 219 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 4414 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 15 HBHAA81 219 Production of IFNgamma FMAT. IFNg plays a central role in the immune system and is considered to be a IFNgamma using proinflammatory cytokine. IFNg promotes TH1 and inhibits TH2 differentiation; promotes IgG2a a T cells and inhibits IgE secretion; induces macrophage activation; and increases MHC expression. Assays for immunomodulatory proteins produced by T cells and NK cells that regulate a variety of inflammatory activities and inhibit TH2 helper cell functions are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, modulate TH2 helper cell function, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as Interferon gamma (IFNg), and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 16 HBIAA59 220 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 17 HBJAC40 221 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 18 HBJCR46 222 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 19 HBJDW56 223 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 20 HBJEL16 224 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 20 HBJEL16 224 Production of Assays for measuring expression of VCAM are well-known in the art and may be used or routinely VCAM in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or endothelial cells antagonists of the invention) to regulate VCAM expression. For example, FMAT may be used to (such as human meaure the upregulation of cell surface VCAM-1 expresssion in endothelial cells. Endothelial cells umbilical vein are cells that line blood vessels, and are involved in functions that include, but are not limited to, endothelial cells angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Exemplary (HUVEC)) endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are available from commercial sources. The expression of VCAM (CD106), a membrane-associated protein, can be upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 20 HBJEL16 224 Upregulation of CD69 FMAT. CD69 is an activation marker that is expressed on activated T cells, B cells, and NK CD69 and cells. CD69 is not expressed on resting T cells, B cells, or NK cells. CD69 has been found to be activation of associated with inflammation. Assays for immunomodulatory proteins expressed in T cells, B cells, T cells and leukocytes are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD69, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Ferenczi et al., J Autoimmun 14(1): 63-78 (200); Werfel et al., Allergy 52(4): 465-469 (1997); Taylor-Fishwick and Siegel, Eur J Immunol 25(12): 3215-3221 (1995); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 21 HBJIG20 225 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 22 HBJKD16 226 Production of MIP-1alpha FMAT. Assays for immunomodulatory proteins produced by activated dendritic cells MIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate chemotaxis, and modulate T cell differentiation. Exemplary assays that test for immunomodulatory proteins evaluate the production of chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a), and the activation of monocytes/macrophages and T cells. Such assays that may be used or routinely modified to test immunomodulatory and chemotaxis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 22 HBJKD16 226 Production of IL-6 FMAT. IL-6 is produced by T cells and has strong effects on B cells. IL-6 participates in IL-4 IL-6 induced IgE production and increases IgA production (IgA plays a role in mucosal immunity). IL-6 induces cytotoxic T cells. Deregulated expression of IL-6 has been linked to autoimmune disease, plasmacytomas, myelomas, and chronic hyperproliferative diseases. Assays for immunomodulatory and differentiation factor proteins produced by a large variety of cells where the expression level is strongly regulated by cytokines, growth factors, and hormones are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation and differentiation and modulate T cell proliferation and function. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-6, and the stimulation and upregulation of T cell proliferation and functional activities. Such assays that may be used or routinely modified to test immunomodulatory and dififerentiation activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Verhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 22 HBJKD16 226 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 23 HBMTY48 227 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 24 HBMUH74 228 Activation of Kinase assay. JNK kinase assays for signal transduction that regulate cell proliferation, activation, JNK Signaling or apoptosis are well known in the art and may be used or routinely modified to assess the ability of Pathway in polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to immune cells promote or inhibit cell proliferation, activation, and apoptosis. Exemplary assays for JNK kinase (such as activity that may be used or routinely modified to test JNK kinase-induced activity of polypeptides eosinophils). of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate signal transduction, cell proliferation, activation, or apoptosis in eosinophils include assays disclosed and/or cited in: Zhang JP, et al., “Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen- activated protein kinase in human eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7 (2000); Hebestreit H, et al., “Disruption of fas receptor signaling by nitric oxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); J Allergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, et al., “In vivo resistance to corticosteroids in bronchial asthma is associated with enhanced phosyphorylation of JUN N-terminal kinase and failure of prednisolone to inhibit JUN N-terminal kinase phosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74 (1999); the contents of each of which are herein incorporated by reference in its entirety. 24 HBMUH74 228 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, a adipocytes key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in adipoocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 25 HBQAB79 229 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 26 HBXCX15 230 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 26 HBXCX15 230 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may be used to identify factors that activate adipocytes. the cAMP signaling pathway. CREB plays a major role in adipogenesis, and is involved in differentiation into adipocytes. CRE contains the binding sequence for the transcription factor CREB (CRE binding protein). Exemplary assays for transcription through the cAMP response element that may be used or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of each of which are herein incorporated by reference in its entirety. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in pre-adipocytes. transcription through the SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. Pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 26 HBXCX15 230 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 26 HBXCX15 230 Activation of This reporter assay measures activation of the NFAT signaling pathway in HMC-1 human mast cell transcription line. Activation of NFAT in mast cells has been linked to cytokine and chemokine production. through NFAT Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) response element response element are well-known in the art and may be used or routinely modified to assess the in immune cells ability of polypeptides of the invention (including antibodies and agonists or antagonists of the (such as mast invention) to regulate NFAT transcription factors and modulate expression of genes involved in cells). immunomodulatory functions. Exemplary assays for transcription through the NFAT response element that may be used or routinely modified to test NFAT-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali et al., J Immunol 165(12): 7215- 7223 (2000); Hutchinson and McCloskey, J Biol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188: 527-537 (1998), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to bind the serum response element response factor and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as T-cells). SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells, such as the MOLT4, that may be used according to these assays are publicly available (e.g., through the ATCC). 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression of in immune cells multiple genes. Exemplary assays for transcription through the STAT6 response element that may (such as natural be used or routinely modified to test STAT6 response element activity of the polypeptides of the killer cells). invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary human T cells, such as the SUPT cell line, that may be used according to these assays are publicly available (e.g., through the ATCC). 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as natural element that may be used or routinely modified to test NFAT-response element activity of killer cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. NK cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human NK cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 26 HBXCX15 230 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate serum response element response factors and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as natural SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention killer cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 27 HCDCY76 231 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or Signaling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 27 HCDCY76 231 Endothelial Cell Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or Apoptosis routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. Induction of apoptosis in endothelial cells supporting the vasculature of tumors is associated with tumor regression due to loss of tumor blood supply. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Endothelial cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary endothelial cells that may be used according to these assays include bovine aortic endothelial cells (bAEC), which are an example of endothelial cells which line blood vessels and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. 28 HCEDR26 232 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 29 HCEEU18 233 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 29 HCEEU18 233 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 30 HCEGX05 234 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 31 HCFLN88 235 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 31 HCFLN88 235 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell in immune cells functions. Exemplary assays for transcription through the cAMP response element that may be used (such as T-cells). or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the HT2 cell line, which is a suspension culture of IL-2 dependent T cells that also respond to IL-4. 31 HCFLN88 235 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. Mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the HT2 cell line, which is an IL-2 dependent suspension culture of T cells that also respond to IL-4. 32 HCHAB84 236 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 33 HCMSX51 237 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 34 HCNCO11 238 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 35 HCNSD29 239 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 36 HCQBH72 240 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that maybe used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 36 HCQBH72 240 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 37 HCQCC96 241 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 37 HCQCC96 241 Regulation of Assays for the regulation of transcription through the PEPCK promoter are well-known in the art transcription and may be used or routinely modified to assess the ability of polypeptides of the invention through the (including antibodies and agonists or antagonists of the invention) to activate the PEPCK promoter PEPCK promoter in a reporter construct and regulate liver gluconeogenesis. Exemplary assays for regulation of in hepatocytes transcription through the PEPCK promoter that may be used or routinely modified to test for PEPCK promoter activity (in hepatocytes) of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., J Biol Chem 275(23): 17814-17820 (2000), the contents of each of which is herein incorporated by reference in its entirety. Hepatocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary liver hepatoma cells that may be used according to these assays include H4lle cells, which contain a tyrosine amino transferase that is inducible with glucocorticoids, insulin, or cAMP derivatives. 37 HCQCC96 241 Activation of Kinase assay. Kinase assays, for examplek Elk-1 kinase assays, for ERK signal transduction that Skeletal Muscle regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signalling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 38 HCQCJ56 242 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 39 HCUDD64 243 Production of GM-CSF FMAT. GM-CSF is expressed by activated T cells, macrophages, endothelial cells, and GM-CSF fibroblasts. GM-CSF regulates differentiation and proliferation of granulocytes- macrophage progenitors and enhances antimicrobial activity in neutrophils, monocytes and macrophage. Additionally, GM-CSF plays an important role in the differentiation of dendritic cells and monocytes, and increases antigen presentation. GM-CSF is considered to be a proinflammatory cytokine. Assays for immunomodulatory proteins that promote the production of GM-CSF are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation and modulate the growth and differentiation of leukocytes. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as GM-CSF, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Ye et al., J Leukoc Biol (58(2): 225-233, the contents of each of which are herein incorporated by reference in its entirety. Natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC) or may be isolated using techniques disclosed herein or otherwise known in the art. Natural killer (NK) cells are large granular lymphocytes that have cytotoxic activity but do bind antigen. NK cells show antibody-independent killing of tumor cells and also recognize antibody bound on target cells, via NK Fc receptors, leading to cell-mediated cytotoxicity. 39 HCUDD64 243 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 40 HCWAE64 244 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription the art and may be used or routinely modified to assess the ability of polypeptides of the invention via DMEF1 (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response response element element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin in adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 40 HCWAE64 244 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may be used to identify factors that activate adipocytes. the cAMP signaling pathway. CREB plays a major role in adipogenesis, and is involved in differentiation into adipocytes. CRE contains the binding sequence for the transcription factor CREB (CRE binding protein). Exemplary assays for transcription through the cAMP response element that may be used or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of each of which are herein incorporated by reference in its entirety. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in pre-adipocytes. transcription through the SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. Pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 40 HCWAE64 244 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorpor- ated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used acc- ording to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 40 HCWAE64 244 Activation of This reporter assay measures activation of the NFAT signaling pathway in HMC-1 human mast cell transcription line. Activation of NFAT in mast cells has been linked to cytokine and chemokine production. through NFAT Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) response element response element are well-known in the art and may be used or routinely modified to assess the in immune cells ability of polypeptides of the invention (including antibodies and agonists or antagonists of the (such as mast invention) to regulate NFAT transcription factors and modulate expression of genes involved in cells). immunomodulatory functions. Exemplary assays for transcription through the NFAT response element that may be used or routinely modified to test NFAT-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali et al., J Immunol 165(12): 7215-7223 (2000); Hutchinson and McCloskey, J Biol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188: 527-537 (1998), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to bind the serum response element response factor and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as T-cells). SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells, such as the MOLT4, that may be used according to these assays are publicly available (e.g., through the ATCC). 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression of in immune cells multiple genes. Exemplary assays for transcription through the STAT6 response element that may (such as natural be used or routinely modified to test STAT6 response element activity of the polypeptides of the killer cells). invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary human T cells, such as the SUPT cell line, that may be used according to these assays are publicly available (e.g., through the ATCC). 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as natural element that may be used or routinely modified to test NFAT-response element activity of killer cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. NK cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human NK cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 40 HCWAE64 244 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate serum response element response factors and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as natural SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention killer cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 41 HDPDJ58 245 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 41 HDPDJ58 245 Upregulation of CD71 FMAT. CD71 is the transferrin receptor. Transferrin is a major iron carrying protein that is CD71 and essential for cell proliferation. CD71 is expressed predominantly on cells that are actively activation of proliferating. Assays for immunomodulatory proteins expressed on activated T cells, B cells, and T cells most proliferating cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD71, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 42 HDPFU43 246 Production of IL-6 FMAT. IL-6 is produced by T cells and has strong effects on B cells. IL-6 participates in IL-4 IL-6 induced IgE production and increases IgA production (IgA plays a role in mucosal immunity). IL-6 induces cytotoxic T cells. Deregulated expression of IL-6 has been linked to autoimmune disease, plasmacytomas, myelomas, and chronic hyperproliferative diseases. Assays for immunomodulatory and differentiation factor proteins produced by a large variety of cells where the expression level is strongly regulated by cytokines, growth factors, and hormones are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation and differentiation and modulate T cell proliferation and function. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-6, and the stimulation and upregulation of T cell proliferation and functional activities. Such assays that may be used or routinely modified to test immunomodulatory and differentiation activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Verhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 42 HDPFU43 246 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to modulate gene expression (commonly via STAT transcription factors) involved in a in immune cells wide variety of cell functions. Exemplary assays for transcription through the GAS response (such as element that may be used or routinely modified to test GAS-response element activity of eosinophils). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995); the contents of each of which are herein incorporated by reference in its entirety. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to activate or inhibit activation of immune cells include assays disclosed and/or cited in: Mayumi M., “EoL-1, a human eosinophilic cell line” Leuk Lymphoma; Jun; 7(3): 243-50 (1992); Bhattacharya S, “Granulocyte macrophage colony-stimulating factor and interleukin-5 activate STAT5 and induce CIS1 mRNA in human peripheral blood eosinophils” Am J Respir Cell Mol Biol; Mar; 24(3): 312-6 (2001); and, Du J, et al., “Engagement of the CrkL adapter in interleukin-5 signaling in eosinophils” J Biol Chem; Oct 20; 275(42): 33167-75 (2000); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are a type of immune cell important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Increases in GAS mediated transcription in eosinophils is typically a result of STAT activation, normally a direct consequence of interleukin or other cytokine receptor stimulation (e.g. IL3, IL5 or GMCSF). 42 HDPFU43 246 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal transduction Skeletal Mucle that regulate glucose metabolism and cell survivial are well-known in the art and may be used or Cell PI3 Kinase routinely modified to assess the ability of polypeptides of the invention (including antibodies and Signalling agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Pathway Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 43 HDPGE24 247 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A.M., et al., Mol Endocrinol, 13(8):1305-17 (1999); Filipsson, K., et al., Ann NY Acad Sci, 865:441-4 (1998); Olson, L.K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4:193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with 5V40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by soniatostatin or glucocorticoids. ATTC#CRL-1777 Refs: Lord and Ashcroft. Biochem. 1. 219: 547-551; Santerre et al. Proc. NatI. Acad. Sci. USA 78: 4339-4343, 1981. 44 HDPIU94 248 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, a hepatocytes key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the mouse 3T3-L1 cell line. 3T3- L1 is a mouse preadipocyte cell line (adherent). It is a continuous substrain of 3T3 fibroblasts developed through clonal isolation. Cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 44 HDPIU94 248 Activation of Assays for the activation of transcription through the NFKB response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through NFKB (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription response element factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription in immune cells through the NFKB response element that may be used or rountinely modified to test NFKB- (such as EOL1 response element activity of polypeptides of the invention (including antibodies and agonists or cells). antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. For example, a reporter assay (which measures increases in transcription inducible from a NFkB responsive element in EOL-1 cells) may link the NFKB element to a repeorter gene and binds to the NFKB transcription factor, which is upregulated by cytokines and other factors. Exemplary immune cells that may be used according to these assays include eosinophils such as the human EOL-1 cell line of eosinophils. Eosinophils are a type of immune cell important in the allergic responses; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Eol-1 is a human eosinophil cell line. 44 HDPIU94 248 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Hepatocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat liver hepatoma cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat liver hepatoma cells that may be used according to these assays include H4lle cells, which are known to respond to glucocorticoids, insulin, or cAMP derivatives. 44 HDPIU94 248 Regulation of Kinase assays, for example an Elk-1 kinase assay for ERK signal transduction that regulates cell proliferation and/ proliferation or differentiation, are well known in the art and may be used or routinely modified to or differentiation assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists in immune cells of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary (such as mast assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced cells). activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Ali H, et al., J Immunol, 165(12): 7215-7223 (2000); Tam SY, et al., Blood, 90(5): 1807-1820 (1997); Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Berra et al., Biochem Pharmacol 60(8): 1171-1178 (2000); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary immune cells that may be used according to these assays include human mast cells such as the HMC-1 cell line. 44 HDPIU94 248 Production of IFNgamma FMAT. IFNg plays a central role in the immune system and is considered to be a IFNgamma using proinflammatory cytokine. IFNg promotes TH1 and inhibits TH2 differentiation; promotes IgG2a a T cells and inhibits IgE secretion; induces macrophage activation; and increases MHC expression. Assays for immunomodulatory proteins produced by T cells and NK cells that regulate a variety of inflammatory activities and inhibit TH2 helper cell functions are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, modulate TH2 helper cell function, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as Interferon gamma (IFNg), and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 45 HDPIY31 249 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Gb luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani K., et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Chin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITTiS Cells. HJTT 15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV4O. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC#CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-55 1; Santerre et al. Proc. Nail. Acad. Sci. USA 78: 4339-4343, 1981. 46 HDPOC24 250 Production of Assay that measures the production of the chemokine interleukin-8 (IL-8) from immune cells (such IL-8 by immune as the EOL-1 human eosinophil cell line) are well known in the art (for example, measurement of cells (such as the IL-8 production by FMAT) and may be used or routinely modified to assess the ability of human EOL-1 polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to eosinophil cells) promote or inhibit. Eosinophils are a type of immune cell important in allergic responses; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. IL8 is a strong immunomodulator and may have a potential proinflammatory role in immunological diseases and disorders (such as allergy and asthma). 46 HDPOC24 250 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 46 HDPOC24 250 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 47 HDPPD93 251 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the AP1 response element are known in the art and transcription may be used or routinely modified to assess the ability of polypeptides of the invention (including through AP1 antibodies and agonists or antagonists of the invention) to modulate growth and other cell functions. response element Exemplary assays for transcription through the AP1 response element that may be used or routinely in immune cells modified to test AP1-response element activity of polypeptides of the invention (including (such as T-cells). antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the HT2 cell line, which is an IL-2 dependent suspension culture cell line that also responds to IL-4. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the AP1 response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through AP1 (including antibodies and agonists or antagonists of the invention) to modulate growth and other cell response element functions. Exemplary assays for transcription through the AP1 response element that may be used in immune cells or routinely modified to test AP1-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays are publicly available (e.g.. through the ATCC). Exemplary human T cells that may be used acc- ording to these assays include the SUPT cell line, which is an IL-2 and IL-4 responsive sus- pension-culture cell line. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the CD28 response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through CD28 (including antibodies and agonists or antagonists of the invention) to stimulate IL-2 expression in T response element cells. Exemplary assays for transcription through the CD28 response element that may be used or in immune cells routinely modified to test CD28-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); McGuire and Iacobelli, J Immunol 159(3): 1319-1327 (1997); Parra et al., J Immunol 166(4): 2437-2443 (2001); and Butscher et al., J Biol Chem 3(1): 552-560 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as T-cells). element that may be used or routinely modified to test NFAT-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Serfling et al., Biochim Biophys Acta 1498(1): 1-18 (2000); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the NFKB response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through NFKB (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription response element factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription in immune cells through the NFKB response element that may be used or rountinely modified to test NFKB- (such as T-cells). response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Gnes 15(2): 105-117 (1997); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 47 HDPPD93 251 Production of Assays for production of IL-10 and activation of T-cells are well known in the art and may be used IL-10 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-10 and/or activation T-cells. of T-cells. Exemplary assays that may be used or routinely modified to assess the ability of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) to modulate IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 47 HDPPD93 251 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as natural element that may be used or routinely modified to test NFAT-response element activity of killer cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. NK cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human NK cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 48 HDPPQ30 252 Production of Endothelial cells, which are cells that line blood vessels, and are involved in functions that include, ICAM in but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell endothelial cells extravasation. Exemplary endothelial cells that may be used in ICAM production assays include (such as human human umbilical vein endothelial cells (HUVEC), and are available from commercial sources. The umbilical vein expression of ICAM (CD54), a intergral membrane protein, can be upregulated by cytokines or other endothelial cells factors, and ICAM expression is important in mediating immune and endothelial cell interactions (HUVEC)) leading to immune and inflammatory responses. Assays for measuring expression of ICAM-1 are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate ICAM-1 expression. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Rolfe BE, et al., Atherosclerosis, 149(1): 99-110 (2000); Panettieri RA Jr, et al., J Immunol, 154(5): 2358-2365 (1995); and, Grunstein MM, et al., Am J Physiol Lung Cell Mol Physiol, 278(6): L1154-L1163 (2000), the contents of each of which is herein incorporated by reference in its entirety. 48 HDPPQ30 252 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 49 HDQHM36 253 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann NY Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 50 HDTFX18 254 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 51 HE2CM39 255 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Thai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 52 HE2HC60 256 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 53 HE2PO93 257 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for P13 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for P13 kinase activity that may be used or routinely modified to test P13 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9):110l-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- Li cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 53 HE2PO93 257 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 54 HE6FU11 258 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: # CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 55 HE6FV29 259 Endothelial Cell Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or Apoptosis routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. Induction of apoptosis in endothelial cells supporting the vasculature of tumors is associated with tumor regression due to loss of tumor blood supply. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Endothelial cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary endothelial cells that may be used according to these assays include bovine aortic endothelial cells (bAEC), which are an example of endothelial cells which line blood vessels and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. 55 HE6FV29 259 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 56 HE8TY46 260 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Hepatocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110(1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat liver hepatoma cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat liver hepatoma cells that may be used according to these assays include H4lle cells, which are known to respond to glucocorticoids, insulin, or cAMP derivatives. 57 HE9EA10 261 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 58 HEBCY54 262 Regulation of Assays for the regulation of transcription through the FAS promoter element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the FAS (including antibodies and agonists or antagonists of the invention) to activate the FAS promoter promoter element element in a reporter construct and to regulate transcription of FAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter is regulated by many transcription factors including SREBP. Insulin increases FAS gene transcription in livers of diabetic mice. This stimulation of transcription is also somewhat glucose dependent. Exemplary assays that may be used or routinely modified to test for FAS promoter element activity (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays, such as H4IIE cells, are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays include rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may be used to identify factors that activate adipocytes. the cAMP signaling pathway. CREB plays a major role in adipogenesis, and is involved in differentiation into adipocytes. CRE contains the binding sequence for the transcription factor CREB (CRE binding protein). Exemplary assays for transcription through the cAMP response element that may be used or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of each of which are herein incorporated by reference in its entirety. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 59 HEBFR46 263 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 59 HEBFR46 263 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the AP1 response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through AP1 (including antibodies and agonists or antagonists of the invention) to modulate growth and other cell response element functions. Exemplary assays for transcription through the AP1 response element that may be used in immune cells or routinely modified to test AP1-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is an IL-2 and IL-4 responsive suspension-culture cell line. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the CD28 response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through CD28 (including antibodies and agonists or antagonists of the invention) to stimulate IL-2 expression in T response element cells. Exemplary assays for transcription through the CD28 response element that may be used or in immune cells routinely modified to test CD28-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); McGuire and Iacobelli, J Immunol 159(3): 1319-1327 (1997); Parra et al., J Immunol 166(4): 2437-2443 (2001); and Butscher et al., J Biol Chem 3(1): 552-560 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as T-cells). element that may be used or routinely modified to test NFAT-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Serfling et al., Biochim Biophys Acta 1498(1): 1-18 (2000); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression of in immune cells multiple genes. Exemplary assays for transcription through the STAT6 response element that may (such as T-cells). be used or routinely modified to test STAT6 response element activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 59 HEBFR46 263 Activation of Assays for the activation of transcription through the NFKB response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through NFKB (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription response element factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription in immune cells through the NFKB response element that may be used or rountinely modified to test NFKB- (such as T-cells). response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Gnes 15(2): 105-117 (1997); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 60 HEBGE07 264 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 60 HEBGE07 264 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incor- porated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 60 HEBGE07 264 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 61 HEGAU15 265 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 ( Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 62 HETCI16 266 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 63 HFCEI04 267 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 64 HFCFE20 268 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 65 HFPCZ55 269 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR. et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 66 HFPDR62 270 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 67 HFPDS07 271 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP, regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may be used to identify factors that activate adipocytes. the cAMP signaling pathway. CREB plays a major role in adipogenesis, and is involved in differentiation into adipocytes. CRE contains the binding sequence for the transcription factor CREB (CRE binding protein). Exemplary assays for transcription through the cAMP response element that may be used or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of each of which are herein incorporated by reference in its entirety. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 67 HFPDS07 271 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Natural Killer regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signaling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary natural killer cells that may be used according to these assays include the human natural killer cell lines (for example, NK-YT cells which have cytolytic and cytotoxic activity) or primary NK cells. 67 HFPDS07 271 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 68 HFVHW43 272 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 69 HFXAV37 273 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 70 HFXFZ46 274 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 71 HGBHP91 275 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous. substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 72 HHEAK45 276 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 73 HHEOW19 277 Regulation of Assays for the regulation (i.e. increases or decreases) of viability and proliferation of cells in vitro viability or are well-known in the art and may be used or routinely modified to assess the ability of polypeptides proliferation of of the invention (including antibodies and agonists or antagonists of the invention) to regulate immune cells viability and proliferation of eosinophil cells and cell lines. For example, the CellTiter-Gloô (such as human Luminescent Cell Viability Assay (Promega Corp., Madison, WI, USA ) can be used to measure the eosinophil EOL-1 number of viable cells in culture based on quantitation of the ATP present which signals the cells). presence of metabolically active cells. Eosinophils are a type of immune cell important in allergic responses; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Eosinophil cell lines that may be used according to these assays are publicly available and/or may be routinely generated. Exemplary eosinophil cells that may be used according to these assays include EOL-1 Cells. 73 HHEOW19 277 Production of TNFa FMAT. Assays for immunomodulatory proteins produced by activated macrophages, T cells, TNF alpha by fibroblasts, smooth muscle, and other cell types that exert a wide variety of inflammatory and dendritic cells cytotoxic effects on a variety of cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate inflammation and cytotoxicity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines such as tumor necrosis factor alpha (TNFa), and the induction or inhibition of an inflammatory or cytotoxic response. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 73 HHEOW19 277 Production of MIP-1alpha FMAT. Assays for immunomodulatory proteins produced by activated dendritic cells MIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate chemotaxis, and modulate T cell differentiation. Exemplary assays that test for immunomodulatory proteins evaluate the production of chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a), and the activation of monocytes/macrophages and T cells. Such assays that may be used or routinely modified to test immunomodulatory and chemotaxis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 73 HHEOW19 277 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 74 HHFFS40 278 Endothelial Cell Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or Apoptosis routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. Induction of apoptosis in endothelial cells supporting the vasculature of tumors is associated with tumor regression due to loss of tumor blood supply. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Endothelial cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary endothelial cells that may be used according to these assays include bovine aortic endothelial cells (bAEC), which are an example of endothelial cells which line blood vessels and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. 74 HHFFS40 278 Production of TNFa FMAT. Assays for immunomodulatory proteins produced by activated macrophages, T cells, TNF alpha by fibroblasts, smooth muscle, and other cell types that exert a wide variety of inflammatory and dendritic cells cytotoxic effects on a variety of cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate inflammation and cytotoxicity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines such as tumor necrosis factor alpha (TNFa), and the induction or inhibition of an inflammatory or cytotoxic response. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Human dendritic cells that may be used according to these assays may be iso- lated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 74 HHFFS40 278 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 75 HHGCS78 279 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 75 HHGCS78 279 Production of Assays for measuring expression of ICAM-1 are well-known in the art and may be used or routinely ICAM-1 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate ICAM-1 expression. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Takacs P, et al, FASEB J, 15(2): 279-281 (2001); and, Miyamoto K, et al., Am J Pathol, 156(5): 1733-1739 (2000), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include microvascular endothelial cells (MVEC). 76 HHPSA85 280 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 77 HHSBI06 281 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 78 HHSBI65 282 Production of Assays for measuring expression of ICAM-1 are well-known in the art and may be used or routinely ICAM-1 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate ICAM-1 expression. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Rolfe BE, et al., Atherosclerosis, 149(1): 99-110 (2000); Panettieri RA Jr, et al., J Immunol, 154(5): 2358-2365 (1995); and, Grunstein MM, et al, Am J Physiol Lung Cell Mol Physiol, 278(6): L1154-L1163 (2000), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include Aortic Smooth Muscle Cells (AOSMC); such as bovine AOSMC. 78 HHSBI65 282 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int. 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: # CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 79 HHSGL28 283 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of T rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 80 HISAT67 284 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 81 HJBCU75 285 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 82 HJMAAO3 286 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 82 HJMAA03 286 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 83 HKABU43 287 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 83 HKABU43 287 Production of Assays for production of IL-10 and activation of T-cells are well known in the art and may be used IL-10 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-10 and/or activation T-cells. of T-cells. Exemplary assays that may be used or routinely modified to assess the ability of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) to modulate IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56(4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 84 HKACI79 288 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary human T cells, such as the SUPT cell line, that may be used according to these assays are publicly available (e.g., through the ATCC). 84 HKACI79 288 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 84 HKACI79 288 Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) expression is restricted to activated T cells. CD152 is a CD152 and negative regulator of T cell proliferation. Reduced CD152 expression has been linked to activation of hyperproliferative and autoimmune diseases. Overexpression of CD152 may lead to impaired T cells immunoresponses. Assays for immunomodulatory proteins important in the maintenance of T cell homeostasis and expressed almost exclusively on CD4+ and CD8+ T cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, maintain T cell homeostasis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD152, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); McCoy et al., Immunol Cell Biol 77(1): 1-10 (1999); Oostervegal et al., Curr Opin Immunol 11(3): 294-300 (1999); and Saito T, Curr Opin Immunol 10(3): 313-321 (1998), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 85 HKIXC44 289 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transfonned with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 86 HKTAB41 290 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 87 HLDQU79 291 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 87 HLDQU79 291 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune transcription through the SRE that may be used or routinely modified to test SRE activity of the cells (such polypeptides of the invention (including antibodies and agonists or antagonists of the invention) as T-cells). include assays disclosed in Berger et al., Gene 66:1-10(1998); Cullen and MaIm, Methods in Enzymol 216:362-368 (1992); Henthom et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 88 HLHAP05 292 Production of MIP-1alpha FMAT. Assays for immunomodulatory proteins produced by activated dendritic cells MIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate chemotaxis, and modulate T cell differentiation. Exemplary assays that test for immunomodulatory proteins evaluate the production of chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a), and the activation of monocytes/macrophages and T cells. Such assays that may be used or routinely modified to test immunomodulatory and chemotaxis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 88 HLHAP05 292 Regulation of Assays for the regulation of transcription through the FAS promoter element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the FAS (including antibodies and agonists or antagonists of the invention) to activate the FAS promoter promoter element element in a reporter construct and to regulate transcription of FAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter is regulated by many transcription factors including SREBP. Insulin increases FAS gene transcription in livers of diabetic mice. This stimulation of transcription is also somewhat glucose dependent. Exemplary assays that may be used or routinely modified to test for FAS promoter element activity (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays, such as H4IIE cells, are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays include rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 89 HLHCS23 293 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 89 HLHCS23 293 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune transcription through the SRE that may be used or routinely modified to test SRE activity of the cells (such polypeptides of the invention (including antibodies and agonists or antagonists of the invention) as T-cells). include assays disclosed in Berger et al., Gene 66:1-10 (1998); Cullen and MaIm, Methods in Enzymol 2 16:362-368 (1992); Henthom et al., Proc NatI Acad Sci USA 85:6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CuLL cell line, which is an IL-2 dependent suspension culture ofT cells with cytotoxic activity 89 HLHCS23 293 Production of IFNgamma FMAT. IFNg plays a central role in the immune system and is considered to be a IFNgamma using proinflammatory cytokine. IFNg promotes TH1 and inhibits TH2 differentiation; promotes IgG2a a T cells and inhibits IgE secretion; induces macrophage activation; and increases MHC expression. Assays for immunomodulatory proteins produced by T cells and NK cells that regulate a variety of inflammatory activities and inhibit TH2 helper cell functions are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, modulate TH2 helper cell function, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as Interferon gamma (IFNg), and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 90 HLICE88 294 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 90 HLICE88 294 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 91 HLMGP50 295 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 92 HLMMX62 296 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 93 HLQCX36 297 Proliferation of Assays for the regulation (i.e. increases or decreases) of viability and proliferation of cells in vitro immune cells are well-known in the art and may be used or routinely modified to assess the ability of polypeptides (such as the of the invention (including antibodies and agonists or antagonists of the invention) to regulate HMC-1 human viability and proliferation of eosinophil cells and cell lines. For example, the CellTiter-Gloô mast cell line) Luminescent Cell Viability Assay (Promega Corp., Madison, WI, USA ) can be used to measure the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Mast cells are found in connective and mucosal tissues throughout the body. Mast cell activation (via immunoglobulin E -antigen, promoted by T helper cell type 2 cytokines) is an important component of allergic disease. Dysregulation of mast cell apoptosis may play a role in allergic disease and mast cell tumor survival. Mast cell lines that may be used according to these assays are publicly available and/or may be routinely generated. Exemplary mast cells that may be used according to these assays include HMC-1, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 93 HLQCX36 297 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 94 HLWAF06 298 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110(1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 95 HLWDB73 299 Activation of Kinase assay. Kinase assays, for examplek Elk-1 kinase assays, for ERK signal transduction that Skeletal Muscle regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signalling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 96 HLYDF73 300 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 97 HLYGB19 301 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 97 HLYGB19 301 Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) expression is restricted to activated T cells. CD152 is a CD152 and negative regulator of T cell proliferation. Reduced CD152 expression has been linked to activation of hyperproliferative and autoimmune diseases. Overexpression of CD152 may lead to impaired T cells immunoresponses. Assays for immunomodulatory proteins important in the maintenance of T cell homeostasis and expressed almost exclusively on CD4+ and CD8+ T cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, maintain T cell homeostasis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD152, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); McCoy et al., Immunol Cell Biol 77(1): 1-10 (1999); Oostervegal et al., Curr Opin Immunol 11(3): 294-300 (1999); and Saito T, Curr Opin Immunol 10(3): 313-321 (1998), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 98 HLYGY91 302 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 98 HLYGY91 302 Production of Assays for production of IL-13 and activation of T-cells are well known in the art and may be used IL-13 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-13 and/or activation T-cells. of T-cells. Exemplary assays for IL-13 production that may be used or routinely modified to test activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays such as disclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13 independently of IL-4 in Experimental asthma” Science; 282: 2261-2263 (1998), and Wills-Karp M, et al., “Interleukin-13: central mediator of allergic asthma” Science; 282: 2258-2261 (1998); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL13, a Th2 type cytokine, is a potent stimulus for mucus production, airway hyper-responsiveness and allergic asthma. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 99 HMDAB29 303 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 99 HMDAB29 303 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 99 HMDAB29 303 Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) expression is restricted to activated T cells. CD152 is a CD152 and negative regulator of T cell proliferation. Reduced CD152 expression has been linked to activation of hyperproliferative and autoimniune diseases. Overexpression of CD152 may lead to impaired T cells immunoresponses. Assays for immunomodulatory proteins important in the maintenance of T cell homeostasis and expressed almost exclusively on CD4+ and CD8+ T cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, maintain T cell homeostasis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD152, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); McCoy et al., Immunol Cell Biol 77 (1): 1-10 (1999); Oostervegal et al., Curr Opin Immunol 11(3): 294-300 (1999); and Saito T, Curr Opin Immunol 10(3): 313-321 (1998), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 100 HMDAD44 304 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 100 HMDAD44 304 Production of TNFa FMAT. Assays for immunomodulatory proteins produced by activated macrophages, T cells, TNF alpha by fibroblasts, smooth muscle, and other cell types that exert a wide variety of inflammatory and dendritic cells cytotoxic effects on a variety of cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate inflammation and cytotoxicity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines such as tumor necrosis factor alpha (TNFa), and the induction or inhibition of an inflammatory or cytotoxic response. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 100 HMDAD44 304 Production of MCP-1 FMAT. Assays for immunomodulatory proteins that are produced by a large variety of cells MCP-1 and act to induce chemotaxis and activation of monocytes and T cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, induce chemotaxis, and modulate immune cell activation. Exemplary assays that test for immunomodulatory proteins evaluate the production of cell surface markers, such as monocyte chemoattractant protein (MCP), and the activation of monocytes and T cells. Such assays that may be used or routinely modified to test immunomodulatory and diffferentiation activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); and Verhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 101 HMEDI90 305 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, a adipocytes key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in adipoocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 102 HMIBF07 306 Production of MCP-1 FMAT. Assays for immunomodulatory proteins that are produced by a large variety of cells MCP-1 and act to induce chemotaxis and activation of monocytes and T cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, induce chemotaxis, and modulate immune cell activation. Exemplary assays that test for immunomodulatory proteins evaluate the production of cell surface markers, such as monocyte chemoattractant protein (MCP), and the activation of monocytes and T cells. Such assays that may be used or routinely modified to test immunomodulatory and diffferentiation activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); and Verhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 102 HMIBF07 306 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 102 HMIBF07 306 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 103 HMICP65 307 Production of Assays for measuring expression of VCAM are well-known in the art and may be used or routinely VCAM in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or endothelial cells antagonists of the invention) to regulate VCAM expression. For example, FMAT may be used to (such as human meaure the upregulation of cell surface VCAM-1 expresssion in endothelial cells. Endothelial cells umbilical vein are cells that line blood vessels, and are involved in functions that include, but are not limited to, endothelial cells angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Exemplary (HUVEC)) endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are available from commercial sources. The expression of VCAM (CD106), a membrane-associated protein, can be upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 103 HMICP65 307 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary human T cells, such as the SUPT cell line, that may be used according to these assays are publicly available (e.g., through the ATCC). 103 HMICP65 307 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 104 HMSHC86 308 Activation of Kinase assay. Kinase assays, for examplek Elk-i kinase assays, for ERK signal transduction that Skeletal Muscle regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signalling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 105 HMUAN45 309 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 106 HMVBC31 310 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 107 HMWBL03 311 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 108 HNFGR08 312 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 109 HNGAK51 313 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 110 HNGDX18 314 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 110 HNGDX18 314 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 110 HNGDX18 314 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 111 HNGGP65 315 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Hepatocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat liver hepatoma cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat liver hepatoma cells that may be used according to these assays include H4lle cells, which are known to respond to glucocorticoids, insulin, or cAMP derivatives. 112 HNGIV64 316 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 112 HNGIV64 316 Production of Assays for production of IL-10 and activation of T-cells are well known in the art and may be used IL-10 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-10 and/or activation T-cells. of T-cells. Exemplary assays that may be used or routinely modified to assess the ability of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) to modulate IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 113 HNGMW45 317 Regulation of Assays for the regulation of transcription through the PEPCK promoter are well-known in the art transcription and may be used or routinely modified to assess the ability of polypeptides of the invention through the (including antibodies and agonists or antagonists of the invention) to activate the PEPCK promoter PEPCK promoter in a reporter construct and regulate liver gluconeogenesis. Exemplary assays for regulation of in hepatocytes transcription through the PEPCK promoter that may be used or routinely modified to test for PEPCK promoter activity (in hepatocytes) of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., J Biol Chem 275(23): 17814-17820 (2000), the contents of each of which is herein incorporated by reference in its entirety. Hepatocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary liver hepatoma cells that may be used according to these assays include H4lle cells, which contain a tyrosine amino transferase that is inducible with glucocorticoids, insulin, or cAMP derivatives. 114 HNGPJ25 318 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 114 HNGPJ25 318 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 114 HNGPJ25 318 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, a adipocytes key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in adipoocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al, Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 115 HNHEN82 319 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 116 HNHFE71 320 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 116 HNHFE71 320 Production of Assays for measuring expression of VCAM are well-known in the art and may be used or routinely VCAM in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or endothelial cells antagonists of the invention) to regulate VCAM expression. For example, FMAT may be used to (such as human meaure the upregulation of cell surface VCAM-1 expresssion in endothelial cells. Endothelial cells umbilical vein are cells that line blood vessels, and are involved in functions that include, but are not limited to, endothelial cells angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Exemplary (HUVEC)) endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are available from commercial sources. The expression of VCAM (CD106), a membrane-associated protein, can be upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 116 HNHFE71 320 Calcium flux in Assays for measuring calcium flux are well-known in the art and may be used or routinely modified immune cells to assess the ability of polypeptides of the invention (including antibodies and agonists or (such as antagonists of the invention) to mobilize calcium. Cells normally have very low concentrations of monocytes) cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux in immune cells (such as monocytes) include assays disclosed in: Chan, CC, et al., J Pharmacol Exp Ther, 269(3): 891-896 (1994); Andersson, K, et al., Cytokine, 12(12): 1784-1787 (2000); Scully, SP, et al., J Clin Invest, 74(2) 589-599 (1984); and, Sullivan, E, et al., Methods Mol Biol, 114: 125-133 (1999), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the THP-1 monocyte cell line. 117 HNHKV56 321 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 118 HOACG07 322 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 119 HODBB70 323 Activation of Assays for the activation of transcription through the cAMP response element are well-known in transcription the art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP and regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell in immune cells functions. Exemplary assays for transcription through the cAMP response element that may be used (such as T-cells). or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 119 HODBB70 323 Regulation of Assays for the regulation of transcription through the PEPCK promoter are well-known in the art transcription and may be used or routinely modified to assess the ability of polypeptides of the invention through the (including antibodies and agonists or antagonists of the invention) to activate the PEPCK promoter PEPCK promoter in a reporter construct and regulate liver gluconeogenesis. Exemplary assays for regulation of in hepatocytes transcription through the PEPCK promoter that may be used or routinely modified to test for PEPCK promoter activity (in hepatocytes) of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., J Biol Chem 275(23): 17814-17820 (2000), the contents of each of which is herein incorporated by reference in its entirety. Hepatocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary liver hepatoma cells that may be used according to these assays include H4lle cells, which contain a tyrosine amino transferase that is inducible with glucocorticoids, insulin, or cAMP derivatives. 120 HOEBK60 324 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 121 HOFAA78 325 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 122 HOFNB74 326 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 123 HORBS82 327 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 123 HORBS82 327 Activation of Kinase assay. JNK kinase assays for signal transduction that regulate cell proliferation, activation, JNK Signaling or apoptosis are well known in the art and may be used or routinely modified to assess the ability of Pathway in polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to immune cells promote or inhibit cell proliferation, activation, and apoptosis. Exemplary assays for JNK kinase (such as activity that may be used or routinely modified to test JNK kinase-induced activity of polypeptides eosinophils). of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate signal transduction, cell proliferation, activation, or apoptosis in eosinophils include assays disclosed and/or cited in: Zhang JP, et al., “Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen- activated protein kinase in human eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7 (2000); Hebestreit H, et al., “Disruption of fas receptor signaling by nitric oxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); J Allergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, et al., “In vivo resistance to corticosteroids in bronchial asthma is associated with enhanced phosyphorylation of JUN N-terminal kinase and failure of prednisolone to inhibit JUN N-terminal kinase phosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74 (1999); the contents of each of which are herein incorporated by reference in its entirety. 123 HORBS82 327 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis of routinely modified to assess the ability of polypeptides of the invention (including antibodies and immune cells agonists or antagonists of the invention) to regulate caspase protease-mediated apoptosis in immune (such as mast cells (such as, for example, in mast cells). Mast cells are found in connective and mucosal tissues cells). throughout the body, and their activation via immunoglobulin E -antigen, promoted by T helper cell type 2 cytokines, is an important component of allergic disease. Dysregulation of mast cell apoptosis may play a role in allergic disease and mast cell tumor survival. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity induced by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Masuda A, et al., J Biol Chem, 276(28): 26107-26113 (2001); Yeatman CF 2nd, et al., J Exp Med, 192(8): 1093-1103 (2000); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary immune cells that may be used according to these assays include mast cells such as the HMC human mast cell line. 123 HORBS82 327 Production of Assays for production of IL-13 and activation of T-cells are well known in the art and may be used IL-13 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-13 and/or activation T-cells. of T-cells. Exemplary assays for IL-13 production that may be used or routinely modified to test activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays such as disclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13 independently of IL-4 in Experimental asthma” Science; 282: 2261-2263 (1998), and Wills-Karp M, et al., “Interleukin-13: central mediator of allergic asthma” Science; 282: 2258-2261 (1998); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL13, a Th2 type cytokine, is a potent stimulus for mucus production, airway hyper-responsiveness and allergic asthma. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 124 HOSDO75 328 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 125 HOUDR07 329 Activation of Kinase assay. Kinase assays, for examplek Elk-1 kinase assays, for ERK signal transduction that Skeletal Muscle regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signalling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 126 HPEAD23 330 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 127 HPFCI36 331 Production of MIP-1alpha FMAT. Assays for immunomodulatory proteins produced by activated dendritic cells MIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate chemotaxis, and modulate T cell differentiation. Exemplary assays that test for immunomodulatory proteins evaluate the production of chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a), and the activation of monocytes/macrophages and T cells. Such assays that may be used or routinely modified to test immunomodulatory and chemotaxis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 127 HPFCI36 331 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 127 HPFCI36 331 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 128 HPFDI37 332 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to modulate gene expression (commonly via STAT transcription factors) involved in a in immune cells wide variety of cell functions. Exemplary assays for transcription through the GAS response (such as element that may be used or routinely modified to test GAS-response element activity of eosinophils). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995); the contents of each of which are herein incorporated by reference in its entirety. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to activate or inhibit activation of immune cells include assays disclosed and/or cited in: Mayumi M., “EoL-1, a human eosinophilic cell line” Leuk Lymphoma; Jun; 7(3): 243-50 (1992); Bhattacharya S, “Granulocyte macrophage colony-stimulating factor and interleukin-5 activate STAT5 and induce CIS1 mRNA in human peripheral blood eosinophils” Am J Respir Cell Mol Biol; Mar; 24(3): 312-6 (2001); and, Du J, et al., “Engagement of the CrkL adapter in interleukin-5 signaling in eosinophils” J Biol Chem; Oct 20; 275(42): 33167-75 (2000); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are a type of immune cell important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Increases in GAS mediated transcription in eosinophils is typically a result of STAT activation, normally a direct consequence of interleukin or other cytokine receptor stimulation (e.g. IL3, IL5 or GMCSF). 128 HPFDI37 332 Regulation of Caspase Apoptosis. Assays for caspase apoptosis are well known in the art and may be used or apoptosis in routinely modified to assess the ability of polypeptides of the invention (including antibodies and pancreatic beta agonists or antagonists of the invention) to promote caspase protease-mediated apoptosis. cells. Apoptosis in pancreatic beta is associated with induction and progression of diabetes. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Tbromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 129 HPIAA80 333 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 129 HPIAA80 333 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, adipocytes a key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in adipoocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 130 HPJCW58 334 Regulation of Assays for the regulation of transcription through the FAS promoter element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the FAS (including antibodies and agonists or antagonists of the invention) to activate the FAS promoter promoter element element in a reporter construct and to regulate transcription of FAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter is regulated by many transcription factors including SREBP. Insulin increases FAS gene transcription in livers of diabetic mice. This stimulation of transcription is also somewhat glucose dependent. Exemplary assays that may be used or routinely modified to test for FAS promoter element activity (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays, such as H4IIE cells, are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays include rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 131 HPRBH85 335 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 132 HPRCA64 336 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 133 HPRCD35 337 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 133 HPRCD35 337 IL-13 in HMC 134 HPTRM02 338 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 134 HPTRM02 338 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 134 HPTRM02 338 Activation of This reporter assay measures activation of the GATA-3 signaling pathway in HMC-1 human mast transcription cell line. Activation of GATA-3 in mast cells has been linked to cytokine and chemokine through GATA-3 production. Assays for the activation of transcription through the GATA3 response element are response element well-known in the art and may be used or routinely modified to assess the ability of polypeptides of in immune cells the invention (including antibodies and agonists or antagonists of the invention) to regulate GATA3 (such as mast transcription factors and modulate expression of mast cell genes important for immune response cells). development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorpor- ated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human mast cells that may be used acc- ording to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 135 HRAAD30 339 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 136 HRADF49 340 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 136 HRADF49 340 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 137 HRDDQ39 341 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 137 HRDDQ39 341 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 137 HRDDQ39 341 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 138 HRDEX93 342 Regulation of Assays for the regulation of transcription through the FAS promoter element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the FAS (including antibodies and agonists or antagonists of the invention) to activate the FAS promoter promoter element element in a reporter construct and to regulate transcription of FAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter is regulated by many transcription factors including SREBP. Insulin increases FAS gene transcription in livers of diabetic mice. This stimulation of transcription is also somewhat glucose dependent. Exemplary assays that may be used or routinely modified to test for FAS promoter element activity (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays, such as H4IIE cells, are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays include rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 139 HROEA08 343 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 140 HRTAP63 344 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 141 HSAVW42 345 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 142 HSAYC41 346 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 143 HSLHX15 347 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 144 HSNBM34 348 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 145 HSSEF77 349 Activation of T- Kinase assay. JNK and p38 kinase assays for signal transduction that regulate cell proliferation, Cell p38 or JNK activation, or apoptosis are well known in the art and may be used or routinely modified to assess Signaling the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the Pathway. invention) to promote or inhibit immune cell (e.g. T-cell) proliferation, activation, and apoptosis. Exemplary assays for JNK and p38 kinase activity that may be used or routinely modified to test JNK and p38 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 145 HSSEF77 349 Insulin Secretion Assays for measuring secretion of insulin are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 145 HSSEF77 349 Production of Assays for measuring expression of VCAM are well-known in the art and may be used or routinely VCAM in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or endothelial cells antagonists of the invention) to regulate VCAM expression. For example, FMAT may be used to (such as human meaure the upregulation of cell surface VCAM-1 expresssion in endothelial cells. Endothelial cells umbilical vein are cells that line blood vessels, and are involved in functions that include, but are not limited to, endothelial cells angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Exemplary (HUVEC)) endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are available from commercial sources. The expression of VCAM (CD106), a membrane-associated protein, can be upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 146 HT4FV41 350 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 147 HT5FX79 351 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 148 HT5GR59 352 Activation of Assays for the activation of transcription through the AP1 response element are known in the art and transcription may be used or routinely modified to assess the ability of polypeptides of the invention (including through AP1 antibodies and agonists or antagonists of the invention) to modulate growth and other cell functions. response element Exemplary assays for transcription through the AP1 response element that may be used or routinely in immune cells modified to test AP1-response element activity of polypeptides of the invention (including (such as T-cells). antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 148 HT5GR59 352 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 149 HTAE178 353 Upregulation of HLA-DR FMAT. MHC class II is essential for correct presentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for T cells immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or other- wise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 150 HTEAG62 354 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 150 HTEAG62 354 Regulation of Assays for the regulation of transcription of Malic Enzyme are well-known in the art and may be transcription of used or routinely modified to assess the ability of polypeptides of the invention (including Malic Enzyme in antibodies and agonists or antagonists of the invention) to regulate transcription of Malic Enzyme, adipocytes a key enzyme in lipogenesis. Malic enzyme is involved in lipogenesisand its expression is stimulted by insulin. ME promoter contains two direct repeat (DR1)- like elements MEp and MEd identified as putative PPAR response elements. ME promoter may also responds to AP1 and other transcription factors. Exemplary assays that may be used or routinely modified to test for regulation of transcription of Malic Enzyme (in adipoocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 151 HTEDJ28 355 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 151 HTEDJ28 355 Activation of Assays for the activation of transcription through the Nuclear Factor of Activated T cells (NFAT) transcription response element are well-known in the art and may be used or routinely modified to assess the through NFAT ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate NFAT transcription factors and modulate expression of genes involved in in immune cells immunomodulatory functions. Exemplary assays for transcription through the NFAT response (such as natural element that may be used or routinely modified to test NFAT-response element activity of killer cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293 (1993), the contents of each of which are herein incorporated by reference in its entirety. NK cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human NK cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 152 HTEDS12 356 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110(1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 153 HTEHA56 357 Activation of Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3 regulate glucose metabolism and cell survival are well-known in the art and may be used or Kinase Signalling routinely modified to assess the ability of polypeptides of the invention (including antibodies and Pathway agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase- induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3- L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 153 HTEHA56 357 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 154 HTEJD29 358 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 155 HTENR63 359 Regulation of Assays for the regulation of transcription through the PEPCK promoter are well-known in the art transcription and may be used or routinely modified to assess the ability of polypeptides of the invention through the (including antibodies and agonists or antagonists of the invention) to activate the PEPCK promoter PEPCK promoter in a reporter construct and regulate liver gluconeogenesis. Exemplary assays for regulation of in hepatocytes transcription through the PEPCK promoter that may be used or routinely modified to test for PEPCK promoter activity (in hepatocytes) of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., J Biol Chem 275(23): 17814-17820 (2000), the contents of each of which is herein incorporated by reference in its entirety. Hepatocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary liver hepatoma cells that may be used according to these assays include H4lle cells, which contain a tyrosine amino transferase that is inducible with glucocorticoids, insulin, or cAMP derivatives. 156 HTGGM44 360 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 157 HTHBZ06 361 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 158 HTLBT80 362 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 158 HTLBT80 362 Activation of Assays for the activation of transcription through the EGR response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the EGR (including antibodies and agonists or antagonists of the invention) to regulate EGR transcription (Early Growth factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription Response) through the EGR response element that may be used or routinely modified to test EGR response element in element activity of polypeptides of the invention (including antibodies and agonists or antagonists of immune cells the invention) include assays disclosed in: Richards JD, et al., J Immunol, 166(6): 3855-3864 (2001); (such as B-cells). Dinkel, A, et al., J Exp Med, 188(12): 2215-2224 (1998); and, Newton, JS, et al., Eur J Immunol 1996 Apr; 26(4): 811-816 (1996), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary epithelial cells that may be used according to these assays include the Raji cell line. 158 HTLBT80 362 Activation of Assays for the activation of transcription through the EGR response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the EGR (including antibodies and agonists or antagonists of the invention) to regulate EGR transcription (Early Growth factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription Response) through the EGR response element that may be used or routinely modified to test EGR response element in element activity of polypeptides of the invention (including antibodies and agonists or antagonists immune cells of the invention) include assays disclosed in: Richards JD, et al., J Immunol, 166(6): 3855-3864 (such as B-cells). (2001); Dinkel, A, et al., J Exp Med, 188(12): 2215-2224 (1998); and, Newton, JS, et al., Eur J Immunol 1996 Apr; 26(4): 811-816 (1996), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary epithelial cells that may be used according to these assays include the Raji cell line. 158 HTLBT80 362 Activation of Assays for the activation of transcription through the NFKB response element are well-known in transcription the art and may be used or routinely modified to assess the ability of polypeptides of the invention through NFKB (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription response element factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription in immune cells through the NFKB response element that may be used or rountinely modified to test NFKB- (such as B-cells). response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gri G, et al., Biol Chem, 273(11): 6431-6438 (1998); Pyatt DW, et al., Cell Biol Toxicol 2000; 16(1): 41-51 (2000); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary immune cells that may be used according to these assays include the Reh B-cell line. 159 HTLDU78 363 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 159 HTLDU78 363 Activation of T- Kinase assay. JNK and p38 kinase assays for signal transduction that regulate cell proliferation, Cell p38 or JNK activation, or apoptosis are well known in the art and may be used or routinely modified to assess Signaling the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the Pathway. invention) to promote or inhibit immune cell (e.g. T-cell) proliferation, activation, and apoptosis. Exemplary assays for JNK and p38 kinase activity that may be used or routinely modified to test JNK and p38 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 159 HTLDU78 363 Production of IL-6 FMAT. IL-6 is produced by T cells and has strong effects on B cells. IL-6 participates in IL-4 IL-6 induced IgE production and increases IgA production (IgA plays a role in mucosal immunity). IL-6 induces cytotoxic T cells. Deregulated expression of IL-6 has been linked to autoimmune disease, plasmacytomas, myelomas, and chronic hyperproliferative diseases. Assays for immunomodulatory and differentiation factor proteins produced by a large variety of cells where the expression level is strongly regulated by cytokines, growth factors, and hormones are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation and differentiation and modulate T cell proliferation and function. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-6, and the stimulation and upregulation of T cell proliferation and functional activities. Such assays that may be used or routinely modified to test immunomodulatory and dififerentiation activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Verhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 160 HTLFA13 364 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 161 HTLGI89 365 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 162 HTNBK13 366 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art viability and and may be used or routinely modified to assess the ability of polypeptides of the invention (includ- proliferation of ing antibodies and agonists or antagonists of the invention) to regulate viability and proliferation pancreatic beta of pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures cells. the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely mod- ified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771- 9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/ or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 162 HTNBK13 366 Production of TNFa FMAT. Assays for immunomodulatory proteins produced by activated macrophages, T cells, TNF alpha by fibroblasts, smooth muscle, and other cell types that exert a wide variety of inflammatory and dendritic cells cytotoxic effects on a variety of cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate inflammation and cytotoxicity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines such as tumor necrosis factor alpha (TNFa), and the induction or inhibition of an inflammatory or cytotoxic response. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human dendritic cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 162 HTNBK13 366 Production of Assays for production of IL-10 and activation of T-cells are well known in the art and may be used IL-10 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-10 and/or activation T-cells. of T-cells. Exemplary assays that may be used or routinely modified to assess the ability of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) to modulate IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 163 HTOAM11 367 Myoblast cell Assays for muscle cell proliferation are well known in the art and may be used or routinely proliferation modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate or inhibit myoblast cell proliferation. Exemplary assays for myoblast cell proliferation that may be used or routinely modified to test activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays disclosed in: Soeta, C., et al. “Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats” Dev Growth Differ Apr; 43(2): 155-64 (2001); Ewton DZ, et al., “IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation” J Endocrinol Mar; 144(3): 539-53 (1995); and, Pampusch MS, et al., “Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells” J Cell Physiol Jun; 143(3): 524-8 (1990); the contents of each of which are herein incorporated by reference in their entirety. Exemplary myoblast cells that may be used according to these assays include the rat myoblast L6 cell line. Rat myoblast L6 cells are an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuse to form multinucleated myotubes and striated fibers after culture in differentiation media. 163 HTOAM11 367 Activation of Assays for the activation of transcription through the AP1 response element are known in the art transcription and may be used or routinely modified to assess the ability of polypeptides of the invention through AP1 (including antibodies and agonists or antagonists of the invention) to modulate growth and other response element cell functions. Exemplary assays for transcription through the AP1 response element that may be in immune cells used or routinely modified to test AP1-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 163 HTOAM11 367 Activation of Assays for the activation of transcription through the cAMP response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through cAMP (including antibodies and agonists or antagonists of the invention) to increase cAMP and regulate response element CREB transcription factors, and modulate expression of genes involved in a wide variety of cell in immune cells functions. Exemplary assays for transcription through the cAMP response element that may be used (such as T-cells). or routinely modified to test cAMP-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 163 HTOAM11 367 Activation of Assays for the activation of transcription through the Gamma Interferon Activation Site (GAS) transcription response element are well-known in the art and may be used or routinely modified to assess the through GAS ability of polypeptides of the invention (including antibodies and agonists or antagonists of the response element invention) to regulate STAT transcription factors and modulate gene expression involved in a wide in immune cells variety of cell functions. Exemplary assays for transcription through the GAS response element that (such as T-cells). may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary mouse T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the CTLL cell line, which is a suspension culture of IL-2 dependent cytotoxic T cells. 163 HTOAM11 367 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 163 HTOAM11 367 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 163 HTOAM11 367 Activation of Assays for the activation of transcription through the CD28 response element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through CD28 (including antibodies and agonists or antagonists of the invention) to stimulate IL-2 expression in response element T cells. Exemplary assays for transcription through the CD28 response element that may be used or in immune cells routinely modified to test CD28-response element activity of polypeptides of the invention (such as T-cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); McGuire and Iacobelli, J Immunol 159(3): 1319-1327 (1997); Parra et al., J Immunol 166(4): 2437-2443 (2001); and Butscher et al., J Biol Chem 3(1): 552-560 (1998), the contents of each of which are herein incorporated by ref- erence in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human T cells that may be used according to these assays include the JURKAT cell line, which is a suspension culture of leukemia cells that produce IL-2 when stimulated. 163 HTOAM11 367 Production of IFNgamma FMAT. IFNg plays a central role in the immune system and is considered to be a IFNgamma using proinflammatory cytokine. IFNg promotes TH1 and inhibits TH2 differentiation; promotes IgG2a a T cells and inhibits IgE secretion; induces macrophage activation; and increases MHC expression. Assays for immunomodulatory proteins produced by T cells and NK cells that regulate a variety of inflammatory activities and inhibit TH2 helper cell functions are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, modulate TH2 helper cell function, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as Interferon gamma (IFNg), and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 164 HTOHO21 368 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110(1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 164 HTOHO21 368 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 165 HTPCO75 369 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 166 HTSFJ32 370 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 166 HTSFJ32 370 Activation of Kinase assay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transduction that regulate glucose metabolism and cell survivial are well-known in the art and may Cell PI3 Kinase be used or routinely modified to assess the ability of polypeptides of the invention (including Signalling antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism Pathway and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by ref- erence in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 167 HTTCB60 371 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 168 HTTEE41 372 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 168 HTTEE41 372 Production of Assays for production of IL-13 and activation of T-cells are well known in the art and may be used IL-13 and or routinely modified to assess the ability of polypeptides of the invention (including antibodies and activation of agonists or antagonists of the invention) to stimulate or inhibit production of IL-13 and/or activation T-cells. of T-cells. Exemplary assays for IL-13 production that may be used or routinely modified to test activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays such as disclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13 independently of IL-4 in Experimental asthma” Science; 282: 2261-2263 (1998), and Wills-Karp M, et al., “Interleukin-13: central mediator of allergic asthma” Science; 282: 2258-2261 (1998); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL13, a Th2 type cytokine, is a potent stimulus for mucus production, airway hyper-responsiveness and allergic asthma. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 169 HTWEH94 373 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 170 HTXFA72 374 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 171 HTXKF95 375 Activation of Kinase assay. Kinase assays, for examplek Elk-1 kinase assays, for ERK signal transduction that Skeletal Muscle regulate cell proliferation or differentiation are well known in the art and may be used or routinely Cell ERK modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Signalling antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Pathway Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 172 HUKDF20 376 Regulation of Assays for the regulation of transcription through the DMEF1 response element are well-known in transcription via the art and may be used or routinely modified to assess the ability of polypeptides of the invention DMEF1 response (including antibodies and agonists or antagonists of the invention) to activate the DMEF1 response element in element in a reporter construct (such as that containing the GLUT4 promoter) and to regulate insulin adipocytes and production. The DMEF1 response element is present in the GLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor and another transcription factor that is required for insulin regulation of Glut4 expression in skeletal muscle. GLUT4 is the primary insulin-responsive glucose transporter in fat and muscle tissue. Exemplary assays that may be used or routinely modified to test for DMEF1 response element activity (in adipocytes and pre-adipocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994); “Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 172 HUKDF20 376 Activation of Assays for the activation of transcription through the AP1 response element are known in the art and transcription may be used or routinely modified to assess the ability of polypeptides of the invention (including through AP1 antibodies and agonists or antagonists of the invention) to modulate growth and other cell functions. response element Exemplary assays for transcription through the AP1 response element that may be used or routinely in immune cells modified to test AP1-response element activity of polypeptides of the invention (including (such as T-cells). antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 172 HUKDF20 376 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate the serum response element response factors and modulate the expression of genes involved in growth. Exemplary assays for in immune cells transcription through the SRE that may be used or routinely modified to test SRE activity of the (such as T-cells). polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66 :1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse T cells that may be used according to these assays include the CTLL cell line, which is an IL-2 dependent suspension culture of T cells with cytotoxic activity. 172 HUKDF20 376 Activation of Assays for the activation of transcription through the NFKB response element are well-known in transcription the art and may be used or routinely modified to assess the ability of polypeptides of the invention through NFKB (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription response element factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription in immune cells through the NFKB response element that may be used or rountinely modified to test NFKB- (such as EOL1 response element activity of polypeptides of the invention (including antibodies and agonists or cells). antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. For example, a reporter assay (which measures increases in transcription inducible from a NFkB responsive element in EOL-1 cells) may link the NFKB element to a repeorter gene and binds to the NFKB transcription factor, which is upregulated by cytokines and other factors. Exemplary immune cells that may be used according to these assays include eosinophils such as the human EOL-1 cell line of eosinophils. Eosinophils are a type of immune cell important in the allergic responses; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Eol-1 is a human eosinophil cell line. 172 HUKDF20 376 Activation of This reporter assay measures activation of the NFkB signaling pathway in Ku812 human basophil transcription cell line. Assays for the activation of transcription through the NFKB response element are well- through NFKB known in the art and may be used or routinely modified to assess the ability of polypeptides of the response element invention (including antibodies and agonists or antagonists of the invention) to regulate NFKB in immune cells transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for (such as transcription through the NFKB response element that may be used or rountinely modified to test basophils). NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), the contents of each of which are herein incorporated by reference in its entirety. Basophils that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary human basophil cell lines that may be used according to these assays include Ku812, originally established from a patient with chronic myelogenous leukemia. It is an immature prebasophilic cell line that can be induced to differentiate into mature basophils. 172 HUKDF20 376 Production of Assays for measuring expression of ICAM-1 are well-known in the art and may be used or routinely ICAM-1 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate ICAM-1 expression. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Takacs P, et al, FASEB J, 15(2): 279-281 (2001); and, Miyamoto K, et al., Am J Pathol, 156(5): 1733-1739 (2000), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include microvascular endothelial cells (MVEC). 172 HUKDF20 376 Activation of Assays for the activation of transcription through the Serum Response Element (SRE) are well- transcription known in the art and may be used or routinely modified to assess the ability of polypeptides of the through serum invention (including antibodies and agonists or antagonists of the invention) to regulate serum response element response factors and modulate the expression of genes involved in growth and upregulate the in immune cells function of growth-related genes in many cell types. Exemplary assays for transcription through the (such as natural SRE that may be used or routinely modified to test SRE activity of the polypeptides of the invention killer cells). (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the content of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the NK-YT cell line, which is a human natural killer cell line with cytolytic and cytotoxic activity. 173 HUSCJ14 377 Regulation of Assays for the regulation of transcription through the FAS promoter element are well-known in the transcription art and may be used or routinely modified to assess the ability of polypeptides of the invention through the FAS (including antibodies and agonists or antagonists of the invention) to activate the FAS promoter promoter element element in a reporter construct and to regulate transcription of FAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter is regulated by many transcription factors including SREBP. Insulin increases FAS gene transcription in livers of diabetic mice. This stimulation of transcription is also somewhat glucose dependent. Exemplary assays that may be used or routinely modified to test for FAS promoter element activity (in hepatocytes) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays, such as H4IIE cells, are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays include rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 173 HUSCJ14 377 Upregulation of CD71 FMAT. CD71 is the transferrin receptor. Transferrin is a major iron carrying protein that is CD71 and essential for cell proliferation. CD71 is expressed predominantly on cells that are actively activation of proliferating. Assays for immunomodulatory proteins expressed on activated T cells, B cells, and T cells most proliferating cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD71, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 174 HUVDJ48 378 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 174 HUVDJ48 378 Activation of Kinase assay. JNK kinase assays for signal transduction that regulate cell proliferation, activation, JNK Signaling or apoptosis are well known in the art and may be used or routinely modified to assess the ability Pathway in of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) immune cells to promote or inhibit cell proliferation, activation, and apoptosis. Exemplary assays for JNK kinase (such as activity that may be used or routinely modified to test JNK kinase-induced activity of polypeptides eosinophils). of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate signal transduction, cell proliferation, activation, or apoptosis in eosinophils include assays disclosed and/or cited in: Zhang JP, et al., “Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7 (2000); Hebestreit H, et al., “Disruption of fas receptor signaling by nitric oxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); J Allergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, et al., “In vivo resistance to corticosteroids in bronchial asthma is associated with enhanced phosyphorylation of JUN N-terminal kinase and failure of prednisolone to inhibit JUN N-terminal kinase phosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74 (1999); the contents of each of which are herein incorporated by reference in its entirety. 175 HBDAB91 379 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 176 HELGG84 380 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 177 HILCA24 381 Regulation of Assays for the regulation of viability and proliferation of cells in vitro are well-known in the art and viability and may be used or routinely modified to assess the ability of polypeptides of the invention (including proliferation of antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pancreatic beta pancreatic beta cells. For example, the Cell Titer-Glo luminescent cell viability assay measures the cells. number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. Exemplary assays that may be used or routinely modified to test regulation of viability and proliferation of pancreatic beta cells by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 177 HILCA24 381 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression in immune cells of multiple genes. Exemplary assays for transcription through the STAT6 response element that (such as T-cells). may be used or routinely modified to test STAT6 response element activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the HT2 cell line, which is an IL-2 dependent suspension culture of T cells that also respond to IL-4. 177 HILCA24 381 Activation of Assays for the activation of transcription through the Signal Transducers and Activators of transcription Transcription (STAT6) response element are well-known in the art and may be used or routinely through STAT6 modified to assess the ability of polypeptides of the invention (including antibodies and agonists or response element antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression in immune cells of multiple genes. Exemplary assays for transcription through the STAT6 response element that (such as T-cells). may be used or routinely modified to test STAT6 response element activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary T cells that may be used according to these assays include the SUPT cell line, which is a suspension culture of IL-2 and IL-4 responsive T cells. 178 HYABC84 382 Stimulation of Assays for measuring calcium flux are well-known in the art and may be used or routinely Calcium Flux in modified to assess the ability of polypeptides of the invention (including antibodies and agonists or pancreatic beta antagonists of the invention) to mobilize calcium. For example, the FLPR assay may be used to cells. measure influx of calcium. Cells normally have very low concentrations of cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 179 HMCAZ04 383 Activation of Kinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERK signal transduction that Adipocyte ERK regulate cell proliferation or differentiation are well known in the art and may be used or routinely Signaling modified to assess the ability of polypeptides of the invention (including antibodies and agonists or Pathway antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 180 HE8FD92 384 Stimulation of Assays for measuring secretion of insulin are well-known in the art and may be used or routinely insulin secretion modified to assess the ability of polypeptides of the invention (including antibodies and agonists or from pancreatic antagonists of the invention) to stimulate insulin secretion. For example, insulin secretion is beta cells. measured by FMAT using anti-rat insulin antibodies. Insulin secretion from pancreatic beta cells is upregulated by glucose and also by certain proteins/peptides, and disregulation is a key component in diabetes. Exemplary assays that may be used or routinely modified to test for stimulation of insulin secretion (from pancreatic cells) by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contents of each of which is herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include rat INS-1 cells. INS-1 cells are a semi-adherent cell line established from cells isolated from an X-ray induced rat transplantable insulinoma. These cells retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167.

Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases. The first column provides a unique clone identifier, “Clone ID NO:”, corresponding to a cDNA clone disclosed in Table 1A and/or Table 1B. The second column provides the unique contig identifier, “Contig ID:” which allows correlation with the information in Table 1B. The third column provides the sequence identifier, “SEQ ID NO:”, for the contig polynucleotide sequences. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five. Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”), as described below.
The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1B, column 3 (e.g., SEQ ID NO:X or the ‘Query’ sequence) was used to search against the NR database. The computer program BLASTX was used to compare a 6-frame translation of the Query sequence to the NR database (for information about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403410 (1990), and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring ‘Subject’) is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring ‘Subject’ is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of Query amino acids in the HSP and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
The PFAM database, PFAM version 2.1, (Sonnhammer, Nucl. Acids Res., 26:320-322, 1998))consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM, that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin, et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1B) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6. Column 7 provides the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
As mentioned, columns 8 and 9 in Table 2, “NT From” and “NT To”, delineate the polynucleotides of “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in ATCC Deposit No:Z. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A and/or 1B.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA ATCC Deposit No:Z (e.g., as set forth in columns 2 and 3 of Table 1A and/or as set forth, for example, in Table 1B, 6, and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
TABLE 2 SEQ ID PFam/NR Score/ cDNA Clone Contig NO: Analysis Accession Percent NT NT ID: ID X Method PFam/NR Description Number Identity From To H6EDM64 841331 11 WUblastx.64 (Q9UID3) ANG2. Q9UID3 90% 928 2451 36% 203 310 36% 931 1038 95% 191 871 HACBJ56 847112 14 WUblastx.64 (Q9D2Q2) 2310079F23RIK PROTEIN. Q9D2Q2 65% 98 286 HADMB15 847116 15 WUblastx.64 (Q9BVH1) SIMILAR TO DLXIN-1. Q9BVH1 100% 8 109 HAJBV67 866415 18 WUblastx.64 (Q9HD45) TRANSMEMBRANE 9 T9S3_HUMAN 100% 13 126 SUPERFAMILY PROTEIN MEMBER 3 PRECU 93% 116 1681 HAOAG15 852204 20 HMMER PFAM: von Willebrand factor type A domain PF00092 180.1 506 1057 2.1.1 WUblastx.64 (O75578) INTEGRIN ALPHA-10 PRECURSOR. ITAG_HUMAN 90% 8 3463 HATCI03 580805 22 WUblastx.64 (Q9H743) CDNA: FLJ21394 FIS, CLONE Q9H743 71% 906 688 COL03536. HBAGD86 838799 23 WUblastx.64 (Q14287) HYPOTHETICAL PROTEIN Q14287 37% 801 559 (FRAGMENT). HBHAA05 603174 24 WUblastx.64 (Q9H387) PR02550. Q9H387 71% 676 386 HBHAA81 846465 25 WUblastx.64 (Q9D1G3) 1110011D13RJK PROTEIN. Q9D1G3 89% 1329 1502 79% 28 1329 HBJAC40 841235 27 WUblastx.64 (Q9P112) CHROMOSOME 16 OPEN READING Q9P112 100% 8 73 FRAME 5. 36% 5 70 57% 11 52 53% 85 180 100% 192 632 HBJCR46 815649 28 HMMER PFAM: WD domain, G-beta repeat PF00400 36.6 790 867 2.1.1 WUblastx.64 (Q9DC22) 1200006M05RIK PROTEIN. Q9DC22 96% 207 611 73% 568 2763 HBJEL16 847030 30 WUblastx.64 (095297) PROTEIN ZERO RELATED 095297 98% 285 491 PROTEIN. HBJIG20 866159 31 HMMER PFAM: Cytochrome c oxidase subunit III PF00510 162.6 321 551 2.1.1 WUblastx.64 (BAA77671) Cytochrome c oxidase subunit 3 BAA77671 81% 9 617 (Fragment HBJKD16 853358 32 WUblastx.64 (Q9NXS4) CDNA FLJ20080 FIS, CLONE Q9NXS4 91% 8 1528 COLO3184. HBMTY48 637521 33 WUblastx.64 (Q9H5N9) CDNA: FLJ23235 FIS, CLONE Q9H5N9 94% 54 941 CAS04980. HBMUH74 866160 34 WUblastx.64 (Q9NVW8) CDNA FLJ10462 FIS, CLONE Q9NVW8 100% 11 427 NT2RP1001494, WEAKLY SIMILAR TO MAL HBQAB79 810542 35 WUblastx.64 (Q9UQ32) AD 3 (FRAGMENT). Q9UQ32 82% 323 204 HBXCX15 637542 36 WUblastx.64 (Q9GMX5) HYPOTHETICAL 12.9 KDA Q9GMX5 41% 726 827 PROTEIN. 52% 578 730 HCDCY76 837972 37 WUblastx.64 frizzled protein 4 - human pir|JC7127|JC7127 100% 1039 527 30% 994 785 79% 567 37 HCEDR26 771144 38 WUblastx.64 (Q9H919) CDNA FLJ13078 FIS, CLONE Q9H919 66% 1157 1095 NT2RP3002002. 66% 1345 1184 HCEEU18 688041 39 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083 49% 186 10 56% 1223 933 HCFLN88 610000 41 WUblastx.64 (Q9BQE9) SIMILAR TO B-CELL Q9BQE9 87% 278 475 CLL/LYMPHOMA 7B (UNKNOWN) (PROTEIN FOR MGC HCHAB84 834326 42 WUblastx.64 (Q9BRV3) STROMAL CELL PROTEIN. Q9BRV3 89% 82 744 HCNSD29 862314 45 WUblastx.64 (O75400) HUNTINGTIN-INTERACTING Q75400 82% 628 1605 PROTEIN HYPA/FBP11 (FRAGMENT). 78% 337 489 HDPDJ58 587265 51 WUblastx.64 hypothetical protein DKFZp434D2328.1 - human pir|T42691|T42691 100% 307 609 (fragment) 87% 621 785 HDPFU43 790189 52 WUblastx.64 (AAH01057) Tyrosylprotein sulfotransferase 2. AAH01057 100% 360 1349 58% 220 348 HDPIU94 813352 54 WUblastx.64 (Q9BVF7) SIMILAR TO HYPOTHETICAL Q9BVF7 99% 63 1703 PROTEIN FLJ10422. HDPIY31 886159 55 WUblastx.64 hypothetical protein DKFZp434N1429.1 - human pir|T46448|T46448 72% 1714 1899 (fragment) HDPOC24 777493 56 WUblastx.64 (Q9H8K1) CDNA FLJ13518 FIS, CLONE Q9H8K1 100% 62 208 PLACE1005799. HDPPQ30 684292 58 WUblastx.64 (Q9H387) PR02550. Q9H387 51% 807 727 79% 1042 815 HDQHM36 852328 59 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083 69% 1129 1257 50% 965 1153 HE2HC60 753265 62 WUblastx.64 (Q9NVC4) CDNA FLJ10814 FIS, CLONE Q9NVC4 88% 125 1300 NT2RP4000984. HE6FU11 827236 64 HMMER PFAM: von Willebrand factor type A domain PF00092 184.7 244 771 2.1.1 WUblastx.64 (095460) MATRILIN-4 PRECURSOR. MTN4_HUMAN 77% 145 789 45% 782 907 41% 791 925 50% 794 907 38% 863 1498 33% 190 741 98% 782 1642 HE8TY46 899528 66 WUblastx.64 (BAB55144) CDNA FLJ14576 fis, clone BAB55144 95% 318 938 NT2RM4001092, w HE9EA10 827796 67 WUblastx.64 laminin alpha-1 chain precursor - human pir|S14458|S14458 99% 761 1891 27% 878 1840 25% 1142 1876 HEBFR46 847064 69 WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS, CLONE Q9NX85 80% 1111 1022 KAIA0536 84% 1265 1110 HEBGE07 798096 70 WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS, CLONE Q9NX85 79% 1851 1720 KAIA0536. HETCI16 844543 72 WUblastx.64 (Q9P0V3) BOG25. Q90V3 99% 3 356 HFCFE20 701985 74 WUblastx.64 (Q9CSE5) EUKARYOTIC TRANSLATION Q9CSE5 89% 438 581 INITIATION FACTOR 3 (FRAGMENT). 54% 1083 1187 HFPDS07 821646 77 WUblastx.64 (O94925) GLUTAMINASE, KIDNEY ISOFORM, GLSK_HUMAN 78% 343 513 MITOCHONDRIAL PRECURS 74% 2 436 HFVHW43 570948 78 WUblastx.64 (Q9BGX4) HYPOTHETICAL 13.8 KDA Q9BGX4 69% 1209 1093 PROTEIN. HGBHP91 693011 81 WUblastx.64 hypothetical protein (L1H 3′region) - human pir|B34087|B34087 52% 541 491 44% 537 34 HHEAK45 765278 82 WUblastx.64 (Q9NPB0) DJ202I21.1 (NOVEL PROTEIN) Q9NPB0 68% 1949 1458 (CDNA FLJ11101 FIS, CLONE PLACE10 HHEOW19 886174 83 WUblastx.64 (018973) RAB5 GDP/GTP EXCHANGE 018973 77% 417 623 FACTOR, RABEX5. 91% 611 715 56% 166 378 92% 129 167 HHFFS40 824059 84 WUblastx.64 (Q9H4A6) GOLGI PROTEIN. Q9H4A6 100% 3 251 HHSBI65 801910 88 WUblastx.64 (Q9H5W9) CDNA:FLJ22888 FIS, CLONE Q9H5W9 100% 270 407 KAT03934. 94% 479 1300 HISAT67 843549 90 WUblastx.64 (Q9UH94) PROLACTIN REGULATORY Q9UH94 88% 219 797 ELEMENT-BINDING PROTEIN (PROLACTIN 91% 788 1447 REGU HJBCU75 638329 91 WUblastx.64 (O45030) STRABISMUS. 045030 44% 199 426 52% 464 964 HJMAA03 824062 92 WUblastx.64 (Q9N032) UNNAMED PROTEIN PRODUCT. Q9N032 71% 415 528 HKABU43 838573 93 WUblastx.64 (AAH03633) Translocase of outer mitochondrial AAH03633 100% 33 62 membr 92% 26 1597 HKACI79 853361 94 WUblastx.64 (Q9BGV8) HYPOTHETICAL 10.0 KDA Q9BGV8 72% 886 1104 PROTEIN. HLDQU79 740755 97 WUblastx.64 (O75477) KE04P. 075477 100% 105 1142 HLDQU79 837599 191 blastx.2 KE04P. sp|O75477|O75477 99% 81 1118 HLHAP05 638476 98 WUblastx.64 (Q9HA67) CDNA FLJ12155 FIS, CLONE Q9HA67 55% 1553 1500 MAMMA1000472. 72% 1650 1585 77% 1807 1646 HLICE88 840321 100 WUblastx.64 fibrinogen gamma-A chain precursor [validated] - pir|A90470|FGHUG 89% 3 584 human HLMGP50 647603 101 WUblastx.64 (Q9GMI7) HYPOTHETICAL 9.0 KDA Q9GMI7 61% 765 709 PROTEIN. 72% 935 807 HLQCX36 584786 103 WUblastx.64 (Q9UI59) PRO0478 PROTEIN. Q9UI59 87% 1100 1216 HLWDB73 838453 105 WUblastx.64 (Q9H7D7) CDNA: FLJ21016 FIS, CLONE Q9H7D7 100% 660 872 CAE05735. 98% 1 657 HLYGB19 838083 107 WUblastx.64 (Q9H0Q1) HYPOTHETICAL 12.3 KDA Q9H0Q1 97% 204 518 PROTEIN. HLYGY91 658703 108 WUblastx.64 (Q9H8N0) CDNA FLJ13386 FIS, CLONE Q9H8N0 94% 221 391 PLACE1001104, WEAKLY SIMILAR TO MYO HMDAB29 584789 109 WUblastx.64 (Q9NX17) CDNA FLJ20489 FIS, CLONE Q9NX17 72% 1186 890 KAT08285. HMEDI90 840077 111 WUblastx.64 (Q9HBA3) RAB3 INTERACTING PROTEIN Q9HBA3 100% 81 794 VARIANT 4 (FRAGMENT). HMICP65 847403 113 WUblastx.64 (Q9HAU9) GUANINE NUCLEOTIDE BINDING Q9HAU9 99% 8 892 PROTEIN BETA SUBUNIT 5L. 22% 269 943 HMSHC86 840402 114 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083 70% 1724 1674 67% 1674 1420 HMUAN45 833072 115 WUblastx.64 (BAB55441) CDNA FLJ14993 fis, clone BAB55441 70% 684 1238 Y79AA1001874, w 65% 239 955 100% 1247 1516 HMVBC31 825598 116 WUblastx.64 (O60725) PROTEIN-S ISOPRENYLCYSTEINE ICMT_HUMAN 80% 747 938 O-METHYLTRANSFERASE (E 87% 121 789 HMWBL03 822861 117 WUblastx.64 (Q9BWT1) C-MYC TARGET JP1. Q9BWT1 85% 137 1240 HNGAK51 603910 119 WUblastx.64 (O60448) NEURONAL THREAD PROTEIN O60448 61% 563 601 AD7C-NTP. 67% 733 915 65% 702 878 74% 714 914 HNGGP65 597449 121 WUblastx.64 (Q9GMU5) HYPOTHETICAL 14.1 KDA Q9GMU5 31% 69 302 PROTEIN. 47% 398 541 HNHFE71 834487 126 WUblastx.64 hypothetical protein DKFZp761L0812.1 - human pir|T47135|T47135 67% 822 583 fragment) HOACG07 792928 128 WUblastx.64 (Q9GZN8) DJ1009E24.3 (A NOVEL PROTEIN) Q9GZN8 99% 183 704 (CDNA FLJ14158 FIS, CLONE NT2R HOEBK60 789396 130 WUblastx.64 (Q9H916) CDNA FL113081 FIS, CLONE Q9H916 98% 132 1916 NT2RP3002033. 100% 14 109 88% 106 159 HOFAA78 836646 131 WUblastx.64 (Q9NXS2) CDNA FLJ20084 FIS, CLONE Q9NXS2 90% 529 792 COL03526. 50% 9 80 88% 29 529 HOFNB74 762821 132 WUblastx.64 (Q99JH1) HYPOTHETICAL 17.7 KDA Q99JH1 72% 44 187 PROTEIN. 97% 199 471 HOSDO75 862049 134 WUblastx.64 (Q9D099) 1110057LI8RIK PROTEIN. Q9D099 89% 11 202 88% 259 630 HOUDR07 745404 135 WUblastx.64 (Q9HBV4) ANGIOPOIETIN-LIKE PROTEIN 9HBV4 87% 170 1384 PP1158. HPFCI36 855966 137 WUblastx.64 (Q9NX47) CDNA FLJ20445 FIS, CLONE Q9NX47 100% 9 320 KAT05170. HPRBH85 695752 141 WUblastx.64 (BAB55300) CDNA FLJ14784 fis, clone BAB55300 62% 2 616 NT2RP4000713. 86% 534 1085 HPRCA64 824074 142 WUblastx.64 (P55161) NCK-ASSOCIATED PROTEIN 1 (NAP1) NCP1_RAT 100% 1021 1926 (P12SNAP1) (MEMBR 85% 387 1019 93% 11 481 HPRCD35 853551 143 WUblastx.64 hypothetical protein DKFZp762L1710.1 - human pir|T50629|T50629 100% 320 613 (fragment) 57% 2 499 HPTRM02 812879 144 WUblastx.64 (Q9UJU6) SRC HOMOLOGY 3 DOMAIN- Q9UJU6 92% 332 940 CONTAINING PROTEIN HIP-55 (DREBRIN F). 97% 2 106 96% 98 190 HRAAD30 866187 145 WUblastx.64 (Q9H6V0) CDNA: FLJ21839 FIS, CLONE Q9H6V0 89% 23 1393 HEP01794. HRADF49 866481 146 WUblastx.64 (Q9H6L1) CDNA: FLJ22169 FIS, CLONE Q9H6L1 90% 13 825 HRC00632. 84% 813 1379 75% 1291 1593 34% 1590 1685 HRDDQ39 840405 147 WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS, CLONE Q9NX85 53% 582 436 KAIA0536. 65% 775 578 HRDEX93 816046 148 WUblastx.64 (Q9UBV8) PEFLIN. Q9UBV8 100% 313 864 HRTAP63 780698 150 WUblastx.64 (Q9Y3C9) CGI-127 PROTEIN. Q9Y3C9 100% 498 860 HSLHX15 777861 153 WUblastx.64 catalase (EC 1.11.1.6) - Campylobacter jejuni pir|I40767|I40767 86% 162 76 HSNBM34 635131 154 WUblastx.64 acyl-CoA dehydrogenase (EC 1.3.99.-) very-long- pir|S54183|S54183 84% 1548 1979 chain specific - human 100% 251 1546 HSSEF77 658725 155 WUblastx.64 (O95637) WW DOMAIN BINDING PROTEIN-1. O95637 42% 10 246 83% 296 829 HT5GR59 801930 158 WUblastx.64 (O60496) DOCKING PROTEIN. O60496 72% 70 1284 HTAEI78 637684 159 WUblastx.64 (Q9UKQ2) ADAM 28 PRECURSOR (EC 3.4.24.-) AD28_HUMAN 90% 85 174 (A DISINTEGRIN AND HTEAG62 812332 160 WUblastx.64 (Q9Y5Z7) HOST CELL FACTOR 2. Q9Y5Z7 60% 1 57 93% 14 2011 30% 107 631 HTEDS12 838621 162 WUblastx.64 (Q9H0K0) HYPOTHETICAL 81.8 KDA Q9H0K0 97% 1029 1391 PROTEIN. 42% 1269 1490 100% 16 1011 HTEHA56 806461 163 WUblastx.64 (Q9H9A0) CDNA FLJ12895 FIS, CLONE Q9H9A0 94% 2 217 NT2RP2004187, WEAKLY SIMILAR TO ZIN 65% 70 468 HTEJD29 695798 164 WUblastx.64 (Q60713) REVERSE TRANSCRIPTASE. Q60713 42% 1115 1285 47% 874 1089 HTENR63 877952 165 WUblastx.64 (Q9HD71) HYPOTHETICAL NUCLEAR Q9HD71 33% 1278 1358 FACTOR SBBI22. 78% 26 1168 HTGGM44 842856 166 WUblastx.64 probable phosphodiesterase I (EC 3.1.4.1) - human pir|T43461|T43461 100% 1400 1924 (fragment) 83% 1925 2488 HTLBT80 840045 168 WUblastx.64 (Q9NQQ7) BA39402.1 (CGI-15 PROTEIN). Q9NQQ7 76% 1214 1405 74% 804 1223 47% 780 845 78% 313 825 HTLFA13 535937 170 WUblastx.64 (Q9UHT1) PRO1902 PROTEIN. Q9UHT1 57% 1118 873 HTLGI89 835069 171 WUblastx.64 (Q9BXS5) CLATHRIN-ASSOCIATED PROTEIN Q9BXS5 98% 104 682 AP47. 99% 675 1370 HTNBKL3 831967 172 WUblastx.64 (Q9Y3M2) HYPOTHETICAL 14.5 KDA Q9Y3M2 81% 123 500 PROTEIN. HTOAM11 664508 173 WUblastx.64 (Q9H5R3) CDNA: FLJ23147 FIS, CLONE Q9H5R3 77% 428 363 LNG09295. 75% 586 425 HTOHO21 732808 174 WUblastx.64 P47 LBC oncogene - human pir|I38434|I38434 97% 581 438 HTPCO75 853645 175 WUblasLx.64 (O00549) ORF2-LIKE PROTEIN (FRAGMENT). O00549 43% 325 26 36% 1318 1253 HTSFJ32 637720 176 WUblastx.64 (Q9WUW2) VESICLE ASSOCIATED Q9WUW2 64% 747 788 MEMBRANE PROTEIN 2B. 94% 448 609 HTTCB60 853401 177 WUblastx.64 (Q9HAW0) RNA POLYMERASE III Q9HAW0 90% 6 881 TRANSCRIPTION INITIATION FACTOR BRFU. HTTEE41 840950 178 WUblastx.64 (P78371) T-COMPLEX PROTEIN 1, BETA TCPB_HUMAN 98% 92 1696 SUBUNIT (TCP-1-BETA) (CC HTWEH94 561680 179 WUblastx.64 (Q9GMX5) HYPOTHETICAL 12.9 KDA Q9GMX5 60% 1150 929 PROTEIN. HTXFA72 853410 180 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083 59% 1688 1557 66% 1839 1681 HTXKF95 834438 181 WUblastx.64 (AAH08360) Similar to hypothetical protein AAH08360 85% 233 553 FLJ22376 100% 2 112 HUSCJ14 894699 183 WUblastx.64 tex261 protein - mouse pir|S47481|S47481 99% 74 661 HUVDJ48 564853 184 WUblastx.64 SHORT ISOFORM OF Q9P2N4 sp_vs|Q9P2N4- 92% 1510 1668 01|Q9P2N4 HBDAB91 864374 185 WUblastx.64 (O00370) PUTATIVE P150. O00370 40% 907 833 35% 849 307 HBDAB91 789532 193 WUblastx.64 (O00370) PUTATIVE P150. O00370 40% 587 513 34% 529 5 HILCA24 869856 187 WUblastx.64 (Q9NUU6) CDNA FLJ11127 FIS, CLONE Q9NUU6 95% 104 1171 PLACE1006225. HILCA24 782450 195 WUblastx.64 (Q9NUU6) CDNA FLJ11127 FIS, CLONE Q9NUU6 73% 103 159 PLACE1006225. 100% 168 1169 HYABC84 865064 188 WUblastx.64 (Q9H429) DJ756N5.2 (A NOVEL PROTEIN Q9H429 92% 163 618 (DKFZP727M231) SIMILAR TO TRP4-AS HYABC84 789854 196 WUblastx.64 (Q99L03) SIMILAR TO TRP4-ASSOCIATED Q99L03 89% 209 553 PROTEIN TAP1 (FRAGMENT). HMCAZ04 668249 189 WUblastx.64 (Q9UQM8) CGI-44 PROTEIN. Q9UQM8 100% 9 1055 HMCAZ04 887445 197 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 107 1456 PROTEIN. HMCAZ04 867910 198 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 106 1455 PROTEIN. HMCAZ04 858210 199 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 106 1455 PROTEIN. HMCAZ04 839783 200 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 106 1455 PROTEIN. HE8FD92 901142 190 WUblastx.64 (Q9UJI9) HYPOTHETICAL 105.9 KDA Q9UJI9 76% 31 480 PROTEIN.

RACE Protocol For Recovery of Full-Length Genes

Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SalI and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.
Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antiqense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol For Generating The 5′ or 3′ End Sequences To Obtain Full Length Genes
Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′ RACE. While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant gene.
The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (e.g., as described in columns 2 and 3 of Table 1A, and/or as set forth in Table 1B, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 1A and Table 7. These deposits are referred to as “the deposits” herein. The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A and/or Table 1B (ATCC Deposit No:Z). A clone which is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables 1A and/or Table 1B or Table 2, by procedures hereinafter further described, and others apparent to those skilled in the art.
Also provided in Table 1A and Table 7 is the name of the vector which contains the cDNA clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene.
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59-(1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or the deposited clone (ATCC Deposit No:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in ATCC Deposit No:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA sequence contained in ATCC Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in ATCC Deposit No:Z.
Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in Table 1C column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, wherein sequentially delineated sequences in the table (i.e. corresponding to those exons located closest to each other) are directly contiguous in a 5′ to 3′ orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, Table 1B, or Table 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, Table 1B, or Table 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1C. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same Clone IQ (see Table 1C, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
Table 3: Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fifth column of Table 1A and/or the fourth column of Table 1B, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a +14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
TABLE 3 SEQ ID cDNA NO: Contig EST Disclaimer Clone ID X ID: Range of a Range of b Accession Numbers H6EDM64 11 841331 1-2596 15-2610 AL529288, AL514648, AL523579, AL523918, AL530571, AL528848, AL523917, AL523578, BE795355, BE614208, AL529287, BE797988, BE747962, BE798201, AL530750, BF689293, BE884814, BF508994, BE798313, BE613450, BE787266, AW131835, AL530749, BG248495, BE386285, BF526775, BE873469, BE299650, AL042569, BE621187, BG168950, AW410458, BE883794, BE869375, BF348689, AW239351, BE737181, BE734276, BF309636, BF129214, BG180549, AW410610, AW601905, BE621858, AA689552, BF310547, AW960649, BF953086, AL045821, BE882424, BF724804, BE019151, AW246108, BG179779, AW374338, AW675186, BE279317, BG011956, A1475847, A1394166, A1142042, AW068652, A1539419, A1970048, A1792316, AA536006, AW272491, BG012645, A1827847, BG254459, A1673493, AW007399, A1719374, AA994188, BG176564, A1707847, AW104963, A1220974, AA022523, BF807054, BG012634, BF803094, N24911, AW665019, A1458806, AA689495, AA480131, AI808412, N41812, W17347, BE772562, BG012642, BF807055, BE772573, BE011957, BE012641, AL514647, F22287, A1160580, A1149344, BE772556, AI870582, BE772568, AW801577, BG176616, AW801325, AW068651, A1197831, BE265961, AA483525, BE772566, BE772574, BE693737, AA687509, BE839398, BF799200, AA687451, A1201450, BF896481, BE772569, BE244158, BE772576, BE826728, A1452812, BE772561, AA317941, AA308425, AA745895, AW751437, BG256219, AA782657, BF373198, AA364848, AL039960, AA405870, AW963550, BE300303, N78953, AA112404, BE826586, A1061434, AI143698, AW087863, AI382254, AW731818, BE788591, AW304748, AI589259, AA357514, BF663656, AW673017, AW664622, AA524482, AW246627, BE831243, BE831271, BG055766, AI749023, AA380438, BF746714, BE839346, AW084279, AAI13160, BF529848, AI160508, BF764174, BF752908, AA053148, AW842671, F32117, AI190107, BF752929, BE547478, AA977756, AA360528, AA022454, BF808843, BF813892, AI917965, BG011699, BG012316, BF373193, BG122581, AA622680, BF688484, BE772558, AA053706, AA733114, BG012318, AW880294, AA482098, BE256450, BE831281, BF765811, BF803085, BE243388, AA774840, AA576098, BE831236, BE772816, AF024631,2, BC007198,1, BC009285.1, AF096303.1, U73627.1, AF061779.1, AC004923.2, AF238378.3, AC000385.1, H6EEU40 12 757048 1-937 15-951 AL534759, AL521087, AL523775, AL518427, AL518354, AL517326, BE741563, BF569745, BF337372, BF570471, BF969174, BG032740, AL536265, BF026597, BE274743, BE546314, AL522079, BE538554, AW960892, BE395781, BE465235, BG167967, BE275462, AI160737, BF804270, BE538514, AA653290, BG163271, AI341701, W94467, AW084148, AI634272, AI634641, AI266283, AI366893, AW409760, AW075307, BF223869, AA604286, AI262840, AA479733, AI985719, W94359, AI271832, AW439127, AI740653, AI500535, AI674680, AW245294, BF032823, BE350203, AI869835, R85540, BF437722, AI394604, AA825592, AW469385, R60570, AI620873, AW470050, AA788601, BE300983, R72347, N77923, AA939017, R72299, R87968, R85120, Z41206, T16501, AA482633, H40664, AA297447, AA081389, R85549, AA558602, AW175922, AA987713, AI628307, AI199953, B0056252, AI382799, AA968853, AAI48651, AI289139, AI281228, AA298765, F09249, W94940, AI282067, AI262509, AI200241, AV735503, AI921784, AI628244, T32046, AA302912, AI417848, AI468747, AW055372, AA857797, AA244103, AI581120, AA679586, AI634273, AA694158, AA946762, AA608791, AI124016, H46898, W46963, AA584396, AI797302, AL518355, AL523776, H27881, AL518428, H84434, N99004, AI540357, D31570, BF765616, AAI32303, AA814926, BE812370, AW404688, AL532367, AL534760, AA887999, AL530884, AL526404, BE937700, R46056, AL517327, AL536266, R40219, AW468110, H24286, AA522908, AI567331, H26975, T81176, AW369400, T80775, AI952287, AW797699, BE782422, AA872110, BE613072, AW269694, BE870596, AA490287, AI336931, BE937686, BE741460, BE937697, AI910098, AI583322, BE962616, BE932414, AL533205, AI905196, AL521088, AA479862, AK000120.1, AL096714.1, BC007519.1, HACAB68 13 584773 1-1286 15-1300 BF967733, BF340072, AW058572, BE877116, BF029667, BE221318, BE042897, BF434234, BE966145, BF593609, AW966641, BE549675, AI692588, BF433926, W68167, AW674743, W67708, BG163487, AI802057, AW051536, AW005086, BE073104, AU145008, AI634647, AI743810, N51396, BE218196, AI857811, AI816124, AI802067, AI095027, BE503637, BF669349, AI925492, BE669954, AI813855, AI811403, BG236435, AA833834, BE073105, AA748470, AW975666, BE502705, N56917, AI146547, AI949209, AI492350, AI190896, BE219670, AI167132, AW013890, AI089941, AI810922, AI804940, AI689151, BF699838, AW873589, AA209320, N62725, AI420094, AI221693, BF130415, AI301467, AA808217, AW5I1885, BE073003, AW166094, AA019916, AI359094, BE073109, AI753256, AW675323, BF671156, AA258518, AA954483, AA324329, BF668455, F13496, AA281446, BF247796, AA487161, BF029971, AA730575, AAI21642, AI123192, R49582, AI887042, AA487312, AA364288, AA385769, AW440846, R60975, AW451535, AA972339, AI091153, T74984, R36295, AW118180, R75731, BE003024, AI459209, BG055090, AW895451, 1180344, AI984894, AA581815, BF877111, AW805837, BE000523, D57701, T03076, AI767454, F10499, Z40296, R43580, AA081798, R49916, BE928534, Z43703, AA493265, AA526871, AU118452, AI144481, BE540542, AL442081.1, AL354793.11, AK001029.1, AF189009.1, AB015344.1, AF293385.1, HACBJ56 14 847112 1-874 15-888 AAI57001, BE348653, AW027639, AA534339, AW001883, AA363258, AW959379, T71037, AW953765, BE048583, BF878388, T67200, AW393348, AW393350, AW384705, AW386713, AAI56760, AW055343, BF892732, BE140594, HADMB15 15 847116 1-316 15-330 AW136268, BG056888, AI131328, AIl74443, AI091646, AW117296, AW168872, AI082447, AI432175, AI290911, AI741489, AI682685, AI142536, BG059892, AW149659, AW071935, AA233541, AI183690, BG056462, AI689641, AA599916, BF196591, BF196843, AAI99743, AW136277, N77910, AA564806, AA243035, AA779709, AV722133, AI032138, AA844525, AI467910, AW965361, AA852418, AI982751, AI282445, AI982761, T03902, AI420648, AW167499, H08108, BE328548, AW068986, C15651, D52660, AW665899, AI246702, AI538705, AI271662, AI435112, AI288692, BE466948, AI690048, D55112, AA779042, AL536118, D53747, D54101, AA486941, D53384, W07076, AA232504, AA486765, BF832290, AI038647, AW497637, BF947006, AU155428, T05461, AL136582.1, BC001207.1, AB040527.1, AB058762.1, AB040528.1, AB040529.1, HAGDW20 16 637489 1-1270 15-1284 AI671549, AA603387, AA614197, AC005629,2, AC006453.3, AC020898,5, AP001610.1, AC025435.5, HAGEQ79 17 828055 1-771 15-785 AI741487, AA779582, BE674646, AW303577, AI305251, BE219521, AI688718, AI936253, AI093754, AW341275, BE222507, AI692909, BF966664, AI493111, BF525487, AW016639, W92767, AU150022, AW341787, AI278427, BF966817, AA044775, AA910036, AI685015, AI285959, AA719683, BE645673, AW196910, AI432636, AI096735, BE618873, BES41159, H91757, AI702190, BG152855, AI244929, AI681847, BE217959, AI498036, BE467879, BE696146, AI810609, R55798, AA897359, BE464034, AA975324, F09971, W73069, AW594097, AA703815, BF224038, F10760, T72606, AA932659, H46138, AA351671, T31206, AA317283, AI867144, AI681277, AV718692, BE938093, AV718489, T31188, AI874073, AV724520, D80253, D80219, AW582752, D80240, D80210, AV719783, D80134, D59275, AV742001, D80391, D51423, AV720464, AV718844, AV720731, D80227, D51250, AV701332, AV699550, D59619, D80193, AV722801, AV699447, AV720220, AW973447, AV742667, AI306486, AV743601, AV745080, AV701443, AV744934, D80949, AV701017, AV742720, AI305252, AV741012, AV701043, AV701248, AV700229, AV743008, D80196, D59927, AV701428, AV719000, AV701004, AV745831, AV744773,D80043, AV701125, AV701154, AV719913, AV699927, AV701431, AV721784, AV700889, AV744771, D80366, M79008, D59787, AV744768, D81026, AV723927, AV701261, AV720203, AV745724, AV720812, AV740535, AV745723, C14227, AV720607, C14014, AV701012, AV701149, AV701118, AV737584, AV745366, AV701163, AV745369, BE702445, AV723097, AV70L166, BE702442, AV744770, AV701422, D50995, AV701344, AV699479, AV699669, AV719822, T11051, AV699866, AV701055, BF224150, AV701227, C75259, D80045, D80168, AV699746, AV746385, AV719324, AV701145, AV718707, AV745488, AV701021, AV701330, AV746335, AV701121, AV742671, AV700357, D80022, D80195, AV700622, AV720211, D58283, AV745392, D59889, C15076, D81030, AV701335, AV746162, D80188, D59467, AV745920, AW959570, D80038, AV701130, F13647, D51799, AV701419, AV718770, AV745847, AV701013, D80378, AV745388, T03269, AV721386, AV745490, AV718800, AV701415, AV718681, AW904616, AV700495, AW949642, AV701151, AV719468, AV738934, C14429, AW978634, AV745853, AV745197, AW949645, AV701231, AV701357, AW965158, AW975618, C14298, AW949633, AW949632, AW949641, D80212, AV699682, AV745621, AW949629, AA285331, AI557751, T11417, AW966531, AV720150, AV701224, AW949653, AW949658, D50979, AW949656, AW966062, D59502, AK022817.1, AL096677.21, AF271371.1, D34614.1, X67155.2, S78798.1, D88547.1, X92518.1, AF058696.1, AB028859.1, AB002449.1, AB035274.1, X98248.2, X60736.1, U79457.1, D50010.1, AB033111.1, AB038216.1, HAJBV67 18 866415 1-2522 15-2536 BG252656, BF732416, AV713753, BE905485, BF062374, BF445098, BF110352, BG252894, BE620095, BG249923, BE867752, AW606977, BG171028, AW576585, BE868698, BF671587, AW860769, BF941584, BF986308, AW305358, BF037687, BE541890, AW958924, AW974216, BF105260, AL048954, BF434917, AA057428, AW860733, BF664978, AI040432, BF984881, BF114918, BE872774, BE349491, AW263003, BF697715, BF382321, BE938703, AI378631, BF447674, AA446149, AA044378, BG114831, BF815345, BF085497, BF815237, BF210190, AA579908, BF132467, AA437015, AW860753, AI741531, AI742016, AI963805, AV748930, AA457625, BF815346, N31845, AI927889, BF699623, AA587067, AA831367, AI038411, AA442844, AI382172, BF084350, AW993684, AW407667, BF029928, AW028681, BE327066, BF887305, AV695738, BE222425, AV696527, BF755168, BE876090, BE167030, AI768063, BE000825, H12700, AV708152, AW001069, H03274, BF063098, BE933732, BF815719, BF594797, AW974217, N93209, N23944, AI290752, BF802746, AA557778, AA604449, Z32781, BE004621, AA910221, AA226865, R78864, BF326913, BG179582, AI370350, BE719765, BE768063, BE932712, AA780882, T31498, AW798498, AI635435, BF088211, BF817478, Z28655, BF943308, Z24930, BE932705, BF126152, AI015125, BF981166, AI684725, T36185, H12701, R37535, AW952059, AI689130, AA296931, AW798657, AW364905, R79351, H03275, BE768230, AW206046, AA081583, AA936681, BG104571, BE176285, AW993023, W69607, R31681, AI479514, BE696398, AA716370, BE463676, AW366456, BE869217, BF064127, BF001446, AW884802, AW999085, BG104993, AI039088, R31723, AW366514, BE871677, AI241206, AI743907, AA306185, BF037794, D61175, AA852523, AW365573, BF799275, AAI29989, BF985004, AW838470, BF802748, R36687, BE001097, AA723997, BE932064, AW366145, BF230069, AW999007, AW993306, BF733961, AA508532, AW972441, AW972636, BE932056, BE086739, AAI64808, BE064535, BF986296, BF095055, AW408116, BE184804, BE184805, BE184738, BE695142, BE184803, BE172976, BE184743, BF984676, BE184732, BF741954, AI366900, AW082623, AW118518, AI698391, BF871314, AI954504, BF753053, AI679312, BE967260, BF207979, AL515195, AW050850, AW089844, AW151136, AL515191, BE965599, AI619607, AI687568, AI540674, AI345688, AL513817, AI590043, BG032036, AA806028, AL043168, AA329665, AI923989, AI670002, AA641818, AI866770, AI679321, AI591420, AI473451, AI445165, AW051088, BF812961, AL514093, AI521560, AI633125, AL514871, AI915291, AW152182, AI247082, AI582932, AI889189, AI587121, BE875959, AA743012, BF814412, AW193894, AL515413, BE911554, AI918449, AF269150.1, AK027788.1, AK000756.1, AF116347,1, AK027438.1, AF160213.1, AF124819.1, BC009311.1, BC001967.1, AB048975.1, AL137478.1, BC002733.1, AB056421.1, AL133560.1, AK027129.1, U42766.1, AL080118.1, BC001969.1, AK026927.1, U38847.1, AB047878.1, AK025857.1, BC004264.1, BC004899.1, AL137529.1, AK000323.1, BC005858.1, M92439.1, AL122100.1, BC006458.1, BC001964.1, AL353956.1, AL137557.1, AF132676.1, AL133640.1, AF061836.1, AL137533.1, AK027164.1, A3406939.1, AL049430.1, AB056427.1, AK027173.1, BC007571.1, BC003122.1, AL136784.1, AF245044.1, BC001215,1, BC004324.1, AF252872.1, AL389935.1, AL136752.1, BC003410.1, AL137560.1, BC008037.1, AL137555.1, AK026649.1, AL136767.1, AK000206.1, BC003658.1, AK026057.1, BC008284.1, AL136786.1, AL110225.1, AL133623.1, AF078844.1, AF353396.1, AB050407,1, AL049938.1, BC007391,1, AF090903.1, AL136747.1, AL050138.1, AL137550.1, D83032,1, BC007053.1, AL512704.1, AK027113.1, AK024588.1, BC001774.1, AL137258.1, AL390184,1, AK000310.1, AL096744.1, BC006136.1, AB060914.1, Y16645.1, BC003619.1, BC008781,1, AL389939.1, AF028823.2, AL110196.1, AK025435.1, AB046642.1, AB050431.1, AK024944.1, AK000414.1, BC000054.1, AB052191.1, AL117435.1, AL110218.1, AK026534.1, BC008780,1, AL136622.1, AL049283.1, BC002697.1, AF069506.1, AF141289.1, BC004958.1, AB063079,1, BC003548.1, BC001056.1, AK025113.1, AJ010277.1, S76508.1, AK000160.1, U72621,3, AK026857,1, AK027096.1, BC003614.1, AL137271.1, AL137459.1, A8063088.1, S77771.1, AF036268,1, BC004556.1, AL157433.1, AK024622.1, AB049852.1, BC004292.1, AX027082.1, AK026749.1, AB063093.1, AB044547.1, BC008078.1, AL110224.1, BC009294.1, BC001082.1, AK027111,1, AL157482.1, AL122104.1, AB050410.1, BC000714,1, AIB063087.1, AL080140.1, AL442082.1, AL137488.1, AF056191.1, AK025465.1, AL122050.1, AL133606.1, AL136882.1, AL133559,1, AC008250.23, BC000725.1, AK027116.1, AK026547.1, AK027121.1, BC007456.1, AF232009.1, AB055352.1, AB056420.1, AL136644.1, BC006525.1, AK025312.1, AL133016.1, AL080074,1, AL512765.1, AL359620.1, BC001844.1, AY026527.1, AL050172.1, BC007499.1, AK026462.1, AL117635.1, AK027114.1, BC003104.1, AL050277.1, BC006091.1, BC008899.1, AB060879,1, AK026959,1, AK000083.1, AK027160,1, AK000618.1, BC007420.1, AL133568.1, AL050393,1, AF227198.1, AK000653.1, AL049347.1, AL162002.1, AL137480.1, AL162079.1, AB062942,1, AL390154.1, AB060897.1, AL110296.1, AL136893.1, AL080148.1, AL122121.1, AL133112,1, AK026593.1, AB049892.1, AL122110.1, AK026542.1, AF100781.1, BC005825.1, AK024992,1, AK026894.1, AL512684.1, X83544.1, BC004290.1, AK026541.1, AF183393.1, AL512746.1, AB051158.1, AF106697.1, AB063071.1, AK000421.1, BC003590.1, AK000257.1, AB060917,1, AF090900.1, BC007680.1, AK027188.1, AL023657.1, AL137292.1, AL133637.1, AL049324,1, S78453.1, BC008416.1, BC008836.1, AJ299431.1, AB047941.1, BC007460.1, AK026613.1, U55017.1, X67688.1, BC004336.1, AL390139.1, AL110221.1, AF262032.1, BC003602.1, BC002476,1, AL110222.1, AL137521.1, BC006181.1, AL137479.1, BC006807.1, HAJCH70 19 827275 1-2168 15-2182 AL159987.19, AL445236.22, AL031294.1, AP001101.2, AC021188.6, AC007677.3, AP001759.1, AC006038.2, AC079468.3, AC016643.6, AC009430.2, AL139824,22, AC007344.3, AL158069,16, AC005939.1, AL138822,13, AL353741.16, AC007225.2, AL009177.1, AC002538.1, AC015801.25; AF108083.1, AL031686.2, AC005486.2, HAOAG15 20 852204 1-5129 15-5143 BE349027, AA460958, AA460959, AI291926, AA461266, N58870, N72734, AI277327, AA461265, H43656, BF927468, H44722, BE835623, BE835624, Z33537, AI903814, BF366858, AW968357, AA465480, AA485068, AA430219, AF112345.1, AF074015.1, AF172723.1, HATCD80 21 826098 1-1795 15-1809 AW936395, AA382841, BF380111, HATCI03 22 580805 1-920 15-934 BF802634, AA744060, AW270385, BF804385, AI696854, AW963489, AA700943, AA583394, AI537407, AI453210, AW571963, AW976024, BF530611, AW468372, AA747757, AA225406, AW958962, BE169870, AW855803, AI554471, BF914416, AA687730, AA559219, AA586667, AW272294, AL137119,26, AJ007690.1, AC002369.1, AC002300.1, AC010494.4, AL445237.16, AC009137.6, Z95114,19, AL135927.14, AC007227.3, AC090950.1, AC005837.1, AC011242.8, AC004867.5, AL161731.20, AC009120.8, AC004821.3, AC011450.4, AL390196.17, AL445248.7, AC005015.2, AC008622,5, AC074 121,16, AC004166, 12, AL035587,5, AL049872.3, AC025594,5, AC074013,5, AP001760.1, AC005080.2, AP001711.1, AP002028.1, U07562.1, AL139330,17, AC020908.6, AL138724,12, AC007011.1, AL354696,11, AL450224.1, AL137802,7, Z97876,1, AL163279.2, AC005098.2, AC004859.2, AC004815.2, AL442167.1, AC005000.2, AC01 1455,6, AL132768,15, AC007991,7, AL355343,18, AC026464.6, AL049538,9, AC017101,10, AC009276,9, AC026432.3, AF168787 .1, AC005899.1, AP000208.1, AF0001 30.1, AC009244,24, AL 132639,4, AC002395.1, AL138787, 11, AF207550.1, AL139353.3, AL132780,5, AL049795,20, AC005067,2, AC053523,5, AC0052 15.1, AC0L 8758.2, AC069548,4, AC005378.2, Z9894 1.1, AC020552,4, AC083868.2, AC002426.1, AP003465.2, AC012379,7, AC007057.3, AC006435,7, AC06794 1,7, AL450226.1, AC07433 1.1, AL5 13366.11, AC007685.2, AL359680,4, Z82190.1, AC005972, 1, AF134726.1, AC083874.2, AC007488.15, AC003101.1, AC004832.3, AC004755.2, Z697 10,1, AC084865.2, AE006466.1, AC007383,4, AK023134.1, AC006088.1, AC0059 19.1, AP000424,3, AC006538.1, AC073834.6, AC011495.6, AL353748.13, AJ251973.1, AC007842.1, AL161937.13, AL163285.2, AC004098.1, AP000096.1, AL359092.14, AL122004.17, AC024239.7, AC068999.15, AJ009616.3, AL136170,12, AL049830.3, U73643.1, AC004836.2, AL356057,12, J00083.1, AF064861.1, AL121891.22, AC011737.10, Z82215.1, U91323.1, AC004531.1, AC067968.1, AL356805.5, Z85986.1, Z85987.13, AC007263,4, AC005412.6, AL121583,25, AP000240.1, AC0LL811.42, AC006388.3, Z82901.1, AL391241 ,21, AC002316.1, U80017.1, AL450344.4, AF0S1976.2, AC012476.8, AC006251.3, AF228703.1, AL049646, 19, AC005746.1, AC005933.1, AP003357.2, AC008403.6, AC00S102,1, AC002470.17, AP001718.1, AL137012.6, AC005516.1, AL135918.6, AC008155.9, AL109976.23, AC005529.7, AL163032.3, Z95152.1, AC008521.5, AC011479.6, AC006333.3, AC006285,11, AC006050.1, AC012507.9, AL158823.11, AC005775.1, AL133295.16, AL121655.1, AL117336.22, AL138702.8, AL109984.14, AL023583,25, AC007845.12, AL118525,17, AF243527.1, AC005874.3, AF134471.1, AC044797.5, AC007318.4, AL355802.13, AL1327 12.4, AP000503.1, AC008266.3, AL033528.19, U47924.1, AC010489.4, AP0G1609.1, AC005702.1, AC008745.6, AP000553.1, AC005231.2, AC000360.35, AC005736.1, AC020942.5, AC026416.4, AF318296.1, AC007066.4, AC004874.1, AC004158.1, AP000873.4, AL137128.4, Z97352.1, AC016257.22, AC006011.2, AC026672A4, AL139044.15, Z93015.9, AP000065.1, AC016816.4, AC008474.7, Z94056.1, HBAGD86 23 838799 1-1699 15-1713 AI65B681, BE466145, AI806836, AI653272, AA004211, BE302094, BF970406, BE018485, AA418617, AA594901, AI580148, BF589715, AI804211, AI669907, AI342168, AI810310, AA506350, AW022528, H10330, AA721162, AA452114, W03931, AW953290, AI262137, R61309, AA680147, N62384, H10331, AI264925, AA765972, BF086698, AW275301, AA485210, C15277, N79353, AA350799, AI867727, AI474438, AI129224, AA093047, D60782, AI535847, AA897480, AA350798,AV714899, AW956763, AV728867, HBHAA05 24 603174 1-676 15-690 AI572680, AW631267, BF970107, AA632355, AI433952, AI753969, AA629668, AA493546, AU158457, BF589864, AL044966, AW5 18882, AI570067, AI828721, BF028225, M77888, AI884404, AI547110, BF724416, AI434103, AV683406, AW836225, BE391183, N55076, AA610644, AV731938, AA313025, AA748071, AV743067, AJ065031, AW148964, AI280566, AI732690, AA601376, AI3 11796, AI268465, T03928, AU158814, AW504667, AW880986, AI819419, AA018258, AA524800, AW971342, AI791659, AW020612, AV759022, AV712092, AA935827, AA773560, AA425283, AI376687, AA493245, AA847341, BF942991, BF944618, AW303052, AI174703, BE392753, BG034698, BG223498, BE152006, AI683079, AA826166, AI590404, AI285651, AC004531.1, AL049780.4, AC006013.3, AC005971.5, AC005522.2, AL138713,11, AC011445.6, AC008403.6, AP001725.1, AC010458.5, AC009412.6, AP001726.1, AP001715.1, AL022323.7, AC008569.6, AL138976.5, AC005015.2, AC005081.3, AL022316.2, AC008738.6, AL590762.1, AL445222.9, AC004967.3, AC004991.1, AC020552.4, AP001716.1, AL109965,34, AC006211.1, AC011514.3, AC011485.6, AJ003147.1, AC018755.3, AC011510.7, AC006057.5, AC016995.4, AI353692.14, AC006334.3, Z97054.1, AC006449.19, AC005940.3, AC004383.1, AL034422.24, AC087071.2, AL133229.40, AL079342.17, AC004051.1, AL359397.3, AC018719.4, AP001712.1, AP001724.1, AC020716.3, AC073316.6, AC024561.4, AC010422.7, AC009488.5, AC020908.6, AC000353.27, AL513366.11, AC006487.8, AC020906.6, AC008066.4, AL354735.14, AC006530.4, U95742.1, AL162426.20, AC007731.14, AP000289.1, AL389925.10, AC005500.2, AC002527.1, AP000042.1, AP000L10.1, AF001549.1, AL135905.6, AC007637.9, AL161732.7, AC067941.7, Z98941.1, AC032011.14, AP000688.1, AC005377.2, AL050335.32, AC090937.1, AC007256.5, AF053356.1, AC079602.15, AC008623.4, AL1 63032.3, AC01 1490.7, AC006040.3, AC00482 1.3, AC007216.2, AC005881.3, AL133545.10, AC008891.7, AL109825.23, AL138756.23, AL355102.5, AC004703.1, AF312032.1, AL445664.14, AL354696.11, AC005529.7, AC005077.5, AL158207.15, AF168787.1, AC003982.1, AC013449.8, AC021019.5, AC005914.1, AC004032.7, AL158052.10, AL353752.6, AB003151.1, AC005052.2, AC005291.1, AC004867.5, AL023583.25, AC020931.5, AC008752.6, AL023693.25, AC004887.2, AL078639.5, AL031651.33, AC005098.2, AC008551.5, AL136304.10, AL023807.6, AL356756.4, AC005921.3, AC005874.3, AF134471.1, AP001714.1, AC004906.3, AP001748.1, AC004166.12, AC020744.4, AL031118.21, AL139109.14, AC011895.4, AC006312.8, AL049537.48, AC007374.6, AP001630.1, AC007383.4, AC010616.5, AL109797.18, AC006970.6, AC005399.19, AC01 8711.4, AL020997.1, AC0873 11.22, AC005632.2, AC005578.1, AC018462.4, AC004522.1, AF243527.1, AC010605.4, AP000563.1, AC026464.6, AP003476.2, AC006511.5, AC004796.2, AC004000.1, AL356113.8, AC0I1005.7, AP00L711.1, AC008857.5, AL138885.21, AC010319.7, AC073347.3, AC002472.6, U52112.1, AP000211.1, AP000133.1, AC009131.6, AC074121.16, AC004263.1, AL449223.7, AJ400877.1, AL161670.4, AC008812.7, AL445071.14, AL117336.22, AC007298.17, AL139343.9, AF207550.1, AL359092.14, AC004963.2, AL096840.25, AG012170.6, AC025593.5, AC022384.4, AL035089.21, AC008969.5, AL3555 12.22, AP001646.4, AC073542.4, AL359382.23, AC011495.6, AC005562.1, AC002565.1, AL034449.1, AL034549.19, AC008892.5, Z98200.8, AC005102.1, AC012450.9, AC006538.1, AL445483.13, AL354720.14, AC069285.8, AL034548.25, AC002432.1, AC008982.5, AC025166.7, AC005618.1, AL109804.41, AC012085.4, AC008481.7, AC020913.6, AC022432.4, AC005520.2, AC005627.2, AL021395, 16, AL1 17380.28, AC008543.7, AL158824.11, AL109935.39, AL161802.15, HBHAA81 25 846465 1-1633 15-1647 AL138080, AL138081, RG056111, AW117532, AI885223, BF724275, AW051808, AI332809, AI564820, N63569, AW207494, AI866785, AW148811, AI954565, AI199326, AI199105, N38888, AI243422, H09138, AI986175, AW381536, Z25110, AI991904, Z18300, F31192, Z28916, AW381528, Z41646, AI954371, N38887, AI033629, Z28784, AW381480, N94825, F25740, F00372, AW380848, F16581, AW381481, AAI97180, AW104762, BF887537, AB042554.1, AB032999.1, AC006059.3, HBLAA59 26 806303 1-2378 15-2392 BG261283, BG029024, AV723100, AV696216, BE931161, BF673030, AV722640, N31939, AI924550, W30681, BF853977, W93554, AV695121, AI693957, AI418000, AI679157, AI271461, AI143084, AI418010, AI186193, BF809099, BF378856, AI199165, AA809478, AI159998, AW250204, AA808416, AW952282, AA593302, AI034450, AI185975, BE677163, AI086931, AI004945, AAI76377, N76113, AA588747, AI095797, W68483, AW243597, AA595636, N68971, AA037099, W77975, W48846, AI079380, AI300827, W56274, AI679162, AI168786, AW105640, W92339, BE151400, AI142671, AA573184, W32053, W05603, W88988, W26295, AW263199, W56352, AW263182, AI346401, T68085, AW665393, N99254, M62046, AI284385, N42776, AA443625, BF476230, W46568, W58618, AW189940, AV685659, AA004754, AA779109, BE185831, AI880425, W46651, BF837568, AI081101, N42802, W89081, AI085087, W73911, BE832538, W68301, R67716, BF081619, W92217, T67937, W58619, BE185943, AI364156, BF911225, R70823, AW805885, AW089262, N31493, AV722639, H97191, AW140040, H03407, AA010449, AW265360, H60356, BF911333, AI253668, AW835116, BE042937, T12138, AI695815, AA004964, AA331001, T31211, N86748, AW304070, Z43338, AA971863, AA007267, R88101, H59084, R79508, H04108, AA315687, AI139849, AW241439, BE073482, AA338165, R66110, AW608546, AA778276, BE009544, AW241496, AI077770, AA887320, W31537, R79238, R70773, AW5 16576, W48613, AA338166, BE815610, AI890222, N76384, AA037713, N55230, AI609982, AW967118, AI824249, R45759, AW366951, BF854364, AV742667, AK025783.1, AL121929.17, HBJAC40 27 841235 1-1753 15-1767 BF345048, AW958511, BF530417, BF975837, BE253816, BG250686, BE887472, BF527935, BE263703, BE909332, BF527878, BE881895, BF194837, BF972454, AW025541, AL048612, BF525861, AI859062, BF835934, AI199762, BF955165, AI052782, H12219, BF955173, AW015231, BE379375, AI681619, BF364391, AI379848, AI363268, BF740272, AA703554, R60286, BF955168, R52740, BE258881, AA448130, R90880, W46453, BF090044, R25794, R22673, R52690, H08245, H08001, AA447988, AA912056, AI245669, R36729, AI290546, H07130, M78974, BE179040, T77037, AA070715, H08144, R58852, H38448, Z44633, H49 103, W46520, R09542, R85342, AI879032, AI590525, R43378, R90851, T31004, R67310, F13258, BE164925, AA364956, F08103, T32340, BE907755, Z40495, C03430, BF884758, R85671, T35911, T30880, F28188, BF115735, AW161049, AW072139, BF820428, R60889, R60792, AA694126, AI086328, BF961135, AI272235, BE258962, AI363035, F04345, R46793, AA095937, BF952087, F10861, H06622, BF841177, BF925698, AA401578, BF820431, BF945162, BF772214, AA095307, R54673, BE677392, AW027215, AI360330, AF072812, T16940, AL035745, R09655, BF945153, BE081049, BE179106, AA382430, W23297, AW162615, AA946598, AI017807, AI220189, AW002147, R85343, H41244, BF527814, AI863690, AA394215, AW961330, AV652536, AV652547, AW963011, AV654689, AW954779, AW958365, AW954506, AV708498, AW957970, AW950179, AW963847, AV709494, AV725063, AV706850, AV725970, AF131218.1, BC002882.1, AL136698.1, AF195661.1, BC007604.1, HBJCR46 28 815649 1-3194 15-3208 BF980168, BF001800, BF221545, BE180558, BG117357, BG165887, BE747286, BE867206, BE559905, BF029089, BE884542, BE180560, BE882087, BF672818, BE180608, BG255311, BF695020, AI765879, AV70L340, AI832097, AV701354, BG164080, BE568492, BF979546, BE268392, BE180559, AI927915, AI675415, BF195785, BF662916, BE673547, AI086866, AI956035, BF671543, AU146956, AA479515, BF063974, W19888, AW992096, BE504075, AW069858, AW992159, AA626631, BE019647, BE221636, AA479513, BF575729, AU144777, AA973047, AA936602, BF671187, BE545703, AV701112, AU144811, AU160319, AI472144, AI263407, BE019684, 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AC020663.1, AC018719.4, AC027319.5, AL118505.17, AC002039.1, HCEGX05 40 827060 1-1291 15-1305 BG115350, AU119350, BE395306, AU149428, AA460507, AA459863, BE044594, AA779792, BE301437, AW579480, AI369811, AU145714, AI143575, AW579479, AI087363, AA888608, AA455331, AA454932, AA975032, AI192991, AI187247, AA457365, AI332380, AW513149, T66101, AA642446, AI423532, T78802, AI357822, AA278914, AI634331, W22924, AA654344, W46308, AA741577, AA635904, AA278754, T90783, AI982596, AI129014, AI961148, AW449931, AW374368, AA741079, AW374371, AI679780, AI751335, AU145338, AA731279, F09505, AA721439, F11860, AA349664, AW374417, AAI32247, T51224, R48887, H22197, W46258, T51338, AW806601, AA832413, AW450706, BE328051, T78418, BG059569, D29092, BF932861, AA767603, AAI32246, AI537429, T98503, F20751, T83300, AW579478, D19816, AI653338, AA933679, AI917488, AW169041, AW338837, BE702499, AW179186, BE872997, AA457414, AW814638, F23623, D29117, BC000143.1, AL133227.15, AK023103.1, AB058737.1.AK022731.1.AK021718.1, AL354720.14, HCFLN88 41 610000 1-1420 15-1434 AL526786, BE622815, BE746913, BG167566, BE612603, BE613343, BE543099, AW328570, AI084727, AW511229, BE879597, AA643500, AI090381, BE876617, AW055002, AI744096, AA714840, AI148138, AA598703, AW340721, BE559510, AI125728, AI830384, AU151986, AI885716, BF432640, AI475597, AW339888, AI377299, AI309247, AW005497, AW771450, BE622365, AI139390, AI422379, N95665, AI144110, BF341713, AI919264, AI475648, AI189926, BF809564, AI559248, BE396630, AI817016, AI640708, AA825735, AV662025, BF526593, AI708507, AA935563, AW939178, AW939138, AW939190, AA961207, N66071, N69365, AA603786, AW939128, AI285062, AI826293, AW951255, BF512843, AA620317, AW605529, N80991, AI285338, AW381729, BF956527, AI031873, AW381749, B0056758, AW371517, AW381756, R78288, AI434319, AI282698, AI343784, AW002119, AW169595, N48713, AW371499, AI537993, N26561, R52366, AI916353, R93746, BE828699, AA835499, AW072300, W70306, AA907306, AW748173, N68294, AI468129, BG032430, H89920, AI080137, AA326474, H69097, AA826574, AW303330, AI858652, AI804227, AA322294, AI342977, AA291513, N35677, AI824463, T86711, H49562, Z19236, AI500205, AW966976, AI187820, R78289, AL525884, W76006, BE828722, N81088, F37932, R31642, AA905077, AA922535, AA877249, BF957167, AA780926, BG055657, AW087260, AI202070, AW129497, AA911554, AA045568, R32359, AA204681, AA057054, AA364763, R63145, AI277392, AI886719, BE616036, AW238934, AI082383, BG164867, BG150873, BE548567, B0035187, N91393, BF957863, H69096, R88359, AW328569, BF743483, BF742356, BC001967.1, BC000956.2, AC005089.2, X89985.1, AJ223979.1, HCHAB84 42 834326 1-1345 15-1359 X84712, BF526942, BF036429, BF035689, BF034330, BE878646, BG117306, BE906856, BE871642, AV703538, AW955111, BE729985, BE958344, BE271782, BF698225, BE568321, BG250080, BF826293, BE156569, AW579884, BF977502, AA587630, BE621946, BF512422, BF695706, AV764128, AA448786, AI032411, AA477231, BF695587, AA641139, BE621432, H02682, H02590, H29948, AW972521, AAI86733, BF909586, R22544, BE673152, AW405966, AI358557, R22543, BG169787, BF813006, BF742389, AW673871, AA327923, AW392393, AA311766, BF916201, BF742334, R70413, AV763358, AA505606, AA477230, AW794624, AW674083, AA595661, AA579044, AW265468, AA807704, AA642809, AW021674, AA618263, AA405570, BG180320, AA533066, AI702049, AI061313, AI254267, AA084439, AA491767, BG059139, AL042667, AL042670, AAI87682, AV763460, BF131490, AA313025, AL121039, AA557945, AW148821, BF901147, AW402784, AA693484, AV758870, AW410844, AW873417, AA601376, AL119909, AI251024, AI444575, BF725761, AW963489, BF857486, AI141202, AA776665, AW069110, AA601290, AW469462, AW270385, AI572680, AW028376, BF817511, AA809116, BF739035, AI064968, AA640310, AA535216, AI679759, AI753113, BF030482, AA600863, AI185160, AW192930, AL138262, BG118544, AW675677, AW023390, AW672927, R67038, AI445699, AI312267, AA659832, AV647070, BF804385, AA610644, AI821901, AV764383, AW105463, BF770715, R83577, AU157209, AI039257, AA602906, AA809546, AW820105, AA502991, AV762112, AI252611, AI567676, AI476049, AA015948, AF126023.1, AF126024.1, BC005943.1, U02057.1, AC002492.1, AL365364.19, AP001760.1, Z68276.1, AC008649.6, AC007842.1, AL157823.9, AL162587.20, AP00L711.1, AC021752.5, AC007405.6, AP000133.1, AP000211.1, AC090514.1, AC004922.2, AC013449.8, AL034451.26, AC008891.7, AC020659.5, AL139039.17, AFI68787.1, AC010463.6, AC003102.1, AL139353.3, AC006261.1, AC009756.9, AC008946.6, AP000563.1, AC002310.1, AC008068.4, U47924.1, Z69719.1, AL050349.27, AC003048.1, AL359983.7, AC009412.6, AL391839.9, AL031846.2, AL121655.1, Z9533 1.2, AL389875.1, AC005207.1, AC020716.3, AC009137.6, AP000252, 1, AC026172.3, AC006111.3, AF001692.1, AC007333.6, AC005821.1, AB038653.1, AL035684.25, AC005081.3, AC008745.6, AL136300.22, AC005902.7, AC073073.2, AL034418.5, AJ295844.1, AL139317.5, AC020904.6, AF139813.1, AC005519.3, AL445483.13, AP003357.2, AC087239.18, AL049869.6, AC020906.6, AC025280.4, L78810.1, AC008567.4, Z83844.5, AC008821.5, AC009220.10, AC073898.1, AC009311.3, AC004542.1, AC002306.1, AL031311.1, AC016683.7, AL158207.15, AC023105.7, AC006449.19, AL139100.9, AC016594.6, AL356805.5, AC008044.4, AL133517.11, AL133541.21, AC016763.8, AC002554.1, AL020993.1, AC008403.6, AC005355.1, AL121675.36, AL359695.6, AC005261.1, AL163032.3, AL513550.9, AP000511.1, AL589677.6, AC026464.6, AL096712.20, AC024561.4, AC011448.3, AL117348.25, AC010627.5, AC073657.5, AC005288.1, AC010271.6, AF001745.1, AL135783.6, AC090950.1, AF334404.1, AC007055.3, AC008771.4, L44140.1, AL031427.15, AC010319.7, AC007637.9, AL158158.14, AL035086.12, AL451125.7, AC018711.4, AC012085.4, AL135752.6, AC009247.12, Z84466.1, AF053356.1, AC008009.4, AC025165.27, AL133211.9, AL158830.17, AC002551.1, AL109984.14, AC002477.1, AL159168.15, AF205588.1, AC007383.4, AP000146.1, AC010491.3, AC006483.3, AE000658.1, AC004883.2, AC025264.16, AL133230.25, AC007390.3, AF042090.1, AL035455.30, AC005280.3, AC008897.7, AC069255.18, AC011495.6, AC005038.5, AC005914.1, AL136228.8, AP001709.1, AC022211.5, AL139113.21, Z97181.1, AL022323.7, AL137244.28, AE006462.1, Z98048.1, AL133453.3, AF111168.2, AC083884.6, AC010422.7, AP001630.1, AC010458.5, AL353716.18, AL109935.39, AL139316.5, Z94160.1, AC008857.5, AL157372.18, AP002007.4, AC005080.2, AC005412.6, AC008481.7, AC009123.6, AP000356.1, AL137787.11, AC004854.2, AL121886.22, AL133375.25, AC003684.1, AP002392.3, AL121594.6, AC027319.5, AC026185.3, AC005759.1, AC004263.1, AC020552.4, AC032011.14, AC004686.1, AL049636.22, AC007957.36, AJ243213.1, AL139343.9, AC091394.2, AL117381.32, AL163636.6, AC005527.3, AC008521.5, AP002427.3, AL354932.26, AC011891.3, AL138836.15, AC005529.7, AC004975.2, AL442166.1, AB023048.1, AC008555.5, AC011445.6, AC005781.1, AC080011.21, AL121712.27, AC020934.7, AC008372.6, AL031587.3, AL121601.13, AL133355.12, AC026445.4, Z84488.1, AL050347.1, AL161747.5, AP000744.4, AC083876.2, AL359236.4, AL133448.4, AL163284.2, AC008543.7, AC007255.4, AL096701.14, AC010203.13, AL157818.12, AL133312.3, AC005180.2, AC010620.4, AF038458.1, AC004659.1, AC007277.2, AC000353.27, HCM5X51 43 788643 1-2239 15-2253 AL520206, AL522291, AL520207, BG115714, B0023953, BF343959, AU133571.BE839880, AW954438, BE264316, BG261277, BE879757, AU131026, BE265959, BE278903, BF725639, AW246741, AA864833, AU148856, EFL11640, AA706935, AA431813, N38742, BE857705, BF476344, AU152863, AU125122, AA480041, AW170367, AI094797, AU154528, N48379, BF913004, N26479, AI803158, AL120744, N35219, AW245159, AI089912, AI927351, N20323, AL046695, AA476664, N35530, AI078494, AA015687, AI016568, BE857202, AI587317, AA446620, AW629254, AI433184, AA548282, W03412, N27597, W00855, AA825427, AI128747, AI082265, H38927, BF970202, R48359, AI569253, N29410, N44883, AW014479, AA934555, N41471, AW974179, R15948, AA573084, AA233832, AA017058, W16680, AI312737, R60804, N67483, R15949, Z43237, BE811896, N45053, BE832888, T11764, Z43099, AA431409, R48260, AI933045, AW874096, AW105691, AA336676, AA044969, BF362640, AW893387, AW892550, AW892516, R60299, N35043, T49574, 141630, AA013473, N35211, AA738419, AA223632, H86402, T49573, AA017209, BF941569, AA635071, AA054652, H86066, AI351292, F02575, N79527, Ti 1765, T35773, AW993110, AW194575, BE893541, AA448030, AI086309, BF737533, AF001690.1, AF029231.1, U96629.1, AB007042.1, AB01 1091.1, T66574, T66575, HCNC011 44 775086 1-732 15-746 BF926420, BF926408, BF875996, AV705104, AV726755, AW964429, AW950395, AV703435, AV707451, AV707628, AW961373, AV705453, AW964210, AW964423, AV704361, AW952896, AW961510, AV726887, AV729 165, AW963643, AV707705, AW963965, AV707556, AV702814, AW963219, AV704916, AV706906, AV703045, AW950229, AV690921, AV704674, AV728297, AV702810, AW960535, AI557262, AW963644, AV708024, AV701594, AV727806, AV727803, AW957298, AV650843, AW957682, AV704283, AV708829, AV701751, AW950078, AW950079, AW949946, AW961329, AW954386, AW954962, AW957974, AW952228, AV725024, AW960663, AW957083, AW950256, AV649266, AV704144, AV703160, AW963857, AW966775, AW958568, AW964298, AW958569, AW966684, AW951998, AV704342, AV703361, AV704848, AV703833, AV703425, AV705771, AV653846, AV728884, AW960406, AW954104, AW945153, AV650865, AV705189, AW958320, AW958316, AV729457, AV728929, AV729285, AV726743, AW949454, AW953619, AW955397, AW949863, AV701787, AW945196, AV708035, AV702901, AW961052, AV701953, AW963108, AV729170, AW958127, AV703284, AV706964, AV728355, AW963612, D50992, AV703367, AV706742, AV703862, AV706047, AV709139, AV652860, AW955394, AV702749, AV708590, AV707020, AW951793, AW951816, AW967188, AV705481, AV706133, AV702435, AV702958, AW955616, AW954194, AW962970, AV726728, AV705420, AV726770, AV695489, AV691061, AW963234, AW955139, AV649942, AW962367, AW964203, AW954242, AW954413, AV727101, AV707189, AW966146, AW951740, AW960600, AW952132, AV703632, AW961431, AW964278, AW958290, AW959722, AV659467, AV706802, AV705998, AV709332, AV705516, AV728913, AV728341, AV709281, AW958045, AW950597, AV707266, AW950411, AW960545, AV705267, AV704712, AV704401, AV702819, AV702458, AV702187, AW965827, AV727386, AW949731, AV707117, AV702298, AV701626, AV727268, AV703465, AW954783, AW953992, AW963581, AW958104, AW950254, AV705154, AW962980, AW957286, AW962378, AW958093, AW963811, AW954221, AV727618, AW963652, AV652536, AW962929, AV704033, AW950520, AW952361, AV701614, AV705518, AV728428, AV729392, AV702035, AV709623, AV702315, AV703266, AV707197, AW945183, AV727756, AV707238, AV706910, AW961377, AW945164, AW950681, AV647033, AV647066, AV647129, AW957628, AW952419, AV707649, AW957779, AV647144, AV702975, D59751, AV650924, AV693604, AW960143, AW963486, AV704876, AV705321, AV652547, AV650877, AW955698, AV707907, AW956167, AW963667, AV703264, AW963401, AV653784, AV727589, AW963641, AW955697, AW955632, AW955629, AV728741, AW961403, AW949521, U94592.1, Z30183.1, HCN5D29 45 862314 1-1714 15-1728 AU130793, AA902780, BG114197, AA675900, BE548792, BE796388, Z78308, BF973800, BF125408, BF382619, BF894864, AA902842, AW083941, BF243278, AW131275, AAI55995, AW771771, AA938206, BE251257, AI745367, AA448317, AW511804, AA448455, AI370549, BE139488, AW176079, AAI56223, H73833, BF940408, 1173162, AW084204, BF062122, AW028149, BF924722, BF433518, AI263130, AA411961, AW071942, BF694503, AA743704, AV764156, BF948901, AW082575, R11580, AA412712, BG153595, BG058948, BF893682, AU130757, BF667868, AF049523.1, BC000273.1, AF049528.1, AK024810.1, U70667.1, AF049524.1, AK023109.1, R34683, R34788, R63327, R63326, R63340, R63341, H15969, H27538, H27547, H27621, H82731, H83344, H83606, H83696, N20620, N32195, N33798, N36103, N36549, N41405, N41578, N44109, W19354, W25310, W38906, W60991, W73124, N89856, AA027859, AA027925, AA034908, AA034975, AAI33603, AAI33602, AAI72294, AA261835, AA262483, AA523928, AA551549, AA563835, AA857095, AA872771, AI095007, AI096629, C05812, C15709, AA247765, AA393650, AA400834, AA487693, AA488710, AA663750, Z21548, AA843596, AA844473, AI041193, AI083985, Z41640, Z46025, Z44537, F03607, D11797, AI262317, AI264408, AI304594, HCQBH72 46 637548 1-1782 15-1796 AA640538, AW974686, BE144592, AA649644, AA649707, R31618, AA652004, R32348, HCQCC96 47 845066 1-2152 15-2166 BP970581, BG117166, AV695085, AV686338, AI341460, AW173384, AV693976, BF032394, B0024316, BE893802, BG254562, AW055235, W39204, BG170478, AW978735, BF572731, AW968956, AI909118, AI909124, AW592429, BG171038, AW118938, AI689438, AI419443, AI801242, AW438695, AI123971, N59864, AA707755, AA974210, AW130020, AA489046, AA768780, AI146982, AW768627, AI093766, AW889585, AW298736, BF111650, AA284319, AA907244, AW874520, AI535676, AW579265, AI955386, AA279581, AA983814, BF185409, BE622718, N59886, AI859864, AI498376, BG171039, BG116650, W01363, AI699807, AA824487, T86598, AA994605, AW044013, T85108, AA489144, AW768600, AA811658, AW271482, BE895361, AI631722, AW021293, R64514, T77523, AA736753, H44608, AI955411, AV689519, N90263, AL119283, H94626, T77559, AL119309, T86597, AI909117, AW376940, N79005, N77027, AW105078, N62828, AI701272, AI334730, T07505, AW848643, AW243861, AI909110, BG007292, BE162291, BE162293, AA532611, AP001816.2, AL022153.1, AC004804.1, AC015982.9, AC006840.17. HCQCJ56 48 832157 1-1273 15-1287 AI674974, AI217307, AA813576, BE302346, AI824976, AA994749, AI244904, AI262935, AA020796, AA234517, BF432061, AA443035, AA463478, AW079079, AA694400, AI005463, AA776532, R00437, R00438, AL513043.7. HCUDD64 49 835082 1-388 15-402 BF109963, AI870761, AI149403, BE675981, AI979111, AI590348, AI769440, AA568609, F04371, R68556, N24429, R85927, AW973928, W02539, BG150863, R79201, N694 12, R79466, T80848, AI494453, R28549, AW440020, AL390151.1. HCWAE64 50 535893 1-457 15-471 AL043265, BE895962, BF091850, BF924502, BF930204, AW973724, BE906549, BF972009, AA558125, BG163769, AW993087, HDPDI58 51 587265 1-1983 15-1997 AW629326, AA642298, AW973571, AW293886, AA714702, AA489697, AW665911, AW119199, AA553561, AW768356, AA744636, AI806778, AA768815, AI221150, AA732540, AV713604, BE247593, T84374, AA354491, T07354, AW273936, AA648816, AI199572, AL133087.1, AC013264.4, AL117381.32, AC019105.7. HDPFU43 52 790189 1-1890 15-1904 BF983796, AV653552, BE259910, AI766649, AV703996, BF304737, BF433988, AA700236, BE178896, AI338643, AI354469, AI823774, AI379434, AI139748, AA934777, AI424295, AI280893, AI360455, AI539184, AI750403, BE184282, BF675256, AI262328, AV653641, AV687758, AV688883, AI538841, AV683306, AV686477, AV687759, AV688680, AA903947, AV689834, AW957949, AI079715, AAI28542, AI160482, AW804386, AA459614, BF770031, AW014830, BF848624, AW859834, AV685347, H94111, BF807109, AW470950, AA316165, AI400889, AA459389, BF924526, AI083688, AA374022, AW086191, R45973, W21315, R63000, BF798131, N93502, BF798175, AA304841, T39902, BF839163, C02619, AI373388, AV752433, AI750402, H94110, AI015368, AI916914, AA531491, AA215914, AW051088, BG180527, AW983783, AI470293, AV681824, AW023338, AW827289, AI929108, BF814357, AI440263, AA579232, AL040694, AI433590, AL042627, AW087445, BF871314, AW162194, AI537677, AI698391, AL037454, BF910810, AI923989, AV738730, BF904265, AV723064, AI345688, BF792445, AI921379, AV657079, AW020397, AV702021, BF814018, AI859991, BF184134, AA808175, AL043975, BE964614, AI446538, AW827118, AW150511, BE908107, BE965121, AV756990, AL514823, BE965758, BE906419, AV714036, AV682345, BE965621, AI623941, AI969655, BF750879, BG031068, BG036067, AI340519, AW452992, AW128931, BG113169, BE972047, BE538997, AL049085, BG260052, BE904851, AV717927, AL110306, AA635382, AW834221, AV706915, AI251221, AW022682, BG165323, BE965432, BE967307, BE965067, AW881086, AI340603, AI560099, AA427700, BE775251, BE965599, AI284509, AI863241, AA857847, AI866465, AI524608, AI538850, AA420722, AI918634, AL039011, BE439835, AW020048, AL080033, AI567351, AW089844, AA613907, AW163554, AI050666, T99953, BG256950, AW075084, BF885000, BG027280, BE878028, BE885490, AI349937, AI334884, AI307708, AL036187, AI963068, AI312325, BF925729, BF218049, AV761001, BG168696, BG179666, AV728833, AI671642, AI801325, AV729462, AI565172, AI307520, AV759235, BG104769, F29308, AA883351, BF966050, AI621341, BG105895, AL119836, AI269323, AI475371, AV718300, AV757781, AI564166, BE964700, AV742848, BE047833, BF835250, AW128855, AW151138, AV685436, AI950892, AI500662, AV681721, AI247193, BF812937, AL514493, BE839731, BF339322, AL120254, AI950664, BE909150, BF822127, AI345608, AI800370, BF983610, BE875407, AV757455, AV722452, BF970162, AA651819, BG260087, AI091468, AW935969, AI536685, BC001057.1, AF049891.1, AL136623.1, AI006198.1, AF061254.1, Z95115.1, AC007429.11, AL355379.5, BC007199.1, BC006412.1, AF143723.1, AL162004.1, AF217982.1, AK026480.1, BC002631.1, BC008488.1, AL133640.1, BC008025.1, AL050149.1, AL133093.2, AL110196.1, AB056809.1, AK026649.1, BC00I418.2, AL359622.1, AL136622.1, BC004905.1, AK024538.1, AL137555.1, AF227198.1, Z37987.1, BC003687.1, BC008387.1, AK024974.1, AL117435.1, BC007346.1, AK000718.1, AK026522.1, AF225424.1, AF125949.1, 578214.1, AL049938.1, AK026528.1, AL050024.1, BC001967.1, AL512733.1, AB056420.1, BC003682.1, AL137529.1, AK026592.1, AL049300.1, AK000618.1, AB048974.1, AC006313.1, AL050277.1, AB05L158.1, BC006480.1, AIB055374.1, AB047801.1, AB055315.1, AL050116.1, AF260566.1, U80742.1, AL389935.1, X72889.1, AB019565.1, AB048953.1, BC005931.1, AJ242859.1, AL133075.1, AK000310.1, AK025254.1, AL162085.1, AL080057.1, BC002481.1, BC006195.1, AL389939.1, AK026086.1, AL137557.1, AB063070.1, AL353957.1, AB063046.1, U55017.1, AF219137.1, AL512719.1, 577771.1, AK000197.1, AF057300.1, AF057299.1, AL137267.1, AL110199.1, AK026534.1, AB055361.1, AK025391.1, AL031984.13, BC000066.1, AL136787.1, Y16645.1, AL390154.1, AL117626.1, AB060908.1, AL162006.1, AL080234.1, BC006525.1, AL442072.1, AL512765.1, AL137527.1, AL122121.1, BC006807.1, X66417.1, Y10080.1, AK026550.1, AL049283.1, AL137478.1, BC008899.1, BC001328.1, AL136786.1, BC002485.1, AK025339.1, BC006103.1, AK026959.1, AL122111.1, AK025092.1, BC001963.1, BC004958.1, AL117649.1, BC000317.1, BC000714.1, BC007998.1, AL023657.1, AK025708.1, AL080156.1, AL110225.1, AK024546.1, AF055917.1, AF205073.1, AL080060.1, AL117648.1, AL137283.1, BC008284.1, AK026642.1, AB063008.1, AK026741.1, AK000212.1, AK027146.1, BC007517.1, AK027160.1, AB055368.1, AL136893.1, AF061795.1, AL117457.1, AF151685.1, AL157431.1, AL133606.1, AL389982.1, AK027868.1, BC002733.1, BC001795.1, AF111112.1, AK026865.1, AK024524.1, AF026030.1, BC008781.1, AB060826.1, AK026353.1, AB060893.1, AL442083.1, AK025414.1, AK027164.1, AB048975.1, AL117460.1, AK025435.1, AB060852.1, AL136768.1, AL050143.1, AB063100.1, AL133113.1, AL136892.1, AL080129.1, AL137480.1, AL080124.1, BC001191.1, AL122110.1, BC008382.1, AL133054.1, AK027113.1, BC002752.1, AK024550.1, BC007255.1, AK026744.1, AB060863.1, AB063074.1, AB055328.1, BC001964.1, AL050155.1, AL137281.1, BC002342.1, AF090896.1, AL049276.1, BC008417.1, AF104032.1, AL133665.1, AL136540.1, BC001082.1, BC007021.1, BC003619.1, BC002643.1, AL049382.1, AL136844.1, AX025209.1, AL137648.1, BC001349.1, AB055366.1, AL049466.1, AK026927.1, AK026434.1, AL050146.1, AL117394.1, AK026855.1, AK025383.1, AK026532.1, BC008365.1, AL049464.1, BC006210.1, BC005858.1, BC002839.1, AK026164.1, AY026527.1, AL049314.1, AL110197.1, AK026762.1, U68233.1, BC002523.1, BC008506.1, AX027188.1, AF262032.1, AL133016.1, AL136864.1, AL137273.1, AL136925.1, AK026526.1, 576508.1, AL136749.1, AK025410.1, BC004926.1, AK024570.1, 561953.1, AK026624.1, AB049892.1, HDPGE24 53 801947 1-2611 15-2625 BE876192, AU145980, BE839859, AW953709, AV651029, AW866434, AW866436, AW866430, AV687299, AA604920, AA604512, AI887664, AW813014, BE839866, AAI64729, AI566037, AA602341, AA602613, AA214047, BE839860, AI355441, AW855356, AA506540, AUI19708, AW855353, AI884345, AV656490, AI049591, AW853687, AW995969, AI963674, BG058784, BF681462, BE811870, AA551394, BE081412, BE672638, AV687875, BE564307, AW935217, BE811892, BE145548, BE563924, AW577107, AV704081, AA631460, AW380640, AA366464, BG012149, BF694965, AW341886, AW866268, AI537997, F29519, AI537504, AI567884, BF874935, AV659506, AW363563, AA631500, AI363970, AA669020, AI270484, H78415, BE709511, AA640505, BE178526, AI989765, AW866337, AW953693, AA484751, AA342969, BF882965, AA484783, AV659374, BE796439, AV695480, AV659391, AW024055, AV659405, AI832956, T81440, AA654981, R70506, BF852810, AA484906, AW997573, AW379425, AI932609, AA631380, AA570339, BE708328, AI597820, BF694852, BE815355, AW934969, AW902128, AV684943, AA366571, BG260565, AA632800, AW007894, AW192258, AI886084, AV764490, H82763, AWI31401, T69164, AA605054, AW573583, AA834697, AW858120, AW893702, AW573573, AW074527, AV714931, AW820698, AI679343, AA558871, BF853927, AW438596, BF883928, Z32833, AA503427, AW393438, AA610678, AA522988, AA483882, R95100, AW893701, T59151, AW965008, AA848158, BE067011, T98359, T68422, AI679520, BF935516, AA528276, BE839943, BE929829, AL120269, AV759172, H02561, AV760701, AW802714, BE541237, N21656, AI457389, BE066950, T30343, AI679960, H78215, AV700663, AW978714, AL135377, AA243867, AW151713, AW102955, BF884208, AW157616, BF846275, BG034591, BF106210, BG0I1353, AAI61288, BF883454, AC000353.27, AF001893.1, AC006121.1, AC005484.2, AP001710.1, AL590762.1, AC009961.11, AL035555.10, AL160411.25, Z83822.1, AC022402.4, AL136139.6, AC005291.1, AC007225.2, AC007021.3, AC018828.3, AC010742.4, AL391259.15, AC090943.1, AC090514.1, Z98946.15, AL450263.15, AL034372.33, AC008873.4, AF002812.3, AF224669.1, AC006030.2, AL031311.1, AIA22020.5, AL049759.10, AL117692.5, Z94801.1, AC008670.4, AL022318.2, AC008892.5, AC027124.4, AP000555.1, AP001169.1, AC018502.5, AC002378.1, AL390074.17, AC083863.2, AC066597.4, AC002091.1, AC079141.7, Z94056.1, AC005157.1, AL354720.14, AC026391.6, AP00L715.1, AC040160.4, AL139109.14, Z97054.1, AC002289.1, AC007450.1, AC007482.7, AE000658.1, AL499604.9, Z84484.1, AP001724.1, AL109865.36, AC066608.5, AC073101.7, AC007850.29, AL137139.9, AL079342.17, AC018642.6, AC007773.1, AC024163.2, AL162231.20, AC007097.4, AL035685.21, AP000352.2, AL021368.1, AF111167.2, AC007363.3, AF088219.1, AC004125.1, AC009137.6, AC018755.3, AL158828.14, AC026398.4, AL356652.19, AC005846.1, AC023510.16, AL355096.4, AC034240.4, AL049713.20, AC011742.3, AL163853.4, AC015982.9, AL138743.5, AC007907.2, AC091492.1, AC021188.6, AC018682.4, AL138878.10, AL390025.1, AC006050.1, AB026898.1, AC011236.8, AC004024.2, AC005214.1, AC002464.1, AL139113.21, AC005046.3, AP000246.1, AP000207.1, AC007563.2, AC005520.2, AL137128.4, AL031670.6, AL442167.1, AL163285.2, AC011816.17, AC024166.3, AC011739.7, AC013734.4, AL021154.1, AC005881.3, AC005697.1, AL022165.1, AL391114.12, AL023513.1, Z98044.13, AC0000.94.3, AP000129.1, AL139415.10, AC011242.8, Z98304.1, AL589693.3, AL391122.9, AL445435.11, AC003071.1, AF131216.1, AL360272.23, AC087071.2, AC003962.1, AL136123.19, AC020908.6, AP001830.4, AC008450.5, AL445669.9, AJ271736.1, AL034550.31, AL118557.5, AC008891.7, AP000782.3, AP000500.1, AC011464.5, AC0L13L1.11, AL158196.24, AC025262.27, AL049869.6, Z93341.5, AC022468.5, Z92542.2, AL034384.1, AP001728.1, AL031387.4, AC002994.2, AL591807.1, AC022407.6, AL160231.4, AC007065.5, AC005539.1, AL050318.13, U91322.1, AL133415.12, AC010252.3, AC068781.18, AL133500.3, AC008011.11, AI400877.1, AL390738.4, AC044797.5, AC006445.11, AP000688.1, AL445071.14, AL133545.10, AL035089.21, Z98884.11, AL355535.14, AC007999.12, AC022007.3, AC083871.2, AP000350.1, AL354696.1 1, U80017.1, AP001671.1, AL121997.7, AC007282.4, AL139330.17, AL049779.6, AL163282.2, U82671.3, AL445928.8, AP001412.2, AC022392.4, AJ400879.1, AL161454.10, AC090957.1, AP001922.4, AC003091.1, AC005971.5, AL121905.23, AL161935.10, AC022267.8, AL158141.14, AC025165.27, AL158198.14, AL160036.12, AL136303.15, AC004707.1, AC012450.9, AE131215.1, AL132657.33, AC006965.3, AL161937.13, AC005969.4, AC018633.2, AC004764.1, AL359846.11, AL118556.4, AF196970.1, AL137787.11, AL360169.17, AC018769.2, AC000052.16, AF190464.1, AL137100.4, AL445248.7, AC008738.6, AC018523.9, AC005670.1, AC010605.4, AC007541.9, AL049795.20, AL096712.20, AC006057.5, AC004019.20, AC006071.1, AC026166.4, AL132640.4, AC022201.4, AC008569.6, AC003049.1, Z84469, 1, HDPIU94 54 813352 1-2182 15-2196 AU140297, AL529544, AL529545, AU124978, AI740820, AU116885, AU126162, AW960772, AI565169, BF111956, BG251247, BG177689, BE780814, AI628285, AA482031, BE784432, AA947029, AW954823, AW190175, AA315300, AU143854, AA707674, AI332610, N50136, AU148736, AU127152, AW768480, BF947113, AA223261, AW955931, AI276839, AAI89165, AA804584, AA767472, AA223378, AA894857, AA252718, R46372, AA939277, N59367, AA219127, AA774827, AV762911, BE546354, N72682, AA219510, AV761697, AA872005, AW188325, W02461, R21326, AI923716, D29223, R68368, BF771937, BE769443, AA322537, R08745, AA417592, R08746, AW952240, AA299861, AW377015, AA337351, H60482, N76470, BF088734, AA218745, AA336556, BE896274, AI810734, AW118290, BF380800, T30177, D29202, BF910258, AA337527, AA336555, R68574, AI167609, AA376922, AW968355, BF351657, AI832198, AW972092, AW968356, AW972093, AW968729, AI432644, AI623302, AW971740, AI432654, AI432650, AI432653, AW081103, AW858522, AW972091, AW969229, BE672759, AW972090, AI432677, AI431230, AI431307, AI431316, AI431328, AI431353, AI431312, AI432655, AI431310, AW128900, AI431238, AL045327, AI431354, AI432666, AA580821, AI431347, AI431315, BF448552, BE672748, AI432661, AL134524, AI431323, AI431337, AI432675, AI431321, AI492519, BE672745, BE672732, AI431246, BE672719, U46344, AI431235, AI431243, AI432647, AI432651, BE672738, AI431255, AI432674, AI431330, AI432649, BE672767, AI791349, AW601637, AI431248, AI431241, AL042842, AI431254, BE672774, AI431357, AL042729, AI432672, AI432665, BE672742, AL042931, BF589777, AI432662, BE672627, AW577201, AI431345, BE672644, AL042655, AI431351, AL042508, AI431231, AI431346, AL042853, AI432676, AI432673, AI432658, AW128884, AI431257, AW577199, AL042533, AL043166, AL047611, AI431340, AL135012, BE672622, BE672792, AI432657, AL042802, AW128846, AI431247, AI432664, AI432645, BE672718, AL042787, AL042515, AL042832, AI431751, AL043295, AI431314, AI492520, HDPIY31 55 886159 1-1964 15-1978 AL533296, AU142272, BE560264, BG117407, BE407326, BG116397, BE314927, BE315405, BF205715, AI935180, BE313422, AW965613, BF315382, AI701496, BF727272, BF115518, AI829152, AW474694, H23483, AL040572, AI005000, AA311926, BE297986, AI298282, BE743406, AI675622, AW845300, BF306252, AA037364, AI097581, BF308743, BE296105, AW148771, AI339720, AA455474, BE670969, H23486, T66744, BE818161, R17317, AI268300, AW070382, AI291626, AW409641, AA834030, AI880154, R17812, R13082, AA324613, H23484, T27067, H86644, BF892771, R46701, AA349448, R20964, AA923102, H05150, H23485, T87222, N77062, Z43932, H23020, AW136846, AI742491, AI372678, T16039, T66743, AI372676, R45314, BF681190, H08379, AW864486, AA732753, BE312682, AW864516, H29561, R22677, AA324777, AA774961, R25545, F10589, AI523364, AA379005, T26481, BF904678, Z39993, H06774, H24087, AA670426, R20154, Z42195, R44466, R18635, F07975, Z44889, R59906, AI560142, R53460, R43016, R20237, AI745398, BF904916, H24300, R41995, R53461, H06899, AW801037, T74057, F07778, H08380, AA323177, BF763326, M85501, F12373, BF689358, H08954, R44142, F04562, F01724, F04134, F04226, T15999, AA037520, AA677085, BF335984, R40512, R20492, AL533261, BF858775, R56265, R13207, AA987631, T34814, R42746, BE747609, F09993, AA099781, R63798, R43692, AA902512, AA455473, R55721, F04028, AI984376, AI243094, AA776086, BE504276, BE155765, T10103, Z38178, T16439, BF851177, T10102, BF851176, T16686, BF335999, F01506, BF851173, BE818200, R14040, BF851221, BE814269, BF851178, BE327839, H06858, AW003241, BE394938, BE393786, BF851170, BF884029, H13338, BF858389, AA234656, AW748209, AI393102, R47781, R50313, R50305, AA976743, BF851229, BF335998, AV727501, BG165051, AA323766, AI758272, BG163618, BG113299, AI922707, BG164558, BF037292, BF341801, AV746964, BF792961, BG030364, AA643623, AI702406, BF970449, AW827289, BF921103, BE895585, AW827227, AW827206, AI471361, AW050578, AW196105, BG249582, AV682575, BG108406, AL036736, BE138658, AI345608, BE620202, AI431909, AI345471, AI805769, AI308032, BG027280, AI344785, AL041772, AI452993, AL134999, BG260037, AW983691, AW071417, AI924686, BF753056, BG112718, AI608936, AI963216, AW082594, AI335426, AI348777, BE965355, BF885000, AV735098, AI620284, R40432, AI269580, AI886124, BF814541, AI802833, AW302992, AI812015, BF343099, BF793644, AI799195, AI824576, AW168031, AL118506.27, AK023132.1, AK024508.1, AL137301.1, AK026542.1, AL389982.1, AK024538.1, AL359596.1, AF260566.1, AL137550.1, AL442072.1, BC008417.1, AL359615.1, BC003687.1, AK000432.1, BC004370.1, AL117585.1, AIB063070.1, AK000647.1, BC008365.1, AF146568.1, AK026784.1, AF003737.1, AB055366.1, AB063084.1, AL110221.1, AK026462.1, AL110225.1, AL136892.1, AK027164.1, AK025967.1, AK026528.1, BC008280.1, AK027204.1, AB019565.1, AL359618.1, AK026629.1, AK025391.1, AL122050.1, AB050510.1, BC002733.1, AB060916.1, AL359941.1, AF217966.1, AL512754.1, AL117460.1, AK025798.1, AB055303.1, AB060887.1, AL136799.1, AF061943.1, AF078844.1, AK024524.1, AL136928.1, AB051158.1, BC00B070.1, AF090943.1, BC005151.1, AF225424.1, A3012755.1, AL080060.1, BC008382.1, AL137271.1, AB056421.1, AL117583.1, AK000083.1, AB060929.1, AL050277.1, BC003548.1, AK026534.1, AF056191.1, AL136805.1, AL512718.1, AF026816.2, AL137556.1, AL049452.1, BC006164.1, AL137560.1, AK026045.1, AB055374.1, 578214.1, AL133557.1, AK026593.1, AL049382.1, AF271350.1, 561953.1, AF090934.1, AX026526.1, AL390154.1, BC001967.1, AK027116.1, AF177336.1, Z82022.1, AF183393.1, AK026630.1, AL389939.1, BC003684.1, AF090896.1, AK026551.1, AL110280.1, AF091084.1, BC007326.1, AF097996.1, BC002839.1, BC008899.1, AK026959.1, AB060863.1, AB063046.1, AL137459.1, AB048954.1, AK027213.1, BC001045.1, AB052200.1, AF125948.1, AL442082.1, AL136845.1, AL122093.1, AB060214.1, BC007199.1, U42766.1, AK026408.1, X72889.1, BC008780.1, AL137526.1, AL512719.1, AB055361.1, AK026762.1, AB060825.1, AL136768.1, AL583915.1, AK025484.1, AL353956.1, AL133565.1, AL512750.1, BC003683.1, AF113222.1, AK026532.1, AL133104.1, AB052191.1, BC009033.1, AK026504.1, AK027096.1, AB048953.1, AF162270.1, AF218014.1, AL136786.1, U39656.1, AB056427.1, AB063008.1, BC006440.1, X69819.1, BC004951.1, AK025414.1, AK027193.1, AL049464.1, AL137538.1, AK000486.1, AL136843.1, AL359601.1, AB055368.1, AK026927.1, BC007198.1, AF111847.1, AL353940.1, U80742.1, AL117394.1, AB056768.1, BC006412.1, BC009341.1, AL137480.1, AL136749.1, AK000615.1, AK026464.1, AB050534.1, AL133067.1, Y16645.1, AL137557.1, AL110196.1, AK000445.1, AB047615.1, AK026642.1, AK025906.1, AX000137.1, AK026947.1, AK025491.1, BC004958.1, AB063079.1, AK025573.1, AB060839.1, AF207829.1, AK025312.1, AL359623.1, AK025524.1, AK025772.1, AL117440.1, AK000391.1, AF090901.1, AL050393.1, X65873.1, BC008983.1, BC006525.1, AK026647.1, AB048964.1, AL080074.1, AK026353.1, AB047904.1, AL049314.1, AL137648.1, AB056420.1, AJ242859.1, BC001963.1, AB060852.1, AL512689.1, AF219137.1, AL050149.1, AF125949.1, AL050146.1, AL136864.1, AL359620.1, BC008488.1, AL050138.1, AL122123.1, AL162002.1, AL122049.1, AB060826.1, AL049300.1, BC005890.1, AK026086.1, AK026480.1, AB062942.1, AB060908.1, AL512733.1, BC006807.1, AL122098.1, AL117457.1, AK026855.1, AL136787.1, BC005678.1, AL080137.1, BC008387.1, BC003122.1, AL117435.1, AK026744.1, AL136844.1, AK026608.1, AL080127.1, AL136789.1, AL162062.1, AF090900.1, AL133016.1, HDP0C24 56 777493 1-1763 15-1777 BE795755, BE799575, BF346049, BE736856, BE735839, BE613439, BE295320, BG165897, BG028052, BE904063, BF525670, BG248216, AI762392, BE798809, BE876262, BE538403, BE541775, BE278995, BE867896, AI928014, BE735450, BE792862, AI302814, AI417544, AI861926, BE868781, AI620234, AI949339, AW292331, AA425932, AI635177, AA716408, BE964527, AI148619, AI188537, AI751315, AW298424, AI570424, BE019043, AI333093, BE563878, BG166956, AI679326, AI339585, BE208614, AI912361, AI199939, AI638579, BF035843, AA575835, AW562325, AW167212, AA613113, AJ403125, BE300704, BG230624, AA631882, AA654351, AI188665, AI744337, AI042080, AA554771, BF205494, AI751314, AI357368, N42873, AI623763, BE205782, BG015408, BF061667, AW296851, N73029, BE548655, AI080401, AI200653, AI346327, AI804793, AA723378, AI219032, A1923960, AW512977, AI334021, BF739379, AI961597, AI933388, AW118019, AI750231, H49782, AW516158, W73379, AI682039, AW068519, AI199855, AI858410, AW275973, AW338079, AA856546, AW071218, AA428801, AI128328, AW026790, BE673142, AW513049, AA582918, BG056078, AA936754, AW513987, AA579898, AW192791, R55015, R80153, BF197452, AI738833, AU157028, AW627680, R62188, BG152556, AW516091, BG057769, AI538819, AW085840, AI500700, AW082065, BE614205, N34466, C75025, R55153, AW630976, R81484, AI687198, BE271478, AA975810, AI207300, BG253177, H13762, BE463629, H13709, AA983929, T97913, AI719056, AI203064, R80154, AI074832, AW104721, R47833, AA852278, AA812419, AA088385, R64576, AI362616, AW068781, W73403, R21634, AA553687, BE544315, D31024, W44598, BF346027, N33449, AA573257, AA857087, R49975, AI915025, AI382413, BF847025, R54690, AW591357, AW953807, N53663, BF752914, AA469380, AA469299, BF111939, AW301188, N50673, AW270044, BE549470, BE540690, AI673113, AI380538, AA364267, W39364, AW572002, AW080124, R81724, N42422, BE785221, D20947, BE966528, W44617, BF829622, W38340, AA948112, BE074077, AL137555.1, AK025951.1, AK025804.1, BC001979.1, AL445222.9, AFL51783.1, AK023580.1. HDPPD93 57 637588 1-687 15-701 AI767544, BF963878, AW391604, AW371053, AW391605, AW380560, BE150935, AW380557, AW609397, AV647627, AW609527, AV647628, BE150882, AA625481, AV647780, AW582425, AW582423, AA053357, Z39028, T31703, AI559952, AI962812, AW023035, AW247321, AW594578, AW204377, AW135110, AW069029, AA737065, R51075, AW294433, AW291912, BF511819, AW292027, AI265783, BF063801, AI274716, AI191509, AA605269, AI768872, BF057534, AI634812, AI698531, AA565825, AI275563, AI366661, BE242780, AI538787, AI203337, AI565080, AW008059, AW088320, AW073352, W73510, AW242351, AI341846, BF222967, AA513453, AI346467, AA622940, AI000935, AA535424, AI022694, BF592958, AA639759, AI679288, AI679864, AI079193, AU147451, N56986, AU148991, AI304785, AAI34162, AW664542, AI337342, AI245649, AI969305, AW582354, AI081970, AI262353, AL079933, AW976197, AI991639, BF349821, AU159502, AW004865, AA665733, AI348128, AI094163, AI184073, AU152120, AU149126, AI359191, BE552234, AA632288, AA814449, AW362622, AU159545, N75380, N49596, BF869959, H48051, N70679, BF987335, AI864296, AA928727, BE963530, AA617935, AI151119, BF680667, AW978110, BE785995, BF913501, AL519088, H56669, BF526020, AI668893, AV655645, AV714752, AW087445, AW104724, AI538716, AL045500, AI564719, AI802542, B0058398, AW026882, BG031815, AI433157, BG179993, BF812961, AI620284, AI524671, AI857296, AI619502, AW118557, AI682841, AI439745, AI934035, AL514129, AL036274, AL036361, BG111590, AI560099, BE048071, BG260037, AI445432, AL036802, BG257535, AV732936, BF970449, BF812938, BG110517, AA640779, AI536685, AI445025, BG180996, AL514983, AL121365, AI521012, AI537677, BE884130, AI475371, BG112718, BG113299, AI702406, BG110684, AI349004, AI580984, AI687127, AL513907, BF032868, AW166970, AI687375, AI625079, AI440239, AI349645, AI635461, AL1 19049, AI433976, AW07 1349, AV753074, AV757018, AI285735, AV758209, BG109270, AI271786, BF792961, AI498579, BF882343, AI281762, AI690480, AI815855, BG170430, AI783504, AL043975, AI636456, AI687728, AV682093, AV708097, AW302988, AI432969, AW148716, AI800384, BG112879, BF924882, AI697137, AL514879, AL036396, AL048656, AI500077, AW827211, AI282655, AI962456, AW268253, AV681618, AV756619, AW086113, BF793244, AW129659, BF885675, AV758822, BF816811, AV758592, AI799470, AI539780, AI828682, AV681869, AI610307, AI612913, AW132056, AI872711, AI818683, AI499393, AI493248, BE887488, AV7 13079, AV652906, AI340519, AI537303, AW07 1417, AI475451, AL040243, BF107577, AW082040, AL036146, BF038106, BG036846, BF726297, AV682849, AK026796.1, AK027770.1, AL391244.1 1, AF251294.1, AL136882.1, AK022241.1, AB051496.1, AK026647.1, BC008387.1, BC006195.1, AF090903.1, AB055361.1, AL1 17394.1, BC008488.1, AB060826.1, AB063070.1, 578214.1, ALL17457.1, AK025339.1, AK025092.1, AF125948.1, AB060912.1, AL359615.1, AL133606.1, AL122050.1, AL049452.1, AL157431.1, BC001967.1, AL049314.1, AL162006.1, AL137459.1, AL512746.1, AL050149.1, AK025958.1, AB055303.1, AB060887.1, AL0501 16.1, AL110225.1, AIB048953.1, AB056420.1, AL050277.1, AL512733.1, BC008365.1, AF106862.1, BC008417.1, AL442082.1, AL117435.1, AK027096.1, AL136749.1, AL133640.1, AL133016.1, BC003687.1, AL136586.1, AL136787.1, AF090934.1, AK025084.1, AK026855.1, AL136789.1, AL512719.1, AL137557.1, AL110196.1, AX026741.1, AL359601.1, AB049758.1, AF090901.1, AL050393.1, AF104032.1, AL162083.1, AK026959.1, AK000083.1, AL133075.1, AL050108.1, AF225424.1, AF090900.1, AL389982.1, BC007021.1, AK026592.1, AK026865.1, AL122121.1, AL122123.1, AK000137.1, AL117460.1, AL110221.1, AL050146.1, AF090943.1, AL133080.1, AF219137.1, AK026045.1, AL359596.1, AK026452.1, AL096744.1, AF090896.1, AB060863.1, AB063046.1, AK0006L8.1, AL080124.1, AL359941.1, AF218014.1, AL512754.1, AL512718.1, AB060916.1, AL049938.1, AL133093.1, AK024538.1, AL049466.1, AF078844.1, AL136892.1, U42766.1, AK027868.1, AK026608.1, AB048954.1, AK026784.1, AL133560.1, AL390167.1, AB055368.1, AB055315.1, AB019565.1, AL050060.1, AL122093.1, AL133565.1, BC003683.1, Y16645.1, AB048964.1, AL389978.1, BC006807.1, AB063008.1, AJ242859.1, AF125949.1, AF111847.1, AL442072.1, AB056768.1, AL137527.1, AL359618.1, AK026744.1, AF146568.1, AK000445.1, AB060825.1, AK025772.1, BC007199.1, U91329.1, AF091084.1, AK025906.1, AL136844.1, AB051158.1, AB047615.1, AL117583.1, AB047801.1, AK026534.1, AL136799.1, AK026542.1, AL353940.1, BC004556.1, BC002733.1, BC008899.1, AK025491.1, AL133557.1, AL136768.1, X82434.1, BC001045.1, AL117585.1, AK000323.1, AL049464.1, AK027113.1, AK026504.1, AF207829.1, AB060908.1, AK000212.1, AB062938.1, AL050138.1, AK000432.1, AK026583.1, BC004951.1, AB055366.1, AK026927.1, AL080137.1, AK000652.1, AB052200.1, AL137550.1, AK025391.1, AK026353.1, AK026532.1, AL049300.1, AK025967.1, AL050024.1, AB060852.1, AL137283.1, AL122098.1, AF097996.1, AL512689.1, AL136928.1, AL049382.1, AK026533.1, AK025414.1, AK026528.1, AB056809.1, AB047904.1, AL136786.1, AB052191.1, BC008070.1, AL136845.1, BC002839.1, AL049430.1, AK000614.1, AF260566.1, AL133113.1, AB056421.1, AK026086.1, AL137271.1, AF177336.1, AK027204.1, BC008485.1, AK027164.1, AF183393.1, AK024588.1, AK024524.1, Z82022.1, AK026947.1, AL122110.1, BC008382.1, AK026526.1, AL512761.1, AL137648.1, AL137463.1, AL049283.1, AK026630.1, AK025484.1, AF271350.1, BC008983.1, AK026480.1, AL359583.1, X72889.1, AK027116.1, AX027213.1, AK026629.1, AB060929.1, AK025524.1, AB050510.1, AK025209.1, AK000647.1, AL512765.1, AK026597.1, AL512684.1, BC003548.1, AL137538.1, AK026642.1, AK000718.1, AL359622.1, AL080127.1, HDPPQ30 58 684292 1-1049 15-1063 AL041375, BE140949, BE077105, BF880881, AA809125, AW177869, AI890297, AW338376, AAI71400, BE328286, AA218684, AV683406, AI310670, AW902110, AA489390, AI523205, AI792092, AI760835, AI821056, AI821805, AI801563, AW901945, AA526542, AW902135, BF876674, AI819419, AW022796, AA084320, AI254267, AW504667, AW162314, BF530611, AA661583, AA515727, AA935827, AI521525, H62123, AW265006, AW021674, AA324108, AI609992, BF812696, AI174703, BG231195, AI310787, AI280535, AA720582, AI753904, AI984168, AA584493, AI349130, AW169183, AI609984, AW264548, BE245594, AI797998, AW020150, AW238341, AI568376, AI610012, AI572680, AI278440, AI277373, AA232928, AW020094, BE676856, AI609974, AA425283, AI224583, AW192419, P23338, AI926102, AW162332, AA555232, AW104161, AA804726, AW021399, BE676988, AA525753, AW971320, BF882222, AAI67656, BF949151, AA689351, AA584241, AL044701, AW511778, AA557945, AW265468, AW328446, BG180320, AA568303, AA493546, AA807684, AA280886, AW855527, AI889995, BE063437, BE154909, AA568311, AI345566, AA599712, AW275432, BF870762, AA810158, AAI82928, R23873, AA492496, BF904892, AI815583, AW674277, AI516537, AA636077, BE244308, AA595661, AW084445, AA657808, AW510403, BG152746, BF800073, AW962791, AAI80056, AW243817, AA604601, P29968, AI344906, AI318548, AW085626, AI369076, AI860423, BF681222, AA610381, AV759517, AI915081, AW157128, R73744, AW571716, BF448553, AW303052, AA507623, AW129188, AV763650, AA703818, AL042667, AL042670, 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AB049848.1, AK000718.1, BC008284.1, AL136786.1, AL137459.1, AB055328.1 AL136892.1, AK025465.1, AL512704.1, AK026533.1, AL512750.1, AL133067.1, U35146.1, AL512746.1, 577771.1, BC002697.1, AF056191.1, AL389982.1, AB019565.1, AL389939.1, BC005858.1, BC004530.1, BC006103.1, BC004256.1, AL359583.1, BC000090.1, BC001098.1, BC004244.1, AL137558.1, AF111847.1, AK026551.1, AL136805.1, 576508.1, BC008455.1, AK026462.1, X72889.1, AF106862.1, AK000418.1, AJ299431.1, AB063008.1, Z82022.1, AF183393.1, AK000391.1, AL359618.1, AL117648.1, BC007326.1, BC002457.1, AF358829.1, AK026855.1, AK000445.1, BC004899.1, AL359600.1, D83989.1, BC004265.1, AL110197.1, AK000083.1, AB047878.1, AB047801.1, AB062938.1, AB063079.1, BC008417.1, U42031.1, AL110221.1, AF090886.1, AL162062.1, AK025312.1, AK027142.1, BC007732.1, AF321617.1, AL137480.1, AK026532.1, AK024524.1, AK026464.1, AK024992.1, BC005151.1, AF073483.1, AK026522.1, BC006408.1, AB060837.1, AL117416.1, BC003569.1, AK027164.1, AK025541.1, AL122100.1, Y14314.1, AK025708.1, BC007534.1, AL353940.1, BC008893.1, ALL10222.1, AF057300.1, AF057299.1, AB051158.1, AK027114.1, BC009311.1, Y16645.1, AB055315.1, AB050510.1, AF069506.1, AB060905.1, AL080154.1, BC008280.1, AK025092.1, AL137538.1, AB063093.1, 569510.1, AB056372.1, BC008282.1, X98834.1, AL133014.1, BC007897.1, AL133113.1, AL049996.1, AL137533.1, AL049460.1, BC004917.1, AL389978.1, AB050411.1, BC002839.1, BC002541.1, AF177336.1, AK026744.1, AB060863.1, AB060916.1, AK026746.1, AB047869.1, AB050431.1, BC004944.1, BC007198.1, AB049758.1, BC006133.1, AK000450.1, AF104032.1, U51587.1, AK025383.1, AL137300.1, AC023880.5, AF271350.1, AL137429.1, BC009033.1, L19437.2, AK024588.1, AL110196.1, BC006508.1, AL162003.1, BC006480.1, M92439.1, AB060211.1, AB056421.1, AF210052.1, AB063046.1, AK000074.1, AB048954.1, AK027182.1, BC008785.1, AK027160.1, U68233.1, AB047904.1, AF125948.1, BC002473.1, AF303581.1, AF178432.1, AL136845.1, AB056768.1, AL080126.1, AL137527.1, HE2P093 63 771655 1-1309 15-1323 BG250792, BF340156, BF691370, BE958544, AI913576, BE892390, BE467084, AI769974, BF978990, AI985726, AI978876, AA805536, BGI14463, BF976892, AI879646, AI640283, BF212660, BE813503, BF212750, BE245388, AI151263, BE813657, AW994769, AI474103, BF326782, AW078667, N66384, BF382578, BE881936, AI268780, AAI37260, BE865738, AW118966, AI690850, BF211945, AAI50376, AW020353, AI218961, AI458422, AI758351, BF030723, AA631892, AI350813, AW079202, AI038862, AAI50275, BF082705, BF240677, AA912346, T17277, BE003683, BE513513, AW468933, AI003046, BE048238, AAI37259, AI208534, BE770261, R77850, AW955572, BF743116, AW183342, AL047998, AA928199, AI282290, AL047999, R77760, BE215622, AL121406, T34660, 1160032, BF336987, AA362660, AA330116, Z40410, AW370452, AI473113, AA306043, AA343807, BF239599, AA621093, BF240906, BF887439, BF886985, R40472, N52225, AW168049, AA789087, BE770101, BF885967, AL534441, AA962715, BF212646, N73192, BE871372, BE715872, AC009709.7, AC007911.8, HB6FU11 64 827236 1-1986 15-2000 AA992948, P1638341, AW134923, P1038302, H54037, P1581139, AJ007581.1, AK027323.1, AF314058.1, AL021578.4, AB027710.1, AB024964.1, HE6FV29 65 588454 1-1512 15-1526 AA984763, AA406303, AA599164, AA600957, BF922107, AL046225, P1623434, BF760969, P1890702, D29050, AL449223.7, AP0Q0114.1, AP000046.1, AP001717.1, AC006039.2, AL078463.11, AL035460.15, AC060232.5, AL161730.9, AF277315.3, AC010L95.8, AC010319.7, AC005099.1, AP003114.1, AC020906.6, AL117377.18, AC004841.2, AL161937.13, AC004951.5, AL161781.12, AL157858.5, AL391260.13, AL158828.14, AP000302.1, AC007193.1, AF123462.1, AC027644.9, AC006254.10, AL035662.65, AL139396.17, AC004084.1, AC015937.7, AF008191.1, AL355916.2, AD00L502.1, AF283320.1, AC005082.3, AC005399.19, AC003030.1, HE8TY46 66 899528 1-2190 15-2204 BG260679, BE744499, BE791844, BE799404, BF341229, BF340249, BG033558, BE787232, BE563298, BE512690, BE727788, BF128559, BE727160, BF792775, BE791996, BF526027, AL520325, BG118406, BF528636, BG120791, Z78324, BG252616, BE267489, AW997217, AA573951, AW378545, BE884919, BE545107, AW161658, BG058810, AL120658, BF954742, AW673944, AW953507, AA479588, AW250895, BE899050, BE531156, AI384065, AW250140, AA777294, BF954739, AW410340, AI955759, BG057374, AW579652, AA512736, AA514652, AI140280, AA889483, AA522793, AW378626, AW273718, AA595719, BE299671, BE272956, AW378630, AI189202, BE901834, H17214, AA888064, AA643992, AW675106, AI375020, AW163090, AW157575, AI308921, AW074078, AI989412, BF876474, AI816518, AA214547, AI751405, AI984530, AW152123, AA578553, W90602, AI688905, AI086778, BF830769, AI564028, AI814124, AI277623, AI000764, BF943828, AW167325, AI591307, AA428453, AV726467, BF839950, AI305195, AA479881, AA448198, BF839936, AI197967, AA351741, W90279, AI186150, BF926156, N38977, AV725693, BF819780, AI150970, BF839952, W26025, AI189201, AI498860, AA477427, AA625566, W26470, BF740682, AA262237, D53953, BF771915, R24861, BF923441, BG167488, W46800, AA401647, N92450, 1120700, AA906675, BF768960, T77973, AA351632, AI760569, BF528593, AI973119, BF848734, BE542451, BF110605, BF927449, BE908963, AW001432, AA741525, AW802164, N48124, AW732250, T40680, AI688597, AI033536, AI648659, AI568284, AI201130, BE245484, W31334, AW087959, AI301841, T68681, BG152674, AW087314, BF923568, AA318045, AI973130, AI493504, T26959, AAI48971, AW732249, H17111, BG055736, AI923243, AA311472, BF992504, BF770005, BF848768, T51763, AA327274, R85673, BE076365, T23497, F32993, AI144182, BF801918, AI887003, BF768181, BF927450, AA213532, T35367, T03584, AI263552, BE264886, BF858836, AA299160, T56823, AA766551, BE258010, AL533953, H52738, BE275083, BF830766, R12841, W22828, T51609, AI452587, AW579657, W28398, AI565665, AI937705, AA262863, AA355737, C03459, AA351740, AFL14054, AI242872, BF802489, R48499, BF811227, T39585, H20701, D29481, AI370713, BF856310, AW087322, AW167588, BF839951, BF876460, Z42014, Z38303, AA678484, 1119947, AA338936, AA351631, W46830, AA961250, AA953364, AA876570, BF843380, R44563, AAI48972, R20751, 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W56301, AW058408, AAI02223, BE30L190, AI091799, R05745, D61582, R01123, AAI02222, AA375163, BG029189, AW293550, AI752483, AA376452, AW275432, BF812696, AI439525, AW151541, AW084324, AL121039, AW265468, AI702049, AW162314, AW327673, AA577706, BE273825, BF940118, AI270280, AW148821, AW162332, AA807704, BG059139, AA661583, AW238137, AA601674, BG180320, AV742390, BE244308, AW410844, AI433952, AI828721, AA631915, AL079734, BG152746, AW473160, AW021399, AW020094, BE677164, AA728954, AI860423, AI039257, BF679568, AW243817, AI049999, AW148964, AI538404, AI826857, AI753131, AI690379, BE676856, AI003469, AV758870, BF214695, AW502688, AW631267, AI904840, AA603359, AI251696, AI819419, AI090377, AI254508, BE176819, AI554399, AAI12864, AI355246, AWI51848, AW962971, AI028148, AI308529, BF868826, BF970107, AA507499, AI751698, AL036896, AC006483.3, BC000787.1, AK024787.1, AC010616.5, AK027150.1, AC004659.1, AL078611.1, AK000385.1, AC005519.3, AC009756.9, AC002543.1, AC005052.2, AL354836.13, AC016995.4, AL023879.1, AC003108.1, AL139824.22, AL121675.36, AL358777.12, AC015651.18, AC011444.5, AC004966.2, AC011526.7, AC010319.7, AL121579.4, AL158040.13, AC007421.12, AC005531.1, AL391259.15, AL096701.14, AC002996.1, AC067945.4, AL109923.29, Z97183.1, AL133458.19, AC010271.6, AF279660.2, AL035086.12, AP000280.2, AL445184.11, AC008440.8, AC008848.7, AL139809.16, AC018663.3, AP000039.1, AP000I07.1, AF195658.1, AP000557.2, AC004974.1, AC010789.9, AC004552.1, AC004985.2, AL160256.21, AC018633.2, AL121897.32, AC090944.1, AC020983.7, AP001715.1, AC007374.6, ALL17382.28, AF207550.1, AC010422.7, AL137852.15, AC008635.6, AL035659.22, AP000463.2, AB017653.1, AL359236.4, AC005358.1, AL035683.9, AL139785.5, AL159168.15, AC000353.27, AC000379.1, L35532.1, AL357972.18, AC011479.6, AL159990.12, AL138849.12, AC008891.7, AP000555.1, AC0L0530.7, AC008744.6, AC025212.5, AL451162.14, AFL67081.1, AC007240.2, AC003007.1, AL512489.11, AC004673.1, AC004752.1, AL138733.15, AL354948.7, AC011740.7, Z85986.1, AC005484.2, AC013355.7, AC016596.5, AL031711.30, U73636.1, AC006064.9, U91327.1, AL031680.20, AC004089.25, AL132640.4, AL109935.39, AC010326.6, AC007676.19, AL133229.40, AL136228.8, AC018797.4, AL033519.42, AL096791.12, AL391139.19, AC002312.1, Z97054.1, Z83840.7, AC004821.3, AC002060.3, AL139184.8, AC005280.3, AF107885.2, AB032485.1, AP000256.1, AF312032.1, AL035705.22, AC012379.7, AC020904.6, AC008521.5, AL356214.20, AF224669.1, AP000691.1, AC018673.4, U07561.1, AC008392.6, AC004263.1, AP002906.2, AL031058.1, AL355480.22, AC006530.4, AC009362.8, AF258545.2, AL049759.10, AL035249.6, AL008582.11, AJ009616.3, AL050404.3, AL122020.5, AP000098.1, AL133163.2, AC025166.7, AC087311.22, AL513366.11, AL353678.11, Z97632.1, AC005041.2, AL121586.31, AC006449.19, AC010412.7, AL355336.15, AL022316.2, AL353748.13, AC010526.7, AC005911.6, AC016025.12, AC084864.2, AC004066.1, AP001748.1, AC026120.33, AL050307.13, AP000361.1, AL157372.18, AL353710.7, AL049780.4, AC011895.4, AC005695.1, AL161937.13, AL354928.9, AC008738.6, AC008372.6, AP000503.1, AC002404.1, AC008482.5, AL121967.11, AC010378.6, AL449209.2, AC026672.44, AL356805.5, AL031662.26, AL449143.18, AL034369.1, AFI34726.1, AL049843.18, AC008623.4, AC072061.8, Z98948.1, AC004662.1, U62317.2, AC012476.8, AC008397.7, Z98752.16, AL161670.4, AC007957.36, AC009399.5, AC008755.6, AF317635.1, AC009812.17, AC002546.1, AC003043.1, AC022211.5, AC007746.3, AL050335.32, AC005015.2, AC022405.5, AC004476.1, AL109804.41, AF001549.1, AC016602.6, AL109799.6, AC008044.4, AC004851.2, AL049795.20, AL162505.20, Z83851.17, AL359541.11, AL031282.1, AC009470.4, AL034422.24, AL133332.12, AC005365.1, AL157789.6, Z98051.6, AC006151.3, AC024561.4, AC009086.5, AC005570.1, AC010679.6, AC009469.4, AC008857.5, AC007845.12, AC005091.1, AL132713.11, L78810, 1, HEBGE07 70 798096 1-1853 15-1867 AI016066, AC010170.3, AC006515.7, AC008641.6, AC019086.7, AC016587.7, AL078581.11, AC037433.6, AL121975.9, AC022267.8, AC009225.3, AC091493.1, AC006257.1, AL160411.25, AL033397.7, AL122023.3, AP001671.1, AL121983.13, AL024506.1, AL135785.10, AC079383.17, AC024085.5, AC020552.4, AC087069.2, AC008115.3, AL162571.9, AC078841.4, AC008543.7, AC083876.2, AC005071.2, AC019041.8, HEGAU15 71 834379 1-1111 15-1125 W92008, W92009, AC009404.5, HETCI16 72 844543 1-2271 15-2285 AW851273, AL043006, AI651386, AW383991, AW373566, BE881467, AI669232, BF345924, BE395168, BE504187, AI681395, AW299401, BE550134, BF093777, BE156260, BE005867, N26576, AI983369, N50035, BF221628, BE156250, AAI56032, W61061, BE005872, BF527978, AI809672, AA932906, AA405124, BE502997, BE772818, BF759946, AW051708, AI609288, BE089879, N63480, BE089880, AA987664, B6054688, AI138308, AI393915, BE156248, BE005860, AA835275, AA909484, W72796, AW440400, AW178796, BE772779, AI286220, BE772827, AI073876, BE772829, AI357767, AI560667, AI264150, AI434188, AA405125, AAI26946, N94778, AW291536, W76118, BF081321, N39638, AI358419, N33885, N31550, W73600, AI393549, AW363497, BF809085, BE086658, AI446713, 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N99875, N77905, AA679441, AI352336, AI188487, AI432136, AA033645, AI266690, AI433965, AA877077, AA055324, W44808, H49413, AA4I1995, AA833544, AI300087, BE972332, AV682350, AA992809, AA814064, AA741027, AV702746, W32538, AA683291, AA621431, AA902136, AA214623, AV647110, AA663647, AA494354, BE245415, AW151578, AW150662, W60562, AA705000, AI950580, N27003, AA345942, 1185708, AAI33758, AI627657, AA224004, W37452, T80362, AA452695, AI146286, H49227, AW074691, BG142207, H47030, F25806, H85509, BE244420, C17218, H24204, AA480190, AV651579, AI272131, T80478, AI381311, W87586, W37696, H18682.1124218, AV647065, N29206, N28458, AW468405, BF081529, W44802, AA927134, AW821055, BF854323, AA083939, F35729, AA682763, R31829, AAI48436, AAI25918, H95144, BE246322, W44717, AW194993, AA729762, H50669, AA343940, BE244987, AAI15539, R26021, N35260, H46491, AI902181, AI149536, R37220, AA325890, AF151885.1, AK025730.1, BC000836.1, AF135161.1, AB062991.1, AC007298.17, AF172940.1, AC022149.3, R26823, R38842, H88357, H88417, H88357, N40125, W05840, W25309, W32702, AA027857, AA027923, AA034476, AAI15050, AAI51732, H5AVW42 151 637660 1-581 15-595 AI336192, AI911235, BE644656, AI459354, BF030919, AI333569, AA652155, AW974708, AI700779, AA932386, AI922689, AI888953, AA848053, AI863382, AI932794, AI554821, AI539687, AW081231, AI587156, AW189415, AI610362, AA225339, AI934011, AI498067, AW168373, BG031338, AI280732, AI590423, AI590686, BF724894, BG036614, AV709522, AI242251, AV756150, AI890907, AI687065, AV682074, AI583065, AI472536, BF811793, BF812960, AW167222, AI636588, AI249946, BF970652, AW169234, AI431909, AI610115, BF981148, AI635016, AI874243, BF970768, BF727034, AI798351, AI580254, AI627988, AL037582, AL037602, AW022682, AI916419, A1872804, AW072719, AW151714, AI613038, AI470293, AV704962, AI588892, BF725644, BE965169, AV757161, AA470491, AW172723, AI281867, AL514791, AW983783, AI802542, AV682875, BG256090, AA502794, AI758437, AI609409, AI798456, AI345551, AI628833, AI590830, AI560683, AW198090, BG105070, AV733470, AI591407, AW090071, AI335426, AI348777, AI521244, AW983832, AI955906, AI288050, BE963244, AW084117, BE546262, BE783819, AL514627, AI274541, AI274745, AW059713, AI612913, AI493576, BG165979, BF726207, AW268302, BF033757, BG001235, AW104196, AI538764, AW983754, AI633125, BF726237, R36271, AL042628, AI539771, BG108268, BF528717, BF970263, BE621472, AI680498, AI269696, AV714710, AI655841, AA910956, AI890223, AI921281, AW169039, AI686073, AI583085, AV682224, AW162189, AW105601, AV648430, AV714100, AI567612, AI697372, AI702073, AW827227, BG179993, AI866082, BE018334, BG114304, AI633000, BG112239, AI950664, AL039086, AL046849, AI816947, AW302954, AV734180, AL036980, AV682791, AW169604, AI249877, AW082088, AL041105, AI866770, AI887772, AA427700, AI376180, AW002174, AW089179, BG036846, BE966927, AA807352, AI241923, AW167918, AV755589, AI864836, BE892503, AI621362, AL048323, AL040827, AI570807, AI634345, AL048340, BE785868, AI608932, BE910373, AI537677, AI879064, 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U38847.1, AF230496.1, AF183393.1, AF090900.1, BC006525.1, AL137533.1, AK026532.1, AK025092.1, AF159615.1, 578214.1, 561953.1, AK000614.1, AK026480.1, AL117578.1, AL023657.1, AF061943.1, X72889.1, AL389935.1, AJ299431.1, AF143723.1, U58996.2, AL133557.1, AF056191.1, AF081197.1, AL122123.1, Y16645.1, AK000083.1, AF057300.1, AF057299.1, AL162083.1, AL122049.1, AL137526.1, AB063070.1, AL080159.1, AL137560.1, BC006103.1, AL117460.1, AL080074.1, AK027160.1, AK026592.1, AK000418.1, AF090943.1, AL359583.1, AF217987.1, AB056420.1, AI242859.1, AL136749.1, AL110171.1, BC008382.1, AL137640.1, Y10936.1, AL137271.1, BC008075.1, AK027204.1, AFL13222.1, BC004958.1, AL359601.1, AL583915.1, AL133606.1, AK026593.1, AL050366.1, AK000718.1, AF162270.1, AF026816.2, AK026642.1, Y10080.1, AK026784.1, AB055368.1, AL117435.1, BC008780.1, AF217966.1, BC006440.1, AB047801.1, AL137529.1, BC007680.1, AK027614.1, AK000486.1, AL080148.1, AK026600.1, AL136805.1, X98834.1, AB049892.1, AL080086.1, BC008488.1, BC003627.1, AK000618.1, AL122050.1, BC009310.1, AL110197.1, AB060908.1, AL136789.1, AF090903.1, AL157482.1, AL512765.1, AK000450.1, AL137273.1, AL136845.1, AL133113.1, AL137292.1, AK027116.1, AF285167.1, AB019565.1, AL512719.1, AL359941.1, AK026533.1, BC009026.1, AL136622.1, AL136615.1, AK026045.1, AF225424.1, AL122106.1, AB056421.1, AK025084.1, AK025209.1, AK026741.1, AK027144.1, AK025708.1, AL442072.1, AL117440.1, AL122093.1, AB062978.1, AL137294.1, AK026542.1, AF061573.2, AK025383.1, AL162004.1, AK026534.1, AL162002.1, AB060912.1, AF090934.1, AL1512718.1, AB048964.1, AB050418.1, AL512761.1, BC004951.1, AF210052.1, AB048954.1, AK026630.1, AB063084.1, AL1133665.1, AL1133075.1, AL049465.1, AF061795.1, AF151685.1, AK025254.1, BC007198.1, AL1157431.1, AL137527.1, BC003122.1, AY033593.1, AL136882.1, AF207829.1, AF081195.1, X53587.1, AL1137276.1, AF271350.1, BC006164.1, U39656.1, AB063008.1, AB050534.1, BC005890.1, AL1353957.1, AF177336.1, AK026885.1, L30117.1, AB060903.1, A8060825.1, AB056809.1, AF262032.1, AL359620.1, AK026551.1, AL1050393.1, BC007021.1, 576508.1, BC008983.1, BC008387.1, AK027114.1, L19437.2, AK026504.1, BC008070.1, AK025967.1, AK026528.1, BC008284.1, AL1136786.1, AL1117585.1, U68233.1, AL1110221.1, AL162062.1, AL050L16.1, AL096744.1, AL1133077.1, AL1110225.1, BC006807.1, AL1050138.1, AJ006417.1, AF106862.1, AL136790.1, BC009341.1, AK026408.1, AL1137429.1, BC009033.1, M64349.1, AF097996.1, AL1389978.1, Z82022.1, BC006180.1, AB060929.1, AL1136843.1, AL1133016.1, BC003684.1, AF125948.1, AF090896.1, AB056768.1, AB060214.1, AL1117432.1, AL110222.1, AL1137521.1, AL137300.1, AL1080060.1, H5AYC41 152 688057 1-200 15-214 AF001545, AA548754, AA909788, AW468262, AI751080, AI251827, AA576709, AI800743, AA875953, AW131183, AW149412, AW339554, AI263391, AW168132, BE300485, AI638563, AI920829, AI751079, AV700614, AI682030, BF688845, AA665727, BC008593.1, AC000159.6, AB007864.1, H5LHX15 153 777861 1-641 15-655 AW068913, AU147309, AW963838, Z21897, AV718844, AV724520, AV742001, AV720464, AV718681, AV720731, AV742667, AV722801, AI051434, D80219, D80253, AV743601, AW973447, AV700229, AV719000, AV70L017, AV701443, D80227, AV718692, AV701248, AV723927, AV745080, AA704077, AV742720, AV701428, AV744934, AV70I166, AV700889, AV743008, D51250, AV718489, AV701043, AV741012, AV719783, AV699447, D80240, D59275, AV721784, AV745724, AV744773, T35625, AV701125, AV701332, AV744771, AV720607, AV699927, AV701261, AV701154, AV701012, AV744768, AV740535, D80043, AV745723, AV743654, AV701118, AV737584, D80210, AV701163, AV745831, D51423, D59787, AV720034, D80134, AV720220, AV699479, D59619, D80391, AV746385, AV701431, D80193, AV745369, AV745488, AV746335, AV744770, AV745366, AV700622, AV720812, AV741888, D59927, D80949, AV745392, D80196, C14227, AV746162, AV719568, AV701004, AW384437, AV701149, D80366, AV738934, D80168, AV720203, AV718707, AV723097, T11051, AV701055, AV701145, D80045, AV699746, D81026, AV699669, AV701227, D50995, AV701335, AV719822, AV718858, AV720150, AV745490, C14014, D59889, AV745621, C75259, AV701121, AV720211, AV701231, AV701422, AV701344, AW063533, AV699550, AV719913, AV701357, AV701021, AV745920, AV700895, AV699682, AL039150, AL038821, AL039085, AL043445, AV701013, AV745388, AV745847, AV719324, AW064110, T24112, T23947, C15076, AL043441, AV701330, AV701151, AL039156, AV745583, AV745411, AV699411, AL043422, D80522, T24119, D80022, AV742996, BF509207, AL039564, AL039538, AL039108, D80038, AL039678, AV699866, AL039074, AL038837, AL039625, AL039648, AI039629, AL037726, AL038531, AV746102, AV758878, AV745853, AL039509, AL039109, AL040992, AL039924, AL039566, D80195, AL039128, AL044407, AL039659, AL036973, T11417, AV717990, AV717964, AV717956, AJ293456, AL045337, AL037051, AL045353, AL039386, AL036725, AL039423, D58283, AV701130, AL045794, AV717952, AV717960, AL045341, AV718021, BF294063, AV735727, AL042909, AV717949, AL039410, AV745917, AV717989, AV717980, AV701782, AV718016, AV717963, AV717965, AV717983, AV717962, AV718018, AV717984, AV718002, AV717988, AV731085, AV742671, D81030, AV745496, AV717955, H00069, AV717968, AV718023, AV718006, AV717959, AV717966, AV717944, AV717972, AV701419, AV721386, AV718020, AV718017, AL038025, D51799, D80188, AW959570, AW969383, AV718014, AV701415, AI557751, AW949645, AV745197, AV717946, AV717941, AC016732.7, AK022198.1, AF271371.1, D34614.1, L27636.1, Z96142.1, X73004.1, M244003.1, AJ244004.1, AJ244005.1, D14548.1, Y11923.1, 578798.1, X67155.2, Y11926.1, D88547.1, X92518.1, 583538.1, M32676.1, AF058696.1, 565373.1, Y11920.1, X73003.1, AB028859.1, AB035274.1, AB002449.1, X98248.2, X60736.1, D50010.1, AB033111.1, AB038216.1, U79457.1, D61405.1, U50871.1, H5NBM34 154 635131 1-2172 15-2186 AL536186, AL531092, AU122269, AU141256, AL517543, AL536185, BE796661, AI052692, BG253425, BE902706, BG259810, BF343446, BE902245, AU121842, BE256726, AL517542, BG120047, AW149519, AI569076, BG250631, AI683339, BE902473, AU139256, AL513575, AI679283, BE049376, BF219821, AL048381, AI065090, BE867031, BE744360, BE256766, AI935198, AA573792, AI963086, BE250714, AW173560, AI690938, AA573194, AW172834, AW192800, BF570159, AW338466, AW173515, AI871886, AW264055, AW083435, AL513576, AI972125, AV692704, AI569959, BE252221, AW081978, BF569657, BE250299, AI355854, AU137971, AI884543, BF971275, BE279755, AU134876, BG119978, AA553785, AL535675, BG256137, AI624040, AW168911, BE251490, AI983677, BE617477, AA573798, AW770904, AA553547, AW338256, BF569362, AAI51761, BE731933, BF914823, AW673207, AW058092, BE378102, BE903055, BF914811, BE328617, AA890369, AW469165, AA614295, BF569993, BE293188, BG024165, AI679858, AA527510, AI954296, AA984269, BG167033, BF972479, BG249971, AW591878, AV698722, BE546239, AU147891, AI826939, AI570855, AA552428, AI905936, AI569573, AL039833, AW250413, BF036826, AI683139, AV693761, AW169566, AL535674, BE962440, AL045941, AA677605, AI336684, BF913847, AW440576, AI205324, AA904129, AI564975, AI755277, BF914809, AI159924, AI521405, AW088700, BE677468, 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AK000652.1, AK026464.1, AK026504.1, AL110196.1, AK000445.1, AK026583.1, AK000647.1, BC008417.1, AL137550.1, AL512754.1, AK025958.1, AK025414.1, AK027204.1, BC006807.1, AB055303.1, AB060887.1, Y14314.1, AK025312.1, AL050149.1, AL133016.1, BC003684.1, AK025772.1, AL442072.1, AL353940.1, AL110225.1, U91329.1, AK027868.1, AF078844.1, AF091084.1, AL080159.1, AL137271.1, AB047941.1, Z82022.1, AK027193.1, AK000083.1, BC002733.1, AK026647.1, AL137488.1, AL136892.1, BC006201.1, AF321617.1, AF026816.2, AK026533.1, AK027113.1, AL049466.1, AL137526.1, AK026526.1, AL049430.1, AL137560.1, AB063008.1, AF217987.1, AK027164.1, BC009212.1, AB062938.1, AK025632.1, AY034001.1, AF061795.1, AF151685.1, BC004556.1, AF090901.1, AL389982.1, AL080124.1, AL080060.1, AB060912.1, X82434.1, AK026353.1, BC008899.1, AK000432.1, AF218014.1, AB047615.1, AK026959.1, BC004951.1, HTGGM44 166 842856 1-3002 15-3016 AW974580, BE764084, AA651951, W01997, H68969, AI459019, H70945, AA486949, T66948, T66949, AA486772, BF329143, BF108414, AW469166, AI568694, T57664, AL133623.1, HTHBZ06 167 832477 1-609 15-623 BG107523, BG180234, BF668800, AL514985, BF339863, AI400160, AI566873, BE909457, AW262875, BE906621, AW470063, AI758577, BE907206, AA777509, AV715444, AWL31846, AA406614, AW087747, AI811951, AI371781, AI742506, AI337891, BE738291, AA934901, N40173, AW157527, AI742505, AI374781, AI081113, AW173107, AI379523, AV756830, AI139790, AAI95689, AI801399, BG054839, AA532727, AA235284, AI087379, AI792601, AI952545, AI245243, BG026067, AI805770, AA600140, AI040546, AV703045, AI753737, AA625963, AW591860, AAI59931, AA477326, AI360032, N40209, AI864174, BF197737, AA430365, AI829158, AI869836, AI955815, AI804015, BF909529, N30689, AA478600, C16344, BE906555, AI640196, AW072764, BF306291, AA905154, AA481723, AA758776, AI371005, R78607, BE783860, AA865424, AW242058, AI185821, H64413, AA302463, H64463, C16267, C15288, AV750104, H92638, AW972807, AI566669, AA302462, AI468749, AW090440, AI336687, AI934133, AW512971, BF542108, C16184, C16080, R78608, F05286, AI269595, AW800298, AA021044, C16548, AA315033, R46768, R35721, BE885010, BF870311, R25875, F02653, BF675020, AA761420, F22486, D57610, F00189, AA906821, BF028115, BF081348, AW367390, BE465797, Z24901, AW963734, C16270, AA527113, AA527036, AA373921, BE074601, AA479641, BE074603, AI680768, AW316622, AW606067, AW606056, AW370132, BF766618, C16603, C16455, C16334, AI273095, AA833555, AA479334, AA657967, AV748779, BE737983, AW972454, AI857650, BE925004, D11915, AA479335, D12263, AAI65042, BF379271, BF087468, H89161, BAL743712, AA732498, AV646354, D12285, AW954020, AW085952, AL514986, AA363869, AV736361, D12284, AA989012, AF006484.1, AB006077.1, AC010904.10, AL139184.8, HTLBT80 168 840045 1-2087 15-2101 AL519674, AL518564, AL530114, AL524125, AL518563, AL524124, AL519673, BG119582, BE736220, BE734743, BE782619, BF795936, BG248163, BE869185, BE893350, BF435225, BE905414, 8G026633, BF033083, BE970046, BE885230, BF692625, BF692541, BE892741, BE393898, BE389940, BG163906, BE313920, BE312030, AI741613, AI762578, AI241474, AI813813, AI922418, AI990378, AA018345, AW631237, AW151233, AI400794, AI420163, BE550276, AI949071, AW963076, AI922430, AA614565, AW051437, AA018346, AA612852, AI000311, AI338519, AI360869, AW613433, AI356485, AW590872, W56183, AA417581, AI214800, AI671156, AA451942, BE242648, AW390145, AA00L0L9, R69763, AI419907, AA768838, AA482598, AI696492, AW473585, AI674961, BE868575, AI247090, H08477, AA576510, BE391031, H06603, AI018102, T56902, W56260, BF372172, AA00L020, AI285366, R87304, BF928779, BG152437, AA814595, AI569050, AI239612, H06633, AI623626, R69764, AA971138, H07140, BE856299, AI357631, AI001995, AW243905, R48323, BF003017, H43938, H52166, AA026966, BF988194, W22979, BF372164, H89737, R87305, BF677308, R48432, AA347636, AW080561, AA593837, AI459770, T56903, H08759, AW750152, AA876261, AA450330, AW797547, BF508949, BE833483, BF512980, AW300516, BE076134, AA514688, AI476002, AW884714, BF992680, AI568120, AA578364, H89800, BE503792, AA344443, T77366, BF8I1395, AV699752, BF748071, AW367168, AA450329, BE833631, AW198236, R30662, BG115081, BGI15274, AW275152, AA216315, AW821860, AA347637, AW953095, BF088397, AF132949.1, AL133227.15, Z55376.1, Z55375.1, HTLDU78 169 637702 1-1304 15-1318 AA417099, AA435761, AA417203, BF748721, AA972917, AI660387, BF748720, AC011444.5, HTLFA13 170 535937 1-1146 15-1160 AA599080, AC018808.4, AC034193.4, AC022007.3, AC090958.1, AC018841.3, AC022234.3, AC007999.12, AC018828.3, AC018758.2, AF168787.1, U91321.1, AC002091.1, AC010530.7, AC004765.2, AL031427.15, AF111168.2, AC005911.6, AC021188.6, AC005783.1, AC010150.3, AC078962.30, AC024028.10, AL121585.22, AL049795.20, AL391834.8, AC005829.1, AL353579.17, AC022384.4, AL049776.3, AC007030.3, AL096700.14, AC002352.1, AC005011.11, AL158830.17, AC006130.1, AL139329.15, AC018500.3, AC005077.5, AL136137.15, AL022315.1, Z85996.1, AAL001752.1, AC025165.27, AC006449.19, AC009144.5, Y18000.1, AC005056.2, AC006211.1, U95742.1, AC010956.12, AL117381.32, AC008162.3, AC022392.4, AL137244.28, AL050335.32, AC011247.10, AC005095.2, AL121601.13, AC005531.1, AC074121.16, AC004491.1, AL049780.4, AL035704.9, AC010422.7, AC018812.5, AC007707.13, AC018644.6, AC008806.4, AL023583.25, AL031985.10, AC018663.3, AC005529.7, AC008755.6, AF190464.1, HTLGI89 171 835069 1-2363 15-2377 AL527649, AL521837, AL521838, AL526812, AL522637, AL516967, BE796246, AL516968, BE733427, AW953470, BE267273, BF984794, BF663431, BE746021, BE744871, BE727677, BE792949, BE891911, BG032127, BE732800, BE880158, BG029699, BF739970, BE547454, BF033906, BF341854, BE410292, BE407731, BF974054, BE730406, BE280141, BE276759, BE396111, BG033296, BE313127, BE266155, BE276474, AW245780, AW245421, BF726872, BG259961, AW170348, AA454052, BE387107, AI125210, BF888054, AW960189, BF109831, BE883630, BE207211, BG024467, AW403899, BF894254, AA789053, AI208601, BF432565, AA031930, AA032048, AI024666, W07699, AW615502, BE296793, AJ001138, AI200867, AI356072, AA622587, AA632148, AA934612, AI369198, AI570447, AA846843, AA758922, BF907531, AI332639, BE276249, BE386995, BE795828, AW271396, AI095065, AI208972, N62476, C06239, AA864754, AI278658, AA431596, AA676722, AW402977, AA099581, BF888062, AI125001, AA609502, BE901965, BE901394, BE797702, BFL10024, BE902040, R87506, AI570705, AA847774, AA062669, AI283903, H80426, AI493468, AA351375, AA766390, AA621386, AUI4I193, AA468656, AL522636, AW960190, AW387425, AA813039, BE733825, H24175, AA361930, AI862627, AA305197, R98946, BE304538, AA975165, N80670, BE019577, AA644232, BE736900, AI034164, AW245820, R23321, AI245639, R78483, BE265415, AW375225, BE266374, BE280632, AAI37081, BE733565, BE901113, AW955959, AW375213, BE267493, AW375220, BE539912, R23244, BE733342, N79196, T16377, T09166, BE799665, BG222879, M85399, R98861, AW080224, AA628355, W73606, AI763081, N50148, AI721021, AW089049, AA226886, AW615653, BE464923, AW089305, AI004405, R87588, T04952, R10049, AW078905, AW083608, H17382, BG223318, AA8I1085, AI245727, AA380030, R78526, H80425, AA226922, AW371367, AW732928, BF931203, R10928, AAI36972, AW377690, AW374893, BE841909, R10879, AA353058, AW374895, AA995577, AI001882, AI015767, AW363526, AA377815, H24067, BG223317, BF931143, BE827975, AA557715, BG122019, AV747893, AA862807, T09167, AW771700, AI338369, AI808072, AA453634, AAI00486, BF987667, BF931921, AA774221, BG059802, BE539623, AAI01981, AA884482, R16694, BF887393, AW749084, BF338616, AA768482, AW473754, AW198229, AA357596, AA379498, AK027528.1, AF290613.1, BC005021.1, BC003387.1, BC003612.1, AF020797.2, AK023863.1, AF225424.1, AF090900.1, AK026865.1, AL133640.1, AK025084.1, AB060916.1, AB063046.1, AL122093.1, AL136749.1, BC008488.1, AL512719.1, AL512754.1, AK026532.1, AK024538.1, AL359601.1, AB055361.1, AK000137.1, AF106862.1, X82434.1, AK025092.1, AL050149.1, AL122121.1, 578214.1, AB063070.1, AL137527.1, AF104032.1, AL512733.1, AL512746.1, AF091084.1, AB060912.1, AL359615.1, AB019565.1, AF090901.1, AL122050.1, AL133560.1, AF090934.1, AB060826.1, AK027096.1, AL359618.1, BC008387.1, BC002733.1, AL162006.1, BC001045.1, AB060852.1, AL442082.1, AK026542.1, AL133606.1, AL049314.1, AK000053.1, AB052200.1, AL136892.1, BC006195.1, BC007021.1, AL117460.1, AK026452.1, AL359941.1, AL162083.1, AK000445.1, AK026045.1, AB060863.1, AB048954.1, AF125948.1, AK026592.1, AB051158.1, AB048953.1, AB047904.1, AL133080.1, BC008899.1, AF218014.1, AB063008.1, AK026959.1, Z82022.1, AB060908.1, AL133075.1, BC003687.1, AF097996.1, BC006807.1, AL137459.1, AJ242859.1, AL117435.1, AL049452.1, BC008417.1, AL137550.1, AF090943.1, AL050277.1, ALL10196.1, AK026741.1, AB060825.1, AL136789.1, AF090903.1, AL136799.1, AL157431.1, AB055315.1, AF078844.1, BC007199.1, AL136928.1, BC001967.1, AK025339.1, AK026608.1, AL359596.1, AL110221.1, AL117457.1, AK026534.1, AL050116.1, AL353940.1, AF090896.1, AL049464.1, AL389978.1, AK026784.1, BC008365.1, AL080124.1, AK000618.1, AL050108.1, AK026533.1, AK026744.1, AB056420.1, AL133016.1, AL137557.1, AF219137.1, AL136586.1, AL049466.1, AK000652.1, AK027868.1, AK026504.1, Y16645.1, AL136844.1, AB062938.1, AB055303.1, AIB055368.1, AB060887.1, AL050146.1, AL050393.1, AL122123.1, AK027113.1, U42766.1, AB047615.1, AB055366.1, AK025491.1, AI117585.1, AF125949.1, AB049758.1, AF111847.1, AL442072.1, AL117394.1.AK026855.1, AK026647.1, AF177336.1, BC003683.1, AK025772.1, AF146568.1, AL389982.1, AL137283.1, AK026583.1, AL117583.1, AL136768.1, AL512718.1, AL133565.1, AL080060.1, AL133557.1, AL096744.1, BC004556.1, AL133093.1, AL136787.1, AK025414.1, AF207829.1, AK000212.1, BC008070.1, AL390167.1, AK024524.1, AL049300.1, AL049938.1, AB048964.1, AK000432.1, AK025958.1, AK027204.1, ALL10225.1, AL080137.1, AL136845.1, AB056768.1, U91329.1, AK025967.1, AL049382.1, AF183393.1, BC004951.1, AK026353.1, AL050138.1, AK026086.1, BC002839.1, AL136786.1, AB056421.1, AK027213.1, AB047801.1, AK026927.1, AL359583.1, BC008485.1, AL512689.1, X65873.1, AK025391.1, AL122098.1, AK000323.1, AL133113.1, AK000614.1, AB052191.1, AL512750.1, AL049430.1, AL137271.1, AL080127.1, AK025524.1, AF260566.1, U80742.1, BC008382.1, AK027164.1, AK026629.1, AX024588.1, AL050024.1, BC008280.1, AK026947.1, AB060929.1, AK000718.1, AL137463.1, AL122110.1, X72889.1, AL137648.1, AX025632.1, AL512765.1, AK000647.1, AL512684.1, AL137521.1, BC008983.1, AL512761.1, AL137538.1, AK026630.1, AB056809.1, AL162062.1, AK025484.1.AK027116.1, BC005168.1, AK026526.1, AL359622.1, HTNBK13 172 831967 1-1146 15-1160 BE799670, BE794458, BF969839, BF116235, BE894258, AI755110, BE693669, AA209372, AA209368, AV702645, AW957276, AV724122, AW517214, AW173346, AAI97278, AI609300, BF726226, AI261762, BE882052, AI400083, AAI12077, AI242204, AAI14827, AA314213, AI741473, AI828740, AI982748, AAI97243, AI140451, BF923463, AA838629, AA854805, AAI14846, N59363, AA931373, AA972617, BF358017, AI687104, AA234016, AA843577, AA625125, AAI33768, AA911212, AI553981, AA304885, AAI33767, R76792, AI559186, AW874604, BF358016, BE180724, AA486696, BG248840, AA733214, R75956, H24319, AA447346, AW839247, AA633116, AI187039, AA877750, AA938362, AA083910, AA384854, N32060, H23039, AA678500, F16874, AA972329, T84723, AW372424, AW372436, AA906710, AW372421, AW372425, AW372444, AW372435, AW372434, AW372427, AW372439, BF897466, AW372423, AW062891, AW372443, AW372420, AW372440, AW372433, AW372437, AW372422, AW372432, AW372442, AW372430, AW372428, AW372419, AW372429, AI383049, AI383050, AW372431, AI702272, AI701769, AW372274, AW372408, AA092723, AA634344, AW805484, AI623944, AI985597, AW073164, AW372407, AA352209, AW749114, AW629894, AA912615, AA825365, AA029876, H55702, BE814640, AA975153.T52666, AI540674, AW073898, AA857847, AA808175, AV721824, AI298026, AW834282, AW952443, AI287233, AI784233, BE966278, AA809897, AL514721, AI698391, AI500714, BE905211, AI978703, BE874163, AA868961, AW051088, AL514025, AI570884, AI250627, AW827249, AI366900, AV713528, AI633125, AI538564, AI915291, AW152182, BE393784, BE843239, AI582932, AI590043, BF970040, BF811804, AI889189, AL514511, BF724894, AA641818, AI866469, BE965208, AI884318, AI638644, AI570056, W74529, AI804983, AL513779, BF814449, AI699823, AI345745, AL514871, AV733582, AI351959, BE896769, AI628337, AL513999, BG026483, AI610714, AW189332, AV726590, BF811802, AI538885, AI274507, AI423198, AV753074, AL043070, AI521005, AI621341, BG029667, AI571966, AI539690, AI500061, AI702527, AI741158, AW020693, BG105241, AI927233, AI979129, AL514279, BF970768, AJ670002, BE881211, BE964961, BE963575, BE967219, AA928539, AV712722, BF089679, AI559524, BE964506, AI583558, AI499570, AW020095, BE784335, AL514455, AI511603, BF750875, AI473536, AL513911, AI922215, AL041150, N33175, AL515033, R41605, AW072349, BF812426, R32821, AV726058, AL513631, AL514357, AL513907, AI815232, BG031068, AL121365, AI859991, BF885082, BE880084, AI872423, AL050345.1, AL021707.2, AL136686.1, AB015347.1, BC008839.1, BC008899.1, AK026959.1, Y10936.1, AK026647.1, AB060893.1, AK027137.1, AL512765.1, AL133637.1, AL389935.1, AL136808.1, AB050431.1, AL110269.1, AL049283.1, AF143723.1, BC002733.1, AF141289.1, AL122100.1, BC001056.1, AL133010.1, AB060877.1, BC002343.1, BC006494.1, AK000250.1, AF184965.1, 578453.1, AB060863.1, BC004925.1, AB047947.1, M85164.1, AK026462.1, BC009311.1, AL049938.1, BC001967.1, AL122045.1, AL080156.1, AL080148.1, AL137480.1, 576508.1, AK024538.1, AF097996.1, BC004899.1, AK026045.1, AK026613.1, AL137284.1, AF155827.1, AL117648.1, BC008063.1, BC005858.1, BC004195.1, BC002372.1, AF058921.1, AF199509.1, AL110218.1, AK000502.1, BC007920.1, BC005825.1, AK024992.1, AL137530.1, AL080110.1, 577771.1, AK024747.1, BC001199.1, BC002697.1, AC020910.5, BC004945.1, X00474.1, AK026744.1, ALL10228.1, AL117394.1, AB047904.1, Z82022.1, BC008285.1, AL136747.1, AK025708.1, AL110225.1, BC004556.1, AL133623.1, AK025889.1, AL137627.1, AJ406939.1, AL049382.1, BC002409.1, AL050280.1, AF090903.1, AL137533.1, BC006287.1, AF227198.1, D83032.1, AF177340.1, BC008920.1, AL136850.1, AK024594.1, BC004530.1, AF069506.1, AF232009.1, BC007926.1, BC004513.1, Y14314.1, AL157482.1, AL133049.1, AL110280.1, AL133665.1, BC006414.1, AB049849.1, BC004991.1, AL110196.1, AL133080.1, AB050410.1, AB047623.1, AX027173.1, AL137574.1, AL137711.1, AB055368.1, AK025312.1, AF274348.1, BC002473.1, AF274347.1, AL133112.1, AK026408.1, BC003658.1, X82434.1, BC009221.1, AF245044.1, BC000253.1, AL080159.1, BC002809.1, BC003614.1, BC004222.1, AK025375.1, AL137523.1, BC005168.1, AK026741.1, BC008037.1, AB056809.1, BC003684.1, AL359941.1, AK026550.1, AK024588.1, AL049339.1, AB055361.1, BC008836.1, AL137560.1, AF217987.1, AL080139.1, AL137529.1, AL389982.1, AK000653.1, BC007499.1, AL137276.1, AF076464.1, AB060912.1, AB048953.1, AB060876.1, AK000I03.1, AL390184.1, AB062938.1, AK025435.1, BC005165.1, AK000257.1, AL136893.1, AB052200.1, AE262032.1, A1136754.1, A1137488.1, A1080126.1, BC004191.1, AL050092.1, AF230402.1, AF098162.1, A1137550.1, AK000690.1, BC005002.1, AK026534.1, A1162083.1, BC008781.1, AK026547.1, BC005805.1, AK026480.1, BC008686.1, AK025339.1, BC003101.1, BC007347.1, A1133075.1, BC002466.1, AK026927.1, AK025857.1, A1136842.1, AL137716.1, AF285167.1, A1162006.1, A1136615.1, BC008918.1, BC006164.1, BC006410.1, AK000630.1, AF183393.1, BC007556.1, AK025254.1, AK027142.1, BC004416.1, AY026527.1, A1133113.1, AL162004.1, AB055315.1, A1356859.12, A1080162.1, A1137526.1, AK000603.1, BC004370.1, BC009310.1, BC006103.1, BC003056.1, BC001652.1, A1117416.1, A1137459.1, BC003569.1, AK000083.1, BC000090.1, AF132730.1, D83989.1, AK027152.1, AK000323.1, A1353956.1, BC001844.1, BC004264.1, BC001969.1, HTOAM11 173 664508 1-1186 15-1200 AV756491, AW500684, AI821931, AI755214, BF725844, AI754567, AI754105, AW576251, AW340905, AL079734, A1040374, BF879045, AAI26763, AI732430, AI380617, T74524, A1120343, BE138594, AA535216, AA533054, AI732458, AAI27499, AI821714, AI792133, AI791913, AA602906, BF725761, AI923052, BF868994, AA714110, BE704103, AI821785, AI358712, AW504168, BF526964, AI755202, H07953, AW961593, AI066646, AW302711, AI144081, AA515733, AA704393, AW407632, AV764259, AW732205, AW973992, BE139267, AW157456, AW957600, AW162697, AV737160, BF917346, AV760941, AI669421, AI357823, AA862227, AA659832, AW275971, AI300054, AW079761, AW502873, BF724838, AA715814, BF977305, AI312309, AA700943, AAL001486.4, AC083868.2, AC006970.6, AC027124.4, AL050335.32, AC009144.5, AC006368.2, AC007011.1, AC005821.1, AC008569.6, AAL002360.4, AC022211.5, A1117334.29, AF038458.1, AC016027.15, AC003101.1, A1159977.10, AF107045.1, AC011464.5, AAL001719.1, AC012627.4, AC016830.5, A1137792.11, AF207550.1, A1121594.6, AC007850.29, AL031845.6, AC073964.3, AC022148.5, AC015853.8, AC008962.8, AC008372.6, AC008551.5, AC003046.3, A1355392.7, AAL000493.1, A1355094.3, AC024163.2, AC020916.7, AC011461.4, AC004840.3, AAL001710.1, AC006120.1, AC004841.2, A1157838.24, AC005932.1, AC002395.1, AC005874.3, AF134471.1, AC008101.15, AC004526.1, A1117381.32, Z83822.1, AC004125.1, AC025593.5, AC005756.1, AL450339.5, AC007227.3, Z83826.12, AC011479.6, AAL002812.3, AC005512.1, AC027130.5, AL035460.15, AC004386.1, AL513008.14, A297357.1, AJ251973.1, AC010463.6, AC084373.24, AF312032.1, AC016770.10, AL136126.34, AL109804.41, AL121890.34, AC011005.7, AC005231.2, AIA45201.14, AC087071.2, AC005702.1, AC006141.2, AAL001670.1, AL078463.11, AL109915.10, Z97632.1, AC005291.1, AC016025.12, AC006468.9, AL133453.3, AL109797.18, AL121601.13, AC005212.1, AC004821.3, AC006285.11, AF254822.1, AAL001759.1, M37468.1, AC009244.24, AC002300.1, AC008969.5, AL160264.22, AC004089.25, AC020947.6, AC016026.13, AL356299.16, AC009996.7, AC016596.5, AC000115.1, AC005015.2, AC008857.5, AC005274.1, AL354932.26, AL022721.1, AC009068.10, AC009812.17, AL135927.14, AC005071.2, AC005051.3, AL136980.5, AF053356.1, AL139396.17, AC002314.1, AL139021.6, AC005225.2, AL096841.6, AL022313.1, AL138756.23, AC090532.1, AC007114.7, AC008753.8, AC006236.1, AAL001717.1, AC011442.5, AC068533.7, AC005793.1, AC008755.6, AC004598.1, AL135752.6, AC018836.4, AC005971.5, AC005914.1, AL049872.3, AL353692.14, AL355497.14, AC002350.1, AL035587.5, U96629.1, AC011497.6, AAL000455.4, AC008124.8, AC005839.1, AF168787.1, AL136137.15, AC007318.4, AL359397.3, AC005091.1, AC078846.2, AC007845.12, AL109976.23, AL138717.6, AL139809.16, AC005695.1, AC009530.5, AL133382.8, AC006345.4, AC004166.12, AC010358.5, AL354836.13, AC006211.1, AL133312.3, AC069255.18, AL353804.22, AC004858.2, AC002429.1, AC008770.6, AL139022.4, AL162390.9, AL008730.1, AC011465.4, AC090941.1, AAL001672.1, AL023284.1, AL096701.14, AF067844.1, AC005041.2, AC022415.5, AC006077.1, Z85996.1, AL353602.10, AAL000555.1, AL352984.4, AC021036.5, AL031658.11, AL034420.16, AC010854.3, AC005215.1, AL133548.6, AL031602.14, AC016769.10, AC011470.5, AC016776.6, AL031276.1, AAL002436.3, AC005606.2, AC012379.7, AC005280.3, AL591076.5, AAL000045.1, AAL000113.1, Z99716.4, AC006026.2, AL590763.1, AL160492.5, AL035410.7, AC034207.4, AC002476.1, AC008812.7, AC013726.7, AC018764.6, AL035455.30, AL390252.9, AL118501.22, AL109743.4, AL136228.8, AC018811.4, AC008267.6, AL133545.10, AL390917.12, AC007057.3, AC026172.3, AC009086.5, AC010319.7, AC004973.1, AC008155.9, AF225899.1, AC007193.1, AL133215.16, HTOHO21 174 732808 1-713 15-727 AI821139, AI791153, BE562162, AW601890, AW801984, BE261016, BE262216, BF316552, BE397707, AW604835, BF508954, AI732110, AA939219, BF311274, AA768121, AF126008.1, U03634.1, AF127481.1, HTPCO75 175 853645 1-1453 15-1467 BF969488, BG109664, BG113966, BF980916, BF970168, BE465437, AA913515, BF667561, N47676, AI984260, AI561226, BE502740, AI016617, BG150307, AW665914, BE081361, BE710057, AA279712, BE220910, AI880662, AI812005, AI223871, AW449123, AA460500, BF184599, N47675, BF243896, AU145432, AA829587, AA931455, T05743, AI685691, BG121044, AA256899, R56024, AA654665, R56025, N51207, BF853453, AI808712, AI765168, BF589254, AW936494, AA910986, BE836669, BF114747, AW377241, AL050136.1, AK021741.1, AC002067.1, Z92542.2, HT5FJ32 176 637720 1-1243 15-1257 BF843130, AU117513, AI681088, AI680701, AI361919, AW157408, C04469, AI955119, AI928912, AI469126, BF116057, AI889802, AV729134, AW162400, BF337597, AW895617, AA863008, AI417558, AW057928, AI815549, AA371959, AW327982, AI678991, H84469, BE382373, R89704, H83576, AV721961, AI753112, AW960529, AW970050, AA416603, AV703569, D53506, AI160829, AA570262, D56033, AI902757, W28382, D55124, AW327983, AV704116, BE771726, AA678993, AA626015, BF677512, AI220523, AI804192, H58710, BF834065, AW961885, C04546, AI816491, BF432083, AW900853, AA974795, AA971398, AA416621, AA916174, AI538211, AA864450, AA909154, AA302583, BF809398, BF196301, AW181916, AU144323, AI123339, AA372034, R37820, H43815, M78721, C04507, H49972, BE932208, AW901234, AW901364, AI565159, AW341471, AA601597, R95024, R94938, H92470, AI799873, D55563, H50010, AA702558, AA574290, AW900864, AW905864, BG015591, R96005, AW903806, R95966, H30648, H20798, T63337, BE243401, AA707707, H27871, BF979913, BF991086, H14147, H30647, H58320, T61965, H56594, H22616, T63962, BE910629, R88595, M78801, H56595, AL036957, BG009728, AA505362, AI915406, BE829368, BE770916, AA977550, BE272026, BF844035, AA910693, AA832013, C20841, BG031911, AF135372.1, AK021522.1, BC002737.1, AJ225044.1, AF240769.1, AL050223.1, M36203.1, M36204.1, HTTCB60 177 853401 1-1490 15-1504 AL527596, AL530567, AI114859, BE407719, AL527597, AL529809, AI830718, AI640304, BF447073, AI862404, AI419019, BE899304, AA496239, AI889002, AL514080, BF000126, AI2003B2, BE048538, AA442132, AI798632, AI140806, AI565622, AU156790, AA834046, AW294987, AI183801, W68566, AI872891, AA456203, AI364465, AJ765479, BE645691, AI468507, BF477789, AI672711, W68567, AI187099, AW135628, AI417504, AI004499, AA815198, AL526637, AI371368, Z19140, AI349321, AI074599, AA442131, AL529295, AA610289, AI620803, W73758, AW880750, BE782057, AI002625, AU136598, BG122646, AA496240, AI298503, AW339508, BE467855, AI310364, AI346793, W73592, AI926388, AW452176, AI884667, AI183908, AW470742, AA593079, N92388, AW771410, AU144823, AW172442, AI086796, AW104502, AW572529, AI381931, AA707604, AI640676, AW571951, AI868834, AI313208, AI861943, AA719132, AW471200, AI990933, AI193543, AI193748, AI921195, AA903834, AW023979, AI825347, AI861932, AI919371, AI248156, AI694324, AA442774, AA701903, BF433373, BF063027, AA885026, AA723857, BF001754, BF588721, AI916136, AI686495, BF062018, AI990711, BF060737, AI202906, AW471024, AI675788, AI910996, AW663382, AW770995, BG057507, BFI10069, BE670537, BF114632, AA427828, AI537838, AI130863, AI651012, AI148378, AA780173, AW079270, AA621634, AI571348, W95356, AA010215, AF206673.2, AK001914.1, AAL000501.1, AF298153.1, AK027296.1, AL110244.1, AB040964.1, AL137610.1, HTEE41 178 840950 1-1959 15-1973 AL533251, AL514520, AL535565.AL519250.AU120401.AL514519.AL513606.AL517678, AI986262, AU139509, AU138912, BF797374, AU124362, AV714807, AI970836, T25350, BG169633, AU126517, B0031251, AA854925, AI683290, AI084631, AU130264, 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BE740789, BF697703, AV759552, BE395111, AI991200, BG178345, BE547992, BG180982, BG170659, AI245997, BE296958, BF218155, BE874752, BG250326, BE537584, BE892651, W37330, AA454974, BE621103, BG249254, AW880574, BE315071, AA724393, AW401755, BF981216, BF724854, BE740318, AW602164, AAI76450, AI798066, BF529155, BF590827, BE938687, AA528038, AAI76930, BE007448, BG260090, AA744235, BE019877, AI755131, BE019722, AA258968, BE007599, AA888876, BE206323, AA454975, W49835, AI658890, AI400439, BE855719, BE965617, AI369432, AI378678, AI814475, BE894966, BE255155, N33371, H30709, AI817038, AI079528, AA988238, AI094675, BE296010, BG055258, BE698523, BF749938, AA781122, BG008471, BE007089, AI078131, BG012658, H28137, AA636058, EF112108, H30791, W52270, AI582733, AI150949, AA410493, AA860936, AAI32898, AI278541, BFL12247, BE044217, BF507690, AA933024, H66966, AA258022, BE247272, AA310300, BE208182, AA256356, H30612, H24913, N47554, AA595919, AA921999, BF772395, AW883922, AI050086, BF820414, AA326738, H66965, W52173, AW630747, AA987810, BF841176, BF841178, H24636, BF886659, AW025934, R85382, AAI76415, AW997264, BE769213, BE873578, AA213621, AA213747, AI090506, AI350930, AI335914, R88068, BE315307, AA306524, R78161, BF772700, BF755261, R25528, BE698518, AA367585, BF880127, BG163239, AW964548, R88419, BE833357, R67073, AW168072, AI866652, R66194, W39693, BF512635, AI921135, R77799, BE742426, R88069, Z25017, BE005706, T52535, AI084635, AW272406, BE005705, AA368103, AA485159, H44289, H21572, AA335920, AAI32726, BE007543, W37836, AI080447, T72649, R23331, AI800373, H11232, AI493270, AA633490, R61159, R48336, R45587, AW591739, BE315089, AA375973, AA091597, AI961784, AW140066, R61873, BF806323, BF342050, Z28722, A1003986, AW608071, T30986, H10607, R48240, AAI43657, T72717, AA378816, AW768907, H28184, AA320516, W45049, BF743131, N47553, BF115823, AA988048, AI698305, AI342699, AA410492, AAI76682, AA366872, BF432748, AW895119, AA329961, AA873382, N51170, AI802557, AW947291, BF432749, AI468036, BE315041, BE814237, H10399, AL045269, AI307370, T52614, AI310496, AW879611, AA463709, AW842163, BE245828, AI364211, AA485063, D20903, BE181425, BE281137, AW998715, AL080223.1, AC007040.2, HUVDJ48 184 564853 1-1813 15-1827 AI479925, AV720735, AI886110, AF261918.1, AB037733.1, HBDAB91 185 864374 1-993 15-1007 AI167963, AA782398, BE043035, AW051006, W07319, R23362, N75825, AW195519, BE858969, AI701657, R45349, AI468816, AW858522, AW577199, AI135012, BE927373, AW601637, BF084778, AL134110, AL134524, AW577201, AL045327, AL045494, AL042523, AW577192, AL045328, BF910726, AL042420, HELGG84 186 851137 1-1095 15-1109 AI822137, AI821793, AI141174, AI742807, AW008096, AI376221, N70665, AI147430, N66810, W05747, AW894967, AI708189, AI792140, N75004, AI597655, AI140241, HILCA24 187 869856 1-1968 15-1982 BE780749, AU137314, AV732875, AW954734, AW138881, BF681107, AI079555, AI624252, AA233208, AU157126, AI734898, AW088851, BE221267, AA314962, AV715966, AA971982, AA233124, AAI29416, AAI33798, AA886808, AA353195, AW132033, T98200, H50558, AI888751, AI818363, BF917932, BE926224, AI784628, H50559, T98201, AK001989.1, AL512750.1, AC010627.5, AC01049 1.3, AC026749.5, AC016656.5, AC016652.5, HYABC84 188 865064 1-1464 15-1478 BE619984, BG180257, BG253753, BE546940, BE795721, BE871790, BE538846, AW953562, AA524254, AW978620, AW970777, AW001609, AI798108, AU159275, AU148477, AA524480, AA476556, AW027610, BE207925, AW166935, AL521960, AI934516, AU149354, AW291597, AW974311, AA443023, AJ186348, AI597811, AI692241, N32579, BE619316, AI689448, AW152379, AI271524, AI093466, AI870536, AI149215, AI432467, AA854903, AA781886, AI199164, AI744310, AL043754, AI372057, BF725035, AI269272, AW770362, AA399350, AI367106, AA463464, AA512239, AW516985, W48832, AWL51757, AI086901, AW571473, AW514611, AA292378, AI079461, BE206384, AA934644, AI269712, AW189899, AA588341, AA676478, AAI26131, AW055258, AW166860, N70058, AA916656, AI874191, AW297040, AW083905, AW664480, AA427894, AA453450, AA843278, AI687474, AA620899, W49813, AA860641, AI692799, BE676833, AA306455, AA292016, T63718, AW236228, AW304861, AW772846, AW468035, BE964404, AI015326, AW662269, AA226903, AW090569, AA304032, AA915898, R56660, AW194056, AA293296, AA047874, H28877, AW076091, AI126745, AA482627, R50813, AA482480, AI674616, BF926901, AA045575, R68388, W37626, H01650, AA598596, AW364253, N59135, AL045883, N23722, AA908894, Z40636, R38774, R68593, AV708320, AI474604, R47766, AI611825, AA022933, BE829256, BF244825, AA888168, F03832, C04718, AA534374, BE297735, R50403, R56826, R81871, T33719, AI569605, N41917, AW731635, AW050454, BF737093, Z44868, AI648412, T06476, AA428005, W37625, BF893080, BF820895, BF769768, F07587, F01530, D54185, AI611777, AA902145, AA338785, Z25279, BE617105, F00464, AA084935, BE241564, R10512, AI627173, BE252263, AA022983, AV743083, BE940174, N75173, BE929227, 1128878, AA094149, AW294418, AI261764, AA449769, T63873, AI885556, AA775981, BE710635, BE936751, BC008836.1, BC001323.1, AL117480.1, AL132825.35, AF055022.1, AL096738.1, HMCAZ04 189 668249 1-1287 15-1301 AL523496, AI804917, AW973481, AV716073, AW024415, AI302184, AV699357, AV700904, AW273859, AL523497, AA948547, BG236229, AW043839, AW516856, AI299954, BF108769, BE819542, AA911904, AA639665, AI133370, BE819457, BE819514, BE839222, AI097457, AI222830, BE819567, AW182738, AI522298, BE819505, AW119093, BF691126, AI305813, AW955102, W96028, BE839045, BE819511, BE831532, BF371410, N50841, BE819590, BE819554, AA527252, AA526991, AA643646, AI751876, AI290542, W94661, BE674172, AI240325, AI290551, AW050812, BF219419, T74688, AV659324, AI300130, AA885922, AAI00857, BE819468, BE772570, AA838720, AI400510, AA664347, C75117, AA056968, N54246, BE819545, AA481324, AA281439, AA486172, AI880359, AA468902, BE939493, AV660065, AV719793, T95295, D20113, BF379099, AA643688, AI394339, BE819461, BEB31675, AW854105, AW176080, T95375, AI890004, N58703, R50203, AW071048, AA280758, T74801, AW054934, T64005, Z19305, BE716219, AI635828, BE301581, AI928438, AA602300, AI685551, AW970142, AA514233, AV716521, BE819546, BG120804, BF027170, AW970145, BF752093, T36144, AI262366, BE789575, AV692708, R40496, BF912723, T86288, AW273588, AI273020, W45353, AA486109, T86387, T90950, T85836, AI635443, BF359777, AA587303, BE839081, AA533897, AW768388, T98225, R10150, T64084, BG169469, BE264000, BE819498, AA513774, AW945448, AA669130, AI872115, BE386262, T27349, BE819456, AA385681, AA253283, AI367142, AW316697, AI758444, W15319, AW467705, AV685386, AI819151, AI571469, BF359773, AV688204, BE546468, BG170954, AAI30233, AI557206, BE831556, BE831640, BE790051, AI613022, AA359423, BE008461, AI401483, AA576059, D82703, BF678342, AA353447, BF000110, BF359762, AA359712, BE018093, AV645442, AV645491, AA703984, AF151802.1, AF042284.1, AC090527.3, AC068722.6, HE8FD92 190 901142 1-3963 15-3977 BG252727, BG260973, BG180624, BG122157, BE887548, AW948984, AW950907, BF965913, AI905469, BE869433, B0032175, AV718334, AV650921, BF793347, BE148077, BF793887, BG120463, BF204516, AW969050, BG121940, AL045343, AV649638, BE621758, AL037725, BE620934, BG169462, AV708063, AL045344, BE892224, AV708871, AV649666, AV653240, AV721974, BF740216, BG110357, AI888249, AV649944, AI814624, BE615208, BE613500, BE540375, AL043485, AA401244, BGL17844, AV705742, AV762287, AW614902, AI768623, AA404260, W95853, BE614234, AW167131, BE542745, BF844095, AW955266, BE551076, AI800419, AL040955, AL040932, BF891913, N32025, BF028570, BF980034, BE147946, AA315005, AL531145, AW020997, AI523819, BE162196, AV689195, AW196856, AL048907, BF378885, BE615086, AW851278, AW948982, AW069571, BF219231, AW664296, BF839881, BE299495, AI859769, AI804043, BE615260, AW439581, AI979070, AW873118, BG028790, AW173627, AW138481, BF836425, BE090183, AW470149, W95142, AI954566, AW966417, AW382691, AA431381, AI923608, AI920788, BF836432, BG028439, AI1074015, AW401596, AI096566, AA777093, AW168867, BE350092, AI872178, AL040146, BF832429, BG179393, AW196804, AJ634706, AI400065, AI983052, AL044108, BF571648, AW874528, AI200933, W94444, AI080172, AI073801, AV728394, BG107927, AA614709, AW963612, AAI34828, BE349345, AA463288, AA625480, AA953123, BE537489, AI589346, AW261884, BF757706, AI026817, AI654244, BE140097, AA932332, AV649957, AW002764, AI435532, N51228, AA725887, AI685954, AI247070, R97631, AI142752, AI569997, W81307, AI473795, AA962014, AA946853, AI916323, AW382688, AW954051, BF725969, AI000904, N69326, AA774786, AI445189, AI348365, BF433144, C17016, BE764894, AW193468, AI274717, AI420632, AI471592, AL040335, N94040, AAI34827, BE156276, W94957, BE151110, AW452484, AV726157, AI077452, AI675925, AW675613, AV694497, AV683792, AA868559, AA504467, AV684722, AI241864, AI826322, AA448916, AA872701, AA398843, BE966521, BF940653, BF804717, BG036364, AI865471, AW474946, AI088270, BE046420, BE764877, W45368, H27416, AV649926, AW020365, AV649742, AW241206, AV649875, BE857361, AA431829, BF923515, AI359458, AI439074, W94259, AL045559, BF998123, T78688, AI174662, AW377563, AV649995, BE764899, AI094039, AW797490, AA463197, AA494404, AI520933, AA609501, AV659915, AV662147, AI125505, BG111536, AI167605, AI149616, AV698723, AA609104, AI913467, BE835924, BF197860, AV651144, AA431425, AA975219, AA635251, BE710504, BE816681, D61350, AI811787, AV734255, AA341676, AV662120, AI368857, AA630621, AF131738.1, AB051480.1, AL117237.1, AL136890.1, AL022240.8, AL359752.11, AL049715.25, AB033071.1, AL050141.1, AK000726.1, Y14436.1, T49441, T49442, T65179, T90631, T79315, T79742, T83158, T85864, R16090, R15724, R18665, R22674, R38982, R39314, R41620, R43379, R41620, R43379, H24497, H29599, H44475, H65354, H65563, N69549, W17223, W40134, W92875, W95598, W95599, AA045010, AAI94855, AA470492, AA470959, AA483167, AA514556, AA557980, AA729817, AA738229, AA805073, AA908302, AA911490, AA938675, AA962188, AA976144, N55792, N74638, N83414, C01293, N88703, C14561, C14674, C15258, C15727, C15842, AA091964, AA094573, AA095891, AA247583, AA449426, AA599645, AA663625, AA665721, AA813724, AI003102, T10450, D31148, F11837, AA694588, AI251461, AI270073, AI280943, AI582507, AI126993, AI203813, AI247468.

Description of Table 4

Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B.2, Column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
TABLE 4 Code Description Tissue Organ Cell Line Disease Vector AR022 a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammary gland a_mammary gland AR025 a_Prostate a_Prostate AR026 a_small intestine a_small intestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood B cells activated Blood B cells activated AR030 Blood B cells resting Blood B cells resting AR031 Blood T cells activated Blood T cells activated AR032 Blood T cells resting Blood T cells resting AR033 brain brain AR034 breast breast AR035 breast cancer breast cancer AR036 Cell Line CAOV3 Cell Line CAOV3 AR037 cell line PA-1 cell line PA-1 AR038 cell line transformed cell line transformed AR039 colon colon AR040 colon (9808co65R) colon (9808co65R) AR041 colon (9809co15) colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9808co64R) AR044 colon cancer 9809co14 colon cancer 9809co14 AR050 Donor II B Cells 24 hrs Donor II B Cells 24 hrs AR051 Donor II B Cells 72 hrs Donor II B Cells 72 hrs AR052 Donor II B-Cells 24 hrs. Donor II B-Cells 24 hrs. AR053 Donor II B-Cells 72 hrs Donor II B-Cells 72 hrs AR054 Donor II Resting B Donor II Resting B Cells Cells AR055 Heart Heart AR056 Human Lung Human Lung (clonetech) (clonetech) AR057 Human Mammary Human Mammary (clontech) (clontech) AR058 Human Thymus Human Thymus (clonetech) (clonetech) AR059 Jurkat (unstimulated) Jurkat (unstimulated) AR060 Kidney Kidney AR061 Liver Liver AR062 Liver (Clontech) Liver (Clontech) AR063 Lymphocytes chronic Lymphocytes chronic lymphocytic leukaemia lymphocytic leukaemia AR064 Lymphocytes diffuse Lymphocytes diffuse large large B cell lymphoma B cell lymphoma AR065 Lymphocytes follicular Lymphocytes follicular lymphoma lymphoma AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) (4004901) AR068 Normal Ovary Normal Ovary 9508G045 9508G045 AR069 Normal Ovary Normal Ovary 9701G208 9701G208 AR070 Normal Ovary Normal Ovary 9806G005 9806G005 AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (9702G001) (9702G001) AR073 Ovarian Cancer Ovarian Cancer (9707G029) (9707G029) AR074 Ovarian Cancer Ovarian Cancer AR075 Ovarian Cancer Ovarian Cancer (9806G019) (9806G019) AR076 Ovarian Cancer Ovarian Cancer (9807G017) (9807G017) AR077 Ovarian Cancer Ovarian Cancer (9809G001) (9809G001) AR078 ovarian cancer 15799 ovarian cancer 15799 AR079 Ovarian Cancer Ovarian Cancer 17717AID 17717AID AR080 Ovarian Cancer Ovarian Cancer 4004664B1 4004664B1 AR081 Ovarian Cancer Ovarian Cancer 4005315A1 4005315A1 AR082 ovarian cancer ovarian cancer 94127303 94127303 AR083 Ovarian Cancer Ovarian Cancer 96069304 96069304 AR084 Ovarian Cancer Ovarian Cancer 9707G029 9707G029 AR085 Ovarian Cancer Ovarian Cancer 9807G045 9807G045 AR086 ovarian cancer ovarian cancer 9809G001 9809G001 AR087 Ovarian Cancer Ovarian Cancer 9905C032RC 9905C032RC AR088 Ovarian cancer 9907 Ovarian cancer 9907 C00 C00 3rd 3rd AR089 Prostate Prostate AR090 Prostate (clonetech) Prostate (clonetech) AR091 prostate cancer prostate cancer AR092 prostate cancer #15176 prostate cancer #15176 AR093 prostate cancer #15509 prostate cancer #15509 AR095 Small Intestine Small Intestine (Clontech) (Clontech) AR096 Spleen Spleen AR097 Thymus T cells Thymus T cells activated activated AR098 Thymus T cells resting Thymus T cells resting AR099 Tonsil Tonsil AR100 Tonsil geminal center Tonsil geminal center centroblast centroblast AR101 Tonsil germinal center Tonsil germinal center B B cell cell AR102 Tonsil lymph node Tonsil lymph node AR103 Tonsil memory B cell Tonsil memory B cell AR104 Whole Brain Whole Brain AR105 Xenograft ES-2 Xenograft ES-2 AR106 Xenograft SW626 Xenograft SW626 AR119 001: IL-2 001: IL-2 AR120 001: IL-2.1 001: IL-2.1 AR121 001: IL-2_b 001: IL-2_b AR124 002: Monocytes 002: Monocytes untreated untreated (1 hr) (1 hr) AR125 002: Monocytes 002: Monocytes untreated untreated (5 hrs) (5 hrs) AR126 002: Control.1C 002: Control.1C AR127 002: IL2.1C 002: IL2.1C AR130 003: Placebo-treated 003: Placebo-treated Rat Rat Lacrimal Gland Lacrimal Gland AR131 003: Placebo-treated 003: Placebo-treated Rat Rat Submandibular Submandibular Gland Gland AR135 004: Monocytes 004: Monocytes untreated untreated (5 hrs) (5 hrs) untreated 1 hr 1 hr AR139 005: Placebo (48 hrs) 005: Placebo (48 hrs) AR140 006: pC4 (24 hrs) 006: pC4 (24 hrs) AR141 006: pC4 (48 hrs) 006: pC4 (48 hrs) AR152 007: PHA(1 hr) 007: PHA(1 hr) AR153 007: PHA(6 HRS) 007: PHA(6 HRS) AR154 007: PMA(6 hrs) 007: PMA(6 hrs) AR155 008: 1449_#2 008: 1449_#2 AR161 01: A - max 24 01: A - max 24 AR162 01: A - max 26 01: A - max 26 AR163 01: A - max 30 01: A - max 30 AR164 01: B - max 24 01: B - max 24 AR165 01: B - max 26 01: B - max 26 AR166 01: B - max 30 01: B - max 30 AR167 1449 Sample 1449 Sample AR168 3T3P10 1.0 uM insulin 3T3P10 1.0 uM insulin AR169 3T3P10 10 nM Insulin 3T3P10 10 nM Insulin AR170 3T3P10 10 uM insulin 3T3P10 10 uM insulin AR171 3T3P10 No Insulin 3T3P10 No Insulin AR172 3T3P4 3T3P4 AR173 Adipose (41892) Adipose (41892) AR174 Adipose Diabetic Adipose Diabetic (41611) (41611) AR175 Adipose Diabetic Adipose Diabetic (41661) (41661) AR176 Adipose Diabetic Adipose Diabetic (41689) (41689) AR177 Adipose Diabetic Adipose Diabetic (41706) (41706) AR178 Adipose Diabetic Adipose Diabetic (42352) (42352) (42366) AR180 Adipose Diabetic Adipose Diabetic (42452) (42452) AR181 Adipose Diabetic Adipose Diabetic (42491) (42491) AR182 Adipose Normal Adipose Normal (41843) (41843) AR183 Adipose Normal Adipose Normal (41893) (41893) AR184 Adipose Normal Adipose Normal (42452) (42452) AR185 Adrenal Gland Adrenal Gland AR186 Adrenal Gland + Whole Adrenal Gland + Whole Brain Brain AR187 B7(1 hr)+ (inverted) B7(1 hr)+ (inverted) AR188 Breast (18275A2B) Breast (18275A2B) AR189 Breast (4004199) Breast (4004199) AR190 Breast (4004399) Breast (4004399) AR191 Breast (4004943B7) Breast (4004943B7) AR192 Breast (4005570B1) Breast (4005570B1) AR193 Breast Cancer Breast Cancer (4004127A30) (4004127A30) AR194 Breast Cancer Breast Cancer (400443A21) (400443A21) AR195 Breast Cancer Breast Cancer (4004643A2) (4004643A2) AR196 Breast Cancer Breast Cancer (4004710A7) (4004710A7) AR197 Breast Cancer Breast Cancer (4004943A21) (4004943A21) AR198 Breast Cancer Breast Cancer (400553A2) (400553A2) (9805C046R) (9805C046R) AR200 Breast Cancer Breast Cancer (9806C012R) (9806C012R) AR201 Breast Cancer (ODQ Breast Cancer (ODQ 45913) 45913) AR202 Breast Cancer Breast Cancer (ODQ45913) (ODQ45913) AR203 Breast Cancer Breast Cancer (ODQ4591B) (ODQ4591B) AR204 Colon Cancer (15663) Colon Cancer (15663) AR205 Colon Cancer Colon Cancer (4005144A4) (4005144A4) AR206 Colon Cancer Colon Cancer (4005413A4) (4005413A4) AR207 Colon Cancer Colon Cancer (4005570B1) (4005570B1) AR208 Control RNA #1 Control RNA #1 AR209 Control RNA #2 Control RNA #2 AR210 Cultured Preadipocyte Cultured Preadipocyte (blue) (blue) AR211 Cultured Preadipocyte Cultured Preadipocyte (Red) (Red) AR212 Donor II B-Cells 24 hrs Donor II B-Cells 24 hrs AR213 Donor II Resting B- Donor II Resting B-Cells Cells AR214 H114EP12 10 nM H114EP12 10 nM Insulin Insulin AR215 H114EP12 (10 nM H114EP12 (10 nM insulin) insulin) AR216 H114EP12 (2.6 ug/ul) H114EP12 (2.6 ug/ul) AR217 H114EP12 (3.6 ug/ul) H114EP12 (3.6 ug/ul) AR218 HUVEC #1 HUVEC #1 AR221 L6 undiff. L6 undiff. AR222 L6 Undifferentiated L6 Undifferentiated AR223 L6P8 + 10 nM Insulin L6P8 + 10 nM Insulin AR224 L6P8 + HS L6P8 + HS AR225 L6P8 10 nM Insulin L6P8 10 nM Insulin AR226 Liver (00-06-A007B) Liver (00-06-A007B) AR227 Liver (96-02-A075) Liver (96-02-A075) AR228 Liver (96-03-A144) Liver (96-03-A144) AR229 Liver (96-04-A138) Liver (96-04-A138) AR230 Liver (97-10-A074B) Liver (97-10-A074B) AR231 Liver (98-09-A242A) Liver (98-09-A242A) AR232 Liver Diabetic (1042) Liver Diabetic (1042) AR233 Liver Diabetic (41616) Liver Diabetic (41616) AR234 Liver Diabetic (41955) Liver Diabetic (41955) AR235 Liver Diabetic (42352R) Liver Diabetic (42352R) AR236 Liver Diabetic (42366) Liver Diabetic (42366) AR237 Liver Diabetic (42483) Liver Diabetic (42483) AR238 Liver Diabetic (42491) Liver Diabetic (42491) AR239 Liver Diabetic (99-09- Liver Diabetic (99-09- A281A) A281A) AR240 Lung Lung AR241 Lung (27270) Lung (27270) AR242 Lung (2727Q) Lung (2727Q) AR243 Lung Cancer Lung Cancer (4005116A1) (4005116A1) AR244 Lung Cancer Lung Cancer (4005121A5) (4005121A5) AR245 Lung Cancer Lung Cancer (4005121A5)) (4005121A5)) AR246 Lung Cancer Lung Cancer (4005340A4) (4005340A4) AR248 Monocyte (CT) Monocyte (CT) AR249 Monocyte (OCT) Monocyte (OCT) AR250 Monocytes (CT) Monocytes (CT) AR251 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR252 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR253 Monocytes (INFG 8-11) Monocytes (INFG 8-11) AR254 Monocytes (O CT) Monocytes (O CT) AR255 Muscle (91-01-A105) Muscle (91-01-A105) AR256 Muscle (92-04-A059) Muscle (92-04-A059) AR257 Muscle (97-11-A056d) Muscle (97-11-A056d) AR258 Muscle (99-06-A210A) Muscle (99-06-A210A) AR259 Muscle (99-07-A203B) Muscle (99-07-A203B) AR260 Muscle (99-7-A203B) Muscle (99-7-A203B) AR261 Muscle Diabetic Muscle Diabetic (42352R) (42352R) AR262 Muscle Diabetic Muscle Diabetic (42366) (42366) AR263 NK-19 Control NK-19 Control AR264 NK-19 IL Treated 72 hrs NK-19 IL Treated 72 hrs AR265 NK-19 UK Treated 72 hrs. NK-19 UK Treated 72 hrs. AR266 Omentum Normal (94- Omentum Normal (94-08- 08-B009) B009) AR267 Omentum Normal (97- Omentum Normal (97-01- 01-A039A) A039A) AR268 Omentum Normal (97- Omentum Normal (97-04- 04-A114C) A114C) AR269 Omentum Normal (97- Omentum Normal (97-06- 06-A117C) A117C) AR270 Omentum Normal (97- Omentum Normal (97-09- 09-B004C) B004C) (17717AID) (17717AID) AR272 Ovarian Cancer Ovarian Cancer (9905C023RC) (9905C023RC) AR273 Ovarian Cancer Ovarian Cancer (9905C032RC) (9905C032RC) AR274 Ovary (9508G045) Ovary (9508G045) AR275 Ovary (9701G208) Ovary (9701G208) AR276 Ovary 9806G005 Ovary 9806G005 AR277 Pancreas Pancreas AR278 Placebo Placebo AR279 rIL2 Control rIL2 Control AR280 RSS288L RSS288L AR281 RSS288LC RSS288LC AR282 Salivary Gland Salivary Gland AR283 Skeletal Muscle Skeletal Muscle AR284 Skeletal Muscle (91- Skeletal Muscle (91-01- 01-A105) A105) AR285 Skeletal Muscle (42180) Skeletal Muscle (42180) AR286 Skeletal Muscle (42386) Skeletal Muscle (42386) AR287 Skeletal Muscle (42461) Skeletal Muscle (42461) AR288 Skeletal Muscle (91-01- Skeletal Muscle (91-01- A105) A105) AR289 Skeletal Muscle (92-04- Skeletal Muscle (92-04- A059) A059) AR290 Skeletal Muscle (96-08- Skeletal Muscle (96-08- A171) A171) AR291 Skeletal Muscle (97-07- Skeletal Muscle (97-07- A190A) A190A) AR292 Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42352) (42352) AR293 Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42366) (42366) Diabetic (42395) (42395) AR295 Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42483) (42483) AR296 Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42491) (42491) AR297 Skeletal Muscle Skeletal Muscle Diabetic Diabetic 42352 42352 AR298 Skeletal Musle (42461) Skeletal Musle (42461) AR299 Small Intestine Small Intestine AR300 Stomach Stomach AR301 T-Cell + HDPBQ71.fc T-Cell + HDPBQ71.fc 1449 16 hrs 1449 16 hrs AR302 T-Cell + HDPBQ71.fc T-Cell + HDPBQ71.fc 1449 6 hrs 1449 6 hrs AR303 T-Cell + IL2 16 hrs T-Cell + IL2 16 hrs AR304 T-Cell + IL2 6 hrs T-Cell + IL2 6 hrs AR306 T-Cell Untreated 16 hrs T-Cell Untreated 16 hrs AR307 T-Cell Untreated 6 hrs T-Cell Untreated 6 hrs AR308 T-Cells 24 hours T-Cells 24 hours AR309 T-Cells 24 hrs T-Cells 24 hrs AR310 T-Cells 24 hrs. T-Cells 24 hrs. AR311 T-Cells 24 hrs T-Cells 24 hrs AR312 T-Cells 4 days T-Cells 4 days AR313 Thymus Thymus AR314 TRE TRE AR315 TREC TREC AR316 Virtual Mixture Virtual Mixture AR317 B lymphocyte, B lymphocyte, AR318 (non-T; non-B) (non-T; non-B) AR326 001-293 RNA (Vector 001-293 RNA (Vector Control) Control) AR328 001: Control.1 001: Control.1 AR355 Acute Lymphocyte Acute Lymphocyte Leukemia Leukemia AR356 AML Patient #11 AML Patient #11 AR357 AML Patient #2 AML Patient #2 AR358 AML Patient #2 SGAH AML Patient #2 SGAH AR359 AML Patient#2 AML Patient#2 AR360 Aorta Aorta AR361 B Cell B Cell AR362 B lymphoblast B lymphoblast AR363 B lymphocyte B lymphocyte AR364 B lymphocytes B lymphocytes AR365 B-cell B-cell AR366 B-Cells B-Cells AR367 B-Lymphoblast B-Lymphoblast AR368 B-Lymphocytes B-Lymphocytes AR369 Bladder Bladder AR370 Bone Marrow Bone Marrow AR371 Bronchial Epithelial Bronchial Epithelial Cell Cell AR372 Bronchial Epithelial Bronchial Epithelial Cells Cells AR373 Caco-2A Caco-2A AR374 Caco-2B Caco-2B AR375 Caco-2C Caco-2C AR376 Cardiac #1 Cardiac #1 AR377 Cardiac #2 Cardiac #2 AR378 Chest Muscle Chest Muscle AR381 Dendritic Cell Dendritic Cell AR382 Dendritic cells Dendritic cells AR383 E. coli E. coli AR384 Epithelial Cells Epithelial Cells AR385 Esophagus Esophagus AR386 FPPS FPPS AR387 FPPSC FPPSC AR388 HepG2 Cell Line HepG2 Cell Line AR389 HepG2 Cell line Buffer HepG2 Cell line Buffer 1 hr. 1 hr. AR390 HepG2 Cell line Buffer HepG2 Cell line Buffer 06 hr. 06 hr AR391 HepG2 Cell line Buffer HepG2 Cell line Buffer 24 hr. 24 hr. AR392 HepG2 Cell line Insulin HepG2 Cell line Insulin 01 hr. 01 hr. AR393 HepG2 Cell line Insulin HepG2 Cell line Insulin 06 hr. 06 hr. AR394 HepG2 Cell line Insulin HepG2 Cell line Insulin 24 hr. 24 hr. AR398 HMC-1 HMC-1 AR399 HMCS HMCS AR400 HMSC HMSC AR401 HUVEC #3 HUVEC #3 AR402 HUVEC #4 HUVEC #4 AR404 KIDNEY NORMAL KIDNEY NORMAL AR405 KIDNEY TUMOR KIDNEY TUMOR AR406 KIDNEY TUMOR AR407 Lymph Node Lymph Node AR408 Macrophage Macrophage AR409 Megakarioblast Megakarioblast AR410 Monocyte Monocyte AR411 Monocytes Monocytes AR413 Myocardium #3 Myocardium #3 AR414 Myocardium #4 Myocardium #4 AR415 Myocardium #5 Myocardium #5 AR416 NK NK AR417 NK cell NK cell AR418 NK cells NK cells AR419 NKYa NKYa AR420 NKYa019 NKYa019 AR421 Ovary Ovary AR422 Patient #11 Patient #11 AR423 Peripheral blood Peripheral blood AR424 Primary Adipocytes Primary Adipocytes AR425 Promyeloblast Promyeloblast AR427 RSSWT RSSWT AR428 RSSWTC RSSWTC AR429 SW 480(G1) SW 480(G1) AR430 SW 480(G2) SW 480(G2) AR431 SW 480(G3) SW 480(G3) AR432 SW 480(G4) SW 480(G4) AR433 SW 480(G5) SW 480(G5) AR434 T Lymphoblast T Lymphoblast AR435 T Lymphocyte T Lymphocyte AR436 T-Cell T-Cell AR438 T-Cell, T-Cell, AR439 T-Cells T-Cells AR440 T-lymphoblast T-lymphoblast AR441 Th 1 Th 1 AR442 Th 2 Th 2 AR443 Th1 Th1 AR444 Th2 Th2 H0002 Human Adult Heart Human Adult Heart Heart Uni-ZAP XR H0004 Human Adult Spleen Human Adult Spleen Spleen Uni-ZAP XR H0008 Whole 6 Week Old Uni-ZAP XR Embryo H0009 Human Fetal Brain Uni-ZAP XR H0012 Human Fetal Kidney Human Fetal Kidney Kidney Uni-ZAP XR H0013 Human 8 Week Whole Human 8 Week Old Embryo Uni-ZAP XR Embryo Embryo H0014 Human Gall Bladder Human Gall Bladder Gall Bladder Uni-ZAP XR H0015 Human Gall Bladder, Human Gall Bladder Gall Bladder Uni-ZAP XR fraction II H0018 Human Greater Human Greater Omentum peritoneum Uni-ZAP XR Omentum, fII remake H0019 Human Fetal Heart Human Fetal Heart Heart pBluescript H0022 Jurkat Cells Jurkat T-Cell Line Lambda ZAP II H0024 Human Fetal Lung III Human Fetal Lung Lung Uni-ZAP XR H0026 Namalwa Cells Namalwa B-Cell Line, Lambda ZAP II EBV immortalized H0028 Human Old Ovary Human Old Ovary Ovary pBluescript H0030 Human Placenta Uni-ZAP XR H0031 Human Placenta Human Placenta Placenta Uni-ZAP XR H0032 Human Prostate Human Prostate Prostate Uni-ZAP XR H0033 Human Pituitary Human Pituitary Uni-ZAP XR H0036 Human Adult Small Human Adult Small Small Int. Uni-ZAP XR Intestine Intestine H0038 Human Testes Human Testes Testis Uni-ZAP XR H0039 Human Pancreas Tumor Human Pancreas Tumor Pancreas disease Uni-ZAP XR H0040 Human Testes Tumor Human Testes Tumor Testis disease Uni-ZAP XR H0041 Human Fetal Bone Human Fetal Bone Bone Uni-ZAP XR H0042 Human Adult Human Adult Pulmonary Lung Uni-ZAP XR Pulmonary H0046 Human Endometrial Human Endometrial Tumor Uterus disease Uni-ZAP XR H0050 Human Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0051 Human Hippocampus Human Hippocampus Brain Uni-ZAP XR H0052 Human Cerebellum Human Cerebellum Brain Uni-ZAP XR H0056 Human Umbilical Vein, Human Umbilical Vein Umbilical vein Uni-ZAP XR Endo. remake Endothelial Cells H0057 Human Fetal Spleen Uni-ZAP XR H0059 Human Uterine Cancer Human Uterine Cancer Uterus disease Lambda ZAP II H0060 Human Macrophage Human Macrophage Blood Cell Line pBluescript H0061 Human Macrophage Human Macrophage Blood Cell Line pBluescript H0063 Human Thymus Human Thymus Thymus Uni-ZAP XR H0064 Human Right Human Brain, right Brain Uni-ZAP XR Hemisphere of Brain hemisphere H0068 Human Skin Tumor Human Skin Tumor Skin disease Uni-ZAP XR H0069 Human Activated T- Activated T-Cells Blood Cell Line Uni-ZAP XR Cells H0071 Human Infant Adrenal Human Infant Adrenal Adrenal gland Uni-ZAP XR Gland Gland H0075 Human Activated T- Activated T-Cells Blood Cell Line Uni-ZAP XR Cells (II) H0081 Human Fetal Epithelium Human Fetal Skin Skin Uni-ZAP XR (Skin) H0082 Human Fetal Muscle Human Fetal Muscle Sk Muscle Uni-ZAP XR H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP XR MEMBRANE BOUND POLYSOMES H0085 Human Colon Human Colon Lambda ZAP II H0086 Human epithelioid Epithelioid Sarcoma, Sk Muscle disease Uni-ZAP XR sarcoma muscle H0087 Human Thymus Human Thymus pBluescript H0090 Human T-Cell T-Cell Lymphoma T-Cell disease Uni-ZAP XR Lymphoma H0095 Human Greater Human Greater Omentum peritoneum Uni-ZAP XR Remake H0098 Human Adult Liver, Human Adult Liver Liver Uni-ZAP XR subtracted H0100 Human Whole Six Human Whole Six Week Embryo Uni-ZAP XR Week Old Embryo Old Embryo H0101 Human 7 Weeks Old Human Whole 7 Week Old Embryo Lambda ZAP II Embryo, subtracted Embryo H0102 Human Whole 6 Week Human Whole Six Week Embryo pBluescript Old Embryo (II), subt Old Embryo H0105 Human Fetal Heart, Human Fetal Heart Heart pBluescript subtracted H0107 Human Infant Adrenal Human Infant Adrenal Adrenal gland pBluescript Gland, subtracted Gland H0117 Human Uterine Cancer, Human Uterine Cancer Uterus pBluescript subtracted H0121 Human Cornea, Human Cornea eye Uni-ZAP XR subtracted H0122 Human Adult Skeletal Human Skeletal Muscle Sk Muscle Uni-ZAP XR Muscle H0123 Human Fetal Dura Human Fetal Dura Mater Brain Uni-ZAP XR Mater H0124 Human Human Sk Muscle disease Uni-ZAP XR Rhabdomyosarcoma Rhabdomyosarcoma H0125 Cem cells Cyclohexamide Treated Blood Cell Line Uni-ZAP XR cyclohexamide treated Cem, Jurkat, Raji, and Supt H0129 Jurkat cells, thiouridine Jurkat Cells Uni-ZAP XR activated, fract II H0130 LNCAP untreated LNCAP Cell Line Prostate Cell Line Uni-ZAP XR H0131 LNCAP + o.3 nM R1881 LNCAP Cell Line Prostate Cell Line Uni-ZAP XR H0132 LNCAP + 30 nM R1881 LNCAP Cell Line Prostate Cell Line Uni-ZAP XR H0134 Raji Cells, Cyclohexamide Treated Blood Cell Line Uni-ZAP XR cyclohexamide treated Cem, Jurkat, Raji, and Supt Sarcoma H0136 Supt Cells, Cyclohexamide Treated Blood Cell Line Uni-ZAP XR cyclohexamide treated Cem, Jurkat, Raji, and Supt H0140 Activated T-Cells, 8 hrs. Activated T-Cells Blood Cell Line Uni-ZAP XR H0141 Activated T-Cells, 12 hrs. Activated T-Cells Blood Cell Line Uni-ZAP XR H0144 Nine Week Old Early 9 Wk Old Early Stage Embryo Uni-ZAP XR Stage Human Human H0147 Human Adult Liver Human Adult Liver Liver Uni-ZAP XR H0149 7 Week Old Early Stage Human Whole 7 Week Old Embryo Uni-ZAP XR Human, subtracted Embryo H0150 Human Epididymus Epididymis Testis Uni-ZAP XR H0151 Early Stage Human Human Fetal Liver Liver Uni-ZAP XR Liver H0154 Human Fibrosarcoma Human Skin Fibrosarcoma Skin disease Uni-ZAP XR H0156 Human Adrenal Gland Human Adrenal Gland Adrenal Gland disease Uni-ZAP XR Tumor Tumor H0159 Activated T-Cells, 8 hrs., Activated T-Cells Blood Cell Line Uni-ZAP XR ligation 2 H0163 Human Synovium Human Synovium Synovium Uni-ZAP XR H0165 Human Prostate Cancer, Human Prostate Cancer, Prostate disease Uni-ZAP XR Stage B2 stage B2 H0166 Human Prostate Cancer, Human Prostate Cancer, Prostate disease Uni-ZAP XR Stage B2 fraction stage B2 H0167 Activated T-Cells, 24 hrs. Activated T-Cells Blood Cell Line Uni-ZAP XR H0169 Human Prostate Cancer, Human Prostate Cancer, Prostate disease Uni-ZAP XR Stage C fraction stage C H0170 12 Week Old Early Twelve Week Old Early Embryo Uni-ZAP XR Stage Human Stage Human H0171 12 Week Old Early Twelve Week Old Early Embryo Uni-ZAP XR Stage Human, II Stage Human Cardiomyopathy, RNA remake H0177 CAMA1Ee Cell Line CAMA1Ee Cell Line Breast Cell Line Uni-ZAP XR H0178 Human Fetal Brain Human Fetal Brain Brain Uni-ZAP XR H0179 Human Neutrophil Human Neutrophil Blood Cell Line Uni-ZAP XR H0181 Human Primary Breast Human Primary Breast Breast disease Uni-ZAP XR Cancer Cancer H0182 Human Primary Breast Human Primary Breast Breast disease Uni-ZAP XR Cancer Cancer H0183 Human Colon Cancer Human Colon Cancer Colon disease Uni-ZAP XR H0187 Resting T-Cell T-Cells Blood Cell Line Lambda ZAP II H0188 Human Normal Breast Human Normal Breast Breast Uni-ZAP XR H0194 Human Cerebellum, Human Cerebellum Brain pBluescript subtracted H0196 Human Human Cardiomyopathy Heart Uni-ZAP XR Cardiomyopathy, subtracted H0197 Human Fetal Liver, Human Fetal Liver Liver Uni-ZAP XR subtracted H0199 Human Fetal Liver, Human Fetal Liver Liver Uni-ZAP XR subtracted, neg clone H0200 Human Greater Human Greater Omentum peritoneum Uni-ZAP XR Omentum, fract II remake, H0201 Human Hippocampus, Human Hippocampus Brain pBluescript subtracted H0202 Jurkat Cells, Cyclohexamide Treated Blood Cell Line Uni-ZAP XR cyclohexamide treated, Cem, Jurkat, Raji, and Supt subtraction H0204 Human Colon Cancer, Human Colon Cancer Colon pBluescript subtracted H0205 Human Colon Cancer, Human Colon Cancer Colon pBluescript H0208 Early Stage Human Human Fetal Lung Lung pBluescript Lung, subtracted H0209 Human Cerebellum, Human Cerebellum Brain Uni-ZAP XR differentially expressed H0212 Human Prostate, Human Prostate Prostate pBluescript subtracted H0213 Human Pituitary, Human Pituitary Uni-ZAP XR subtracted H0216 Supt cells, Cyclohexamide Treated Blood Cell Line pBluescript cyclohexamide treated, Cem, Jurkat, Raji, and Supt subtracted H0220 Activated T-Cells, 4 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR subtracted H0222 Activated T-Cells, 8 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR subtracted H0224 Activated T-Cells, 12 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR subtracted H0225 Activated T-Cells, Activated T-Cells Blood Cell Line Uni-ZAP XR 12 hrs, differentially expressed H0229 Early Stage Human Early Stage Human Brain Brain Lambda ZAP II Brain, random primed H0230 Human Human Cardiomyopathy Heart disease Uni-ZAP XR Cardiomyopathy, diff exp H0231 Human Colon, Human Colon pBluescript subtraction H0235 Human colon cancer, Human Colon Cancer, Liver pBluescript metaticized to liver, metasticized to liver subtraction H0239 Human Kidney Tumor Human Kidney Tumor Kidney disease Uni-ZAP XR H0241 C7MCF7 cell line, C7MCF7 Cell Line, Breast Cell Line Uni-ZAP XR subtraction H0242 Human Fetal Heart, Human Fetal Heart Heart pBluescript Differential (Fetal- 0 Specific) H0244 Human 8 Week Whole Human 8 Week Old Embryo Uni-ZAP XR Embryo, subtracted Embryo H0246 Human Fetal Liver- Human Fetal Liver Liver Uni-ZAP XR Enzyme subtraction H0249 HE7, subtracted by Human Whole 7 Week Old Embryo Uni-ZAP XR hybridization with E7 Embryo cDNA H0250 Human Activated Human Monocytes Uni-ZAP XR Monocytes H0251 Human Human Chondrosarcoma Cartilage disease Uni-ZAP XR Chondrosarcoma H0252 Human Osteosarcoma Human Osteosarcoma Bone disease Uni-ZAP XR H0253 Human adult testis, Human Adult Testis Testis Uni-ZAP XR large inserts H0254 Breast Lymph node Breast Lymph Node Lymph Node Uni-ZAP XR cDNA library H0255 breast lymph node Breast Lymph Node Lymph Node Lambda ZAP II CDNA library H0256 HL-60, unstimulated Human HL-60 Cells, Blood Cell Line Uni-ZAP XR unstimulated H0257 HL-60, PMA 4H HL-60 Cells, PMA Blood Cell Line Uni-ZAP XR stimulated 4H H0261 H. cerebellum, Enzyme Human Cerebellum Brain Uni-ZAP XR subtracted H0263 human colon cancer Human Colon Cancer Colon disease Lambda ZAP II H0264 human tonsils Human Tonsil Tonsil Uni-ZAP XR H0265 Activated T-Cell T-Cells Blood Cell Line Uni-ZAP XR (12 hs)/Thiouridine H0266 Human Microvascular HMEC Vein Cell Line Lambda ZAP II Endothelial Cells, fract. A H0267 Human Microvascular HMEC Vein Cell Line Lambda ZAP II Endothelial Cells, fract. B H0268 Human Umbilical Vein HUVE Cells Umbilical vein Cell Line Lambda ZAP II Endothelial Cells, fract. A H0269 Human Umbilical Vein HUVE Cells Umbilical vein Cell Line Lambda ZAP II Endothelial Cells, fract. B H0270 HPAS (human pancreas, Human Pancreas Pancreas Uni-ZAP XR subtracted) H0271 Human Neutrophil, Human Neutrophil - Blood Cell Line Uni-ZAP XR Activated Activated H0272 HUMAN TONSILS, Human Tonsil Tonsil Uni-ZAP XR FRACTION 2 H0274 Human Adult Spleen, Human Adult Spleen Spleen Uni-ZAP XR fractionII H0275 Human Infant Adrenal Human Infant Adrenal Adrenal gland pBluescript Gland, Subtracted Gland H0280 K562 + PMA (36 hrs) K562 Cell line cell line Cell Line ZAP Express H0284 Human OB MG63 Human Osteoblastoma Bone Cell Line Uni-ZAP XR control fraction I MG63 cell line H0286 Human OB MG63 Human Osteoblastoma Bone Cell Line Uni-ZAP XR treated (10 nM E2) MG63 cell line fraction I H0288 Human OB HOS Human Osteoblastoma Bone Cell Line Uni-ZAP XR control fraction I HOS cell line H0290 Human OB HOS treated Human Osteoblastoma Bone Cell Line Uni-ZAP XR (1 nM E2) fraction I HOS cell line (10 nM E2) fraction I HOS cell line H0293 WI 38 cells Uni-ZAP XR H0294 Amniotic Cells - TNF Amniotic Cells - TNF Placenta Cell Line Uni-ZAP XR induced induced H0295 Amniotic Cells - Amniotic Cells - Primary Placenta Cell Line Uni-ZAP XR Primary Culture Culture H0305 CD34 positive cells CD34 Positive Cells Cord Blood ZAP Express (Cord Blood) H0306 CD34 depleted Buffy CD34 Depleted Buffy Coat Cord Blood ZAP Express Coat (Cord Blood) (Cord Blood) H0309 Human Chronic Synovium, Chronic Synovium disease Uni-ZAP XR Synovitis Synovitis/Osteoarthritis H0316 HUMAN STOMACH Human Stomach Stomach Uni-ZAP XR H0318 HUMAN B CELL Human B Cell Lymphoma Lymph Node disease Uni-ZAP XR LYMPHOMA H0327 human corpus colosum Human Corpus Callosum Brain Uni-ZAP XR H0328 human ovarian cancer Ovarian Cancer Ovary disease Uni-ZAP XR H0329 Dermatofibrosarcoma Dermatofibrosarcoma Skin disease Uni-ZAP XR Protuberance Protuberans H0331 Hepatocellular Tumor Hepatocellular Tumor Liver disease Lambda ZAP II H0333 Hemangiopericytoma Hemangiopericytoma Blood vessel disease Lambda ZAP II H0334 Kidney cancer Kidney Cancer Kidney disease Uni-ZAP XR H0339 Duodenum Duodenum Uni-ZAP XR H0340 Corpus Callosum Corpus Collosum-93052 Uni-ZAP XR H0341 Bone Marrow Cell Line Bone Marrow Cell Line Bone Marrow Cell Line Uni-ZAP XR (RS4;11) RS4;11 H0343 stomach cancer (human) Stomach Cancer - 5383A disease Uni-ZAP XR (human) H0344 Adipose tissue (human) Adipose - 6825A (human) Uni-ZAP XR H0345 SKIN Skin - 4000868H Skin Uni-ZAP XR H0346 Brain-medulloblastoma Brain (Medulloblastoma)- Brain disease Uni-ZAP XR 9405C006R cDNA library H0350 Human Fetal Liver, Human Fetal Liver, mixed Liver Uni-ZAP XR mixed 10 & 14 week 10&14 Week H0351 Glioblastoma Glioblastoma Brain disease Uni-ZAP XR H0352 wilm''s tumor Wilm''s Tumor disease Uni-ZAP XR H0354 Human Leukocytes Human Leukocytes Blood Cell Line pCMVSport 1 H0355 Human Liver Human Liver, normal Adult pCMVSport 1 H0357 H. Normalized Fetal Human Fetal Liver Liver Uni-ZAP XR Liver, II H0361 Human rejected kidney Human Rejected Kidney disease pBluescript H0366 L428 cell line L428 ZAP Express H0369 H. Atrophic Atrophic Endometrium and Uni-ZAP XR Endometrium myometrium H0370 H. Lymph node breast Lymph node with Met. disease Uni-ZAP XR Cancer Breast Cancer H0373 Human Heart Human Adult Heart Heart pCMVSport 1 H0374 Human Brain Human Brain pCMVSport 1 H0375 Human Lung Human Lung pCMVSport 1 H0379 Human Tongue, frac 1 Human Tongue pSport1 H0380 Human Tongue, frac 2 Human Tongue pSport1 H0381 Bone Cancer Bone Cancer disease Uni-ZAP XR H0383 Human Prostate BPH, Human Prostate BPH Uni-ZAP XR re-excision H0385 H. Leukocytes, Kozak Human Leukocytes Blood Cell Line pCMVSport 1 H0388 Human Rejected Human Rejected Kidney disease pBluescript Kidney, 704 re-excision H0390 Human Amygdala Human Amygdala disease pBluescript Depression, re-excision Depression H0391 H. Meniingima, M6 Human Meningima brain pSport1 H0392 H. Meningima, M1 Human Meningima brain pSport1 H0393 Fetal Liver, subtraction Human Fetal Liver Liver pBluescript II H0399 Human Kidney Cortex, Human Kidney Cortex Lambda ZAP II re-rescue H0400 Human Striatum Human Brain, Striatum Brain Lambda ZAP II Depression, re-rescue Depression H0401 Human Pituitary, Human Pituitary pBluescript subtracted V H0402 CD34 depleted Buffy CD34 Depleted Buffy Coat Cord Blood ZAP Express Coat (Cord Blood), re- (Cord Blood) excision H0403 H. Umbilical Vein HUVE Cells Umbilical vein Cell Line Uni-ZAP XR Endothelial Cells, IL4 induced H0406 H Amygdala Human Amygdala Uni-ZAP XR Depression, subtracted Depression H0408 Human kidney Cortex, Human Kidney Cortex pBluescript subtracted H0409 H. Striatum Depression, Human Brain, Striatum Brain pBluescript subtracted Depression H0411 H Female Bladder, Human Female Adult Bladder pSport1 Adult Bladder H0412 Human umbilical vein HUVE Cells Umbilical vein Cell Line pSport1 endothelial cells, IL-4 induced H0413 Human Umbilical Vein HUVE Cells Umbilical vein Cell Line pSport1 Endothelial Cells, uninduced H0414 Ovarian Tumor I, Ovarian Tumor, OV5232 Ovary disease pSport1 OV5232 H0415 H. Ovarian Tumor, II, Ovarian Tumor, OV5232 Ovary disease pCMVSport 2.0 OV5232 H0416 Human Neutrophils, Human Neutrophil - Blood Cell Line pBluescript Activated, re-excision Activated subtracted VIII H0419 Bone Cancer, re- Bone Cancer Uni-ZAP XR excision H0421 Human Bone Marrow, Bone Marrow pBluescript re-excision H0422 T-Cell PHA 16 hrs T-Cells Blood Cell Line pSport1 H0423 T-Cell PHA 24 hrs T-Cells Blood Cell Line pSport1 H0424 Human Pituitary, subt Human Pituitary pBluescript IX H0427 Human Adipose Human Adipose, left pSport1 hiplipoma H0428 Human Ovary Human Ovary Tumor Ovary pSport1 H0431 H. Kidney Medulla, re- Kidney medulla Kidney pBluescript excision H0433 Human Umbilical Vein HUVE Cells Umbilical vein Cell Line pBluescript Endothelial cells, frac B, re-excision H0435 Ovarian Tumor 10-3-95 Ovarian Tumor, OV350721 Ovary pCMVSport 2.0 H0436 Resting T-Cell T-Cells Blood Cell Line pSport1 Library, II H0437 H Umbilical Vein HUVE Cells Umbilical vein Cell Line Lambda ZAP II Endothelial Cells, frac A, re-excision H0438 H. Whole Brain #2, re- Human Whole Brain #2 ZAP Express excision H0439 Human Eosinophils Eosinophils pBluescript H0441 H. Kidney Cortex, Kidney cortex Kidney pBluescript subtracted H0444 Spleen metastic Spleen, Metastic malignant Spleen disease pSport1 melanoma melanoma H0445 Spleen, Chronic Human Spleen, CLL Spleen disease pSport1 lymphocytic leukemia H0450 CD34+cells, II CD34 positive cells pCMVSport 2.0 H0453 H. Kidney Pyramid, Kidney pyramids Kidney pBluescript subtracted H0455 H. Striatum Depression, Human Brain, Striatum Brain pBluescript subt Depression H0456 H Kidney Cortex, Human Kidney Cortex pBluescript subtracted III H0457 Human Eosinophils Human Eosinophils pSport1 H0458 CD34+ cell, I, frac II CD34 positive cells pSport1 H0459 CD34+ cells, II, CD34 positive cells pCMVSport 2.0 FRACTION 2 H0461 H. Kidney Medulla, Kidney medulla Kidney pBluescript subtracted H0477 Human Tonsil, Lib 3 Human Tonsil Tonsil pSport1 H0478 Salivary Gland, Lib 2 Human Salivary Gland Salivary gland pSport1 H0483 Breast Cancer cell line, Breast Cancer Cell line, pSport1 MDA 36 MDA 36 H0484 Breast Cancer Cell line, Breast Cancer Cell line, pSport1 angiogenic Angiogenic, 36T3 H0485 Hodgkin''s Lymphoma I Hodgkin''s Lymphoma I disease pCMVSport 2.0 H0486 Hodgkin''s Lymphoma Hodgkin''s Lymphoma II disease pCMVSport 2.0 II H0487 Human Tonsils, lib I Human Tonsils pCMVSport 2.0 H0488 Human Tonsils, Lib 2 Human Tonsils pCMVSport 2.0 H0492 HL-60, RA 4h, HL-60 Cells, RA stimulated Blood Cell Line Uni-ZAP XR Subtracted for 4H H0494 Keratinocyte Keratinocyte pCMVSport 2.0 H0497 HEL cell line HEL cell line HEL 92.1.7 pSport1 H0505 Human Astrocyte Human Astrocyte pSport1 H0506 Ulcerative Colitis Colon Colon pSport1 H0509 Liver, Hepatoma Human Liver, Hepatoma, Liver disease pCMVSport 3.0 patient 8 Patient # 8 H0518 pBMC stimulated w/ pBMC stimulated with poly pCMVSport 3.0 poly I/C I/C H0519 NTERA2, control NTERA2, Teratocarcinoma pCMVSport 3.0 cell line H0520 NTERA2 + retinoic NTERA2, Teratocarcinoma pSport1 acid, 14 days cell line H0521 Primary Dendritic Cells, Primary Dendritic cells pCMVSport 3.0 lib 1 H0522 Primary Dendritic Primary Dendritic cells pCMVSport 3.0 cells, frac 2 H0529 Myoloid Progenitor Cell TF-1 Cell Line; Myoloid pCMVSport 3.0 Line progenitor cell line H0530 Human Dermal Human Dermal Endothelial pSport1 Endothelial Cells; untreated Cells, untreated H0535 Human ovary tumor cell Ovarian Tumor, OV350721 Ovary disease pSport1 OV350721 H0538 Merkel Cells Merkel cells Lymph node pSport1 H0539 Pancreas Islet Cell Pancreas Islet Cell Tumour Pancreas disease pSport1 Tumor H0540 Skin, burned Skin, leg burned Skin pSport1 H0542 T Cell helper I Helper T cell pCMVSport 3.0 H0543 T cell helper II Helper T cell pCMVSport 3.0 H0544 Human endometrial Human endometrial stromal pCMVSport 3.0 stromal cells cells H0545 Human endometrial Human endometrial stromal pCMVSport 3.0 stromal cells-treated cells-treated with proge with progesterone H0546 Human endometrial Human endometrial stromal pCMVSport 3.0 stromal cells-treated cells-treated with estra with estradiol teratocarcinoma cell cell line line + retinoic acid (14 days) H0549 H. Epididiymus, caput Human Epididiymus, caput Uni-ZAP XR & corpus and corpus H0550 H. Epididiymus, cauda Human Epididiymus, cauda Uni-ZAP XR H0551 Human Thymus Stromal Human Thymus Stromal pCMVSport 3.0 Cells Cells H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 Rejected Kidney, lib 4 Human Rejected Kidney Kidney disease pCMVSport 3.0 H0556 Activated T- T-Cells Blood Cell Line Uni-ZAP XR cell(12 h)/Thiouridine- re-excision H0559 HL-60, PMA 4H, re- HL-60 Cells, PMA Blood Cell Line Uni-ZAP XR excision stimulated 4H H0560 KMH2 KMH2 pCMVSport 3.0 H0561 L428 L428 pCMVSport 3.0 H0562 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized c5-11-26 H0563 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized 50021F H0565 HUman Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized 100024F H0566 Human Fetal Human Fetal Brain pCMVSport 2.0 Brain, normalized c50F H0567 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized A5002F H0569 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized CO H0570 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized C500H H0571 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 H0572 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalized AC5002 H0574 Hepatocellular Tumor; Hepatocellular Tumor Liver disease Lambda ZAP II re-excision H0575 Human Adult Human Adult Pulmonary Lung Uni-ZAP XR Pulmonary; re-excision H0576 Resting T-Cell; re- T-Cells Blood Cell Line Lambda ZAP II excision H0580 Dendritic cells, pooled Pooled dendritic cells pCMVSport 3.0 H0581 Human Bone Marrow, Human Bone Marrow Bone Marrow pCMVSport 3.0 treated H0583 B Cell lymphoma B Cell Lymphoma B Cell disease pCMVSport 3.0 H0584 Activated T-cells, 24 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR re-excision H0585 Activated T-Cells, 12 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR re-excision H0586 Healing groin wound, healing groin wound, 6.5 groin disease pCMVSport 3.0 6.5 hours post incision hours post incision - 2/ H0587 Healing groin wound; Groin-2/19/97 groin disease pCMVSport 3.0 7.5 hours post incision H0589 CD34 positive cells CD34 Positive Cells Cord Blood ZAP Express (cord blood), re-ex H0590 Human adult small Human Adult Small Small Int. Uni-ZAP XR intestine, re-excision Intestine H0591 Human T-cell T-Cell Lymphoma T-Cell disease Uni-ZAP XR lymphoma; re-excision H0592 Healing groin wound - HGS wound healing disease pCMVSport 3.0 zero hr post-incision project; abdomen (control) H0593 Olfactory Olfactory epithelium from pCMVSport 3.0 epithelium; nasalcavity roof of left nasal cacit H0594 Human Lung Cancer; re- Human Lung Cancer Lung disease Lambda ZAP II H0595 Stomach cancer Stomach Cancer - 5383A disease Uni-ZAP XR (human); re-excision (human) H0596 Human Colon Human Colon Cancer Colon Lambda ZAP II Cancer; re-excision H0597 Human Colon; re- Human Colon Lambda ZAP II excision H0598 Human Stomach; re- Human Stomach Stomach Uni-ZAP XR excision H0599 Human Adult Heart; re- Human Adult Heart Heart Uni-ZAP XR excision H0600 Healing Abdomen Abdomen disease pCMVSport 3.0 wound; 70&90 min post incision H0601 Healing Abdomen Abdomen disease pCMVSport 3.0 Wound; 15 days post incision H0602 Healing Abdomen Abdomen disease pCMVSport 3.0 Wound; 21&29 days post incision H0604 Human Pituitary, re- Human Pituitary pBluescript excision H0606 Human Primary Breast Human Primary Breast Breast disease Uni-ZAP XR Cancer; re-excision Cancer H0607 H. Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 50A3 H0611 H. Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 500 B H0613 H. Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 5B H0615 Human Ovarian Cancer Ovarian Cancer Ovary disease Uni-ZAP XR Reexcision H0616 Human Testes, Human Testes Testis Uni-ZAP XR H0617 Human Primary Breast Human Primary Breast Breast disease Uni-ZAP XR Cancer Reexcision Cancer H0618 Human Adult Testes, Human Adult Testis Testis Uni-ZAP XR Large Inserts, Reexcision H0619 Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0620 Human Fetal Kidney; Human Fetal Kidney Kidney Uni-ZAP XR Reexcision H0622 Human Pancreas Human Pancreas Tumor Pancreas disease Uni-ZAP XR Tumor; Reexcision H0623 Human Umbilical Vein; Human Umbilical Vein Umbilical vein Uni-ZAP XR Reexcision Endothelial Cells H0624 12 Week Early Stage Twelve Week Old Early Embryo Uni-ZAP XR Human II; Reexcision Stage Human H0625 Ku 812F Basophils Line Ku 812F Basophils pSport1 H0626 Saos2 Cells; Untreated Saos2 Cell Line; Untreated pSport1 H0627 Saos2 Cells; Vitamin Saos2 Cell Line; Vitamin pSport1 D3 Treated D3 Treated H0628 Human Pre- Human Pre-Differentiated Uni-ZAP XR Differentiated Adipocytes Adipocytes H0630 Human Human Normalized pCMVSport 1 Leukocytes, normalized leukocyte control #4 H0631 Saos2, Dexamethosome Saos2 Cell Line; pSport1 Treated Dexamethosome Treated H0632 Hepatocellular Hepatocellular Tumor Liver Lambda ZAP II Tumor; re-excision H0633 Lung Carcinoma A549 TNFalpha activated A549-- disease pSport1 TNFalpha activated Lung Carcinoma H0634 Human Testes Tumor, Human Testes Tumor Testis disease Uni-ZAP XR re-excision Cells, re-excision H0637 Dendritic Cells From Dentritic cells from CD34 pSport1 CD34 Cells cells H0638 CD40 activated CD40 activated monocyte pSport1 monocyte dendridic dendridic cells cells H0640 Ficolled Human Stromal Ficolled Human Stromal Other Cells, Untreated Cells, Untreated H0641 LPS activated derived LPS activated monocyte pSport1 dendritic cells derived dendritic cells H0642 Hep G2 Cells, lambda Hep G2 Cells Other library H0643 Hep G2 Cells, PCR Hep G2 Cells Other library H0644 Human Placenta (re- Human Placenta Placenta Uni-ZAP XR excision) H0645 Fetal Heart, re-excision Human Fetal Heart Heart Uni-ZAP XR H0646 Lung, Cancer (4005313 Metastatic squamous cell pSport1 A3): Invasive Poorly lung carcinoma, poorly di Differentiated Lung Adenocarcinoma, H0647 Lung, Cancer (4005163 Invasive poorly disease pSport1 B7): Invasive, Poorly differentiated lung Diff. Adenocarcinoma, adenocarcinoma Metastatic H0648 Ovary, Cancer: Papillary Cstic neoplasm of disease pSport1 (4004562 B6) Papillary low malignant potentia Serous Cystic Neoplasm, Low Malignant Pot H0649 Lung, Normal: Normal Lung pSport1 (4005313 B1) H0651 Ovary, Normal: Normal Ovary pSport1 (9805C040R) H0652 Lung, Normal: Normal Lung pSport1 (4005313 B1) H0653 Stromal Cells Stromal Cells pSport1 H0656 B-cells (unstimulated) B-cells (unstimulated) pSport1 H0657 B-cells (stimulated) B-cells (stimulated) pSport1 H0658 Ovary, Cancer 9809C332-Poorly Ovary & disease pSport1 (9809C332): Poorly differentiate Fallopian Tubes differentiated adenocarcinoma H0659 Ovary, Cancer Grade II Papillary Ovary disease pSport1 (15395A1F): Grade II Carcinoma, Ovary Papillary Carcinoma H0660 Ovary, Cancer: Poorly differentiated disease pSport1 (15799A1F) Poorly carcinoma, ovary differentiated carcinoma H0661 Breast, Cancer: Breast cancer disease pSport1 (4004943 A5) H0662 Breast, Normal: Normal Breast - Breast pSport1 (4005522B2) #4005522(B2) H0663 Breast, Cancer: Breast Cancer - Breast disease pSport1 (4005522 A2) #4005522(A2) H0664 Breast, Cancer: Breast Cancer Breast disease pSport1 (9806C012R) H0665 Stromal cells 3.88 Stromal cells 3.88 pSport1 H0666 Ovary, Cancer: Ovarian Cancer, Sample disease pSport1 (4004332 A2) #4004332A2 H0667 Stromal Stromal cell(HBM 3.18) pSport1 cells(HBM3.18) H0668 stromal cell clone 2.5 stromal cell clone 2.5 pSport1 H0670 Ovary, Cancer(4004650 Ovarian Cancer - pSport1 Differentiated Micropapillary Serous Carcinoma H0671 Breast, Cancer: Breast Cancer- Sample # pSport1 (9802C02OE) 9802C02OE H0672 Ovary, Cancer: Ovarian Ovary pSport1 (4004576 A8) Cancer(4004576A8) H0673 Human Prostate Cancer, Human Prostate Cancer, Prostate Uni-ZAP XR Stage B2; re-excision stage B2 H0674 Human Prostate Cancer, Human Prostate Cancer, Prostate Uni-ZAP XR Stage C; re-excission stage C H0675 Colon, Cancer: Colon Cancer 9808C064R pCMVSport 3.0 (9808C064R) H0676 Colon, Cancer: Colon Cancer 9808C064R pCMVSport 3.0 (9808C064R)-total RNA H0677 TNFR degenerate oligo B-Cells PCRII H0682 Serous Papillary serous papillary pCMVSport 3.0 Adenocarcinoma adenocarcinoma (9606G304SPA3B) H0683 Ovarian Serous Serous papillary pCMVSport 3.0 Papillary adenocarcinoma, stage 3C Adenocarcinoma (9804G01 H0684 Serous Papillary Ovarian Cancer-9810G606 Ovaries pCMVSport 3.0 Adenocarcinoma H0685 Adenocarcinoma of Adenocarcinoma of Ovary, pCMVSport 3.0 Ovary, Human Cell Human Cell Line, # Line, # OVCAR-3 OVCAR- H0686 Adenocarcinoma of Adenocarcinoma of Ovary, pCMVSport 3.0 Ovary, Human Cell Human Cell Line, # SW- Line 626 H0687 Human normal Human normal Ovary pCMVSport 3.0 H0688 Human Ovarian Human Ovarian pCMVSport 3.0 Cancer(#9807G017) cancer(#9807G017), mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport 3.0 #9806G019 H0690 Ovarian Cancer, # Ovarian Cancer, pCMVSport 3.0 9702G001 #9702G001 H0692 BLyS Receptor from B Cell Lymphoma B Cell pCMVSport 3.0 Expression Cloning H0693 Normal Prostate Normal Prostate Tissue # pCMV Sport 3.0 #ODQ3958EN ODQ3958EN H0694 Prostate gland Prostate gland, prostate gland pCMVSport 3.0 adenocarcinoma adenocarcinoma, mod/diff, gleason H0695 mononucleocytes from mononucleocytes from pCMVSport 3.0 patient patient at Shady Grove Hospit N0006 Human Fetal Brain Human Fetal Brain N0007 Human Hippocampus Human Hippocampus S0001 Brain frontal cortex Brain frontal cortex Brain Lambda ZAP II S0002 Monocyte activated Monocyte-activated blood Cell Line Uni-ZAP XR S0003 Human Osteoclastoma Osteoclastoma bone disease Uni-ZAP XR S0005 Heart Heart-left ventricle Heart pCDNA S0007 Early Stage Human Human Fetal Brain Uni-ZAP XR Brain S0010 Human Amygdala Amygdala Uni-ZAP XR S0011 STROMAL - Osteoclastoma bone disease Uni-ZAP XR OSTEOCLASTOMA S0014 Kidney Cortex Kidney cortex Kidney Uni-ZAP XR S0015 Kidney medulla Kidney medulla Kidney Uni-ZAP XR S0016 Kidney Pyramids Kidney pyramids Kidney Uni-ZAP XR S0022 Human Osteoclastoma Osteoclastoma Stromal Uni-ZAP XR unamplified S0024 Human Kidney Medulla - Human Kidney Medulla unamplified S0026 Stromal cell TF274 stromal cell Bone marrow Cell Line Uni-ZAP XR S0027 Smooth muscle, serum Smooth muscle Pulmanary Cell Line Uni-ZAP XR treated artery S0028 Smooth muscle, control Smooth muscle Pulmanary Cell Line Uni-ZAP XR artery S0029 brain stem Brain stem brain Uni-ZAP XR S0031 Spinal cord Spinal cord spinal cord Uni-ZAP XR S0032 Smooth muscle-ILb Smooth muscle Pulmanary Cell Line Uni-ZAP XR induced artery S0036 Human Substantia Nigra Human Substantia Nigra Uni-ZAP XR S0037 Smooth muscle, IL1b Smooth muscle Pulmanary Cell Line Uni-ZAP XR induced artery S0038 Human Whole Brain #2 - Human Whole Brain #2 ZAP Express Oligo dT >1.5 Kb S0040 Adipocytes Human Adipocytes from Uni-ZAP XR Osteoclastoma S0044 Prostate BPH prostate BPH Prostate disease Uni-ZAP XR S0045 Endothelial cells-control Endothelial cell endothelial cell- Cell Line Uni-ZAP XR lung S0046 Endothelial-induced Endothelial cell endothelial cell- Cell Line Uni-ZAP XR lung S0049 Human Brain, Striatum Human Brain, Striatum Uni-ZAP XR S0050 Human Frontal Cortex, Human Frontal Cortex, disease Uni-ZAP XR Schizophrenia Schizophrenia S0051 Human Human Hypothalamus, disease Uni-ZAP XR Hypothalmus, Schizophrenia Schizophrenia S0052 neutrophils control human neutrophils blood Cell Line Uni-ZAP XR S0053 Neutrophils IL-1 and human neutrophil induced blood Cell Line Uni-ZAP XR S0106 STRIATUM BRAIN disease Uni-ZAP XR DEPRESSION S0110 Brain Amygdala Brain disease Uni-ZAP XR Depression S0112 Hypothalamus Brain Uni-ZAP XR S0114 Anergic T-cell Anergic T-cell Cell Line Uni-ZAP XR S0116 Bone marrow Bone marrow Bone marrow Uni-ZAP XR S0118 Smooth muscle control 2 Smooth muscle Pulmanary Cell Line Uni-ZAP XR artery S0126 Osteoblasts Osteoblasts Knee Cell Line Uni-ZAP XR S0132 Epithelial-TNFa and Airway Epithelial Uni-ZAP XR INF induced S0134 Apoptotic T-cell apoptotic cells Cell Line Uni-ZAP XR S0140 eosinophil-IL5 induced eosinophil lung Cell Line Uni-ZAP XR S0142 Macrophage-oxLDL macrophage-oxidized LDL blood Cell Line Uni-ZAP XR treated S0144 Macrophage (GM-CSF Macrophage (GM-CSF Uni-ZAP XR treated) treated) S0146 prostate-edited prostate BPH Prostate Uni-ZAP XR S0148 Normal Prostate Prostate prostate Uni-ZAP XR S0150 LNCAP prostate cell LNCAP Cell Line Prostate Cell Line Uni-ZAP XR line S0152 PC3 Prostate cell line PC3 prostate cell line Uni-ZAP XR S0176 Prostate, normal, Prostate prostate Uni-ZAP XR subtraction I S0182 Human B Cell 8866 Human B-Cell 8866 Uni-ZAP XR S0188 Prostate, BPH, Lib 2 Human Prostate BPH disease pSport1 S0190 Prostate BPH, Lib 2, Human Prostate BPH pSport1 subtracted S0192 Synovial Fibroblasts Synovial Fibroblasts pSport1 (control) S0194 Synovial hypoxia Synovial Fibroblasts pSport1 stimulated S0206 Smooth Muscle- Smooth muscle Pulmanary Cell Line pBluescript HASTE normalized artery S0208 Messangial cell, frac 1 Messangial cell pSport1 S0210 Messangial cell, frac 2 Messangial cell pSport1 S0212 Bone Marrow Stromal Bone Marrow Stromal pSport1 Cell, untreated Cell, untreated S0214 Human Osteoclastoma, Osteoclastoma bone disease Uni-ZAP XR re-excision S0216 Neutrophils IL-1 and human neutrophil induced blood Cell Line Uni-ZAP XR LPS induced S0218 Apoptotic T-cell, re- apoptotic cells Cell Line Uni-ZAP XR excision S0220 H. hypothalamus, frac Hypothalamus Brain ZAP Express A; re-excision S0222 H. Frontal H. Brain, Frontal Cortex, Brain disease Uni-ZAP XR cortex, epileptic; re- Epileptic excision S0242 Synovial Fibroblasts Synovial Fibroblasts pSport1 (I11/TNF), subt S0250 Human Osteoblasts II Human Osteoblasts Femur disease pCMVSport 2.0 S0260 Spinal Cord, re-excision Spinal cord spinal cord Uni-ZAP XR S0276 Synovial hypoxia-RSF Synovial fobroblasts Synovial tissue pSport1 subtracted (rheumatoid) S0278 H Macrophage (GM- Macrophage (GM-CSF Uni-ZAP XR CSF treated), re- treated) excision S0280 Human Adipose Tissue, Human Adipose Tissue Uni-ZAP XR re-excision S0282 Brain Frontal Cortex, Brain frontal cortex Brain Lambda ZAP II re-excision S0294 Larynx tumor Larynx tumor Larynx, vocal disease pSport1 S0298 Bone marrow Bone marrow Bone marrow pSport1 stroma, treated stroma, treatedSB S0300 Frontal Frontal Lobe Brain Uni-ZAP XR lobe, dementia; re- dementia/Alzheimer''s excision S0306 Larynx normal #10 261-273 Larynx normal pSport1 S0308 Spleen/normal Spleen normal pSport1 S0310 Normal trachea Normal trachea pSport1 S0312 Human Human osteoarthritic disease pSport1 osteoarthritic; fraction II cartilage S0314 Human Human osteoarthritic disease pSport1 osteoarthritis; fraction I cartilage S0318 Human Normal Human Normal Cartilage pSport1 Cartilage Fraction II S0322 Siebben Polyposis Siebben Polyposis pSport1 S0328 Palate carcinoma Palate carcinoma Uvula disease pSport1 S0330 Palate normal Palate normal Uvula pSport1 S0332 Pharynx carcinoma Pharynx carcinoma Hypopharynx pSport1 S0338 Human Osteoarthritic Human osteoarthritic disease pSport1 Cartilage Fracion III cartilage S0340 Human Osteoarthritic Human osteoarthritic disease pSport1 Cartilage Fraction IV cartilage S0342 Adipocytes; re-excision Human Adipocytes from Uni-ZAP XR Osteoclastoma S0344 Macrophage-oxLDL; macrophage-oxidized LDL blood Cell Line Uni-ZAP XR re-excision treated S0346 Human Amygdala; re- Amygdala Uni-ZAP XR excision S0348 Cheek Carcinoma Cheek Carcinoma disease pSport1 S0350 Pharynx Carcinoma Pharynx carcinoma Hypopharynx disease pSport1 S0352 Larynx Carcinoma Larynx carcinoma disease pSport1 S0356 Colon Carcinoma Colon Carcinoma Colon disease pSport1 S0358 Colon Normal III Colon Normal Colon pSport1 S0360 Colon Tumor II Colon Tumor Colon disease pSport1 S0364 Human Quadriceps Quadriceps muscle pSport1 S0366 Human Soleus Soleus Muscle pSport1 S0370 Larynx carcinoma II Larynx carcinoma disease pSport1 S0372 Larynx carcinoma III Larynx carcinoma disease pSport1 S0374 Normal colon Normal colon pSport1 S0376 Colon Tumor Colon Tumor disease pSport1 S0378 Pancreas normal PCA4 Pancreas Normal PCA4 No pSport1 No S0380 Pancreas Tumor PCA4 Pancreas Tumor PCA4 Tu disease pSport1 Tu S0382 Larynx carcinoma IV Larynx carcinoma disease pSport1 S0384 Tongue carcinoma Tongue carcinoma disease pSport1 S0388 Human Human Hypothalamus, disease Uni-ZAP XR Hypothalamus, schizoph Schizophrenia renia, re-excision S0390 Smooth muscle, control; Smooth muscle Pulmanary Cell Line Uni-ZAP XR re-excision artery S0392 Salivary Gland Salivary gland; normal pSport1 S0394 Stomach; normal Stomach; normal pSport1 S0398 Testis; normal Testis; normal pSport1 S0400 Brain; normal Brain; normal pSport1 S0402 Adrenal Gland, normal Adrenal gland; normal pSport1 S0404 Rectum normal Rectum, normal pSport1 S0406 Rectum tumour Rectum tumour pSport1 S0408 Colon, normal Colon, normal pSport1 S0410 Colon, tumour Colon, tumour pSport1 S0412 Temporal cortex- Temporal cortex, alzheimer disease Other Alzheizmer; subtracted Alzheimer Subtracted Subtracted S0418 CHME Cell Line; treated CHME Cell Line; treated pCMVSport 3.0 5 hrs S0420 CHME Cell CHME Cell line, untreatetd pSport1 Line, untreated S0422 Mo7e Cell Line GM- Mo7e Cell Line GM-CSF pCMVSport 3.0 CSF treated (1 ng/ml) treated (1 ng/ml) S0424 TF-1 Cell Line GM- TF-1 Cell Line GM-CSF pSport1 CSF Treated Treated S0426 Monocyte activated; re- Monocyte-activated blood Cell Line Uni-ZAP XR excision S0428 Neutrophils control; re- human neutrophils blood Cell Line Uni-ZAP XR excision S0430 Aryepiglottis Normal Aryepiglottis Normal pSport1 S0432 Sinus piniformis Sinus piniformis Tumour pSport1 Tumour S0434 Stomach Normal Stomach Normal disease pSport1 S0436 Stomach Tumour Stomach Tumour disease pSport1 S0438 Liver Normal Met5No Liver Normal Met5No pSport1 S0440 Liver Tumour Met 5 Tu Liver Tumour pSport1 S0442 Colon Normal Colon Normal pSport1 S0444 Colon Tumor Colon Tumour disease pSport1 S0448 Larynx Normal Larynx Normal pSport1 S0450 Larynx Tumour Larynx Tumour pSport1 S0452 Thymus Thymus pSport1 S0456 Tongue Normal Tongue Normal pSport1 S0458 Thyroid Normal Thyroid normal pSport1 (SDCA2 No) S0460 Thyroid Tumour Thyroid Tumour pSport1 S0468 Ea.hy.926 cell line Ea.hy.926 cell line pSport1 S0472 Lung Mesothelium PYBT pSport1 S0474 Human blood platelets Platelets Blood platelets Other excission S3012 Smooth Muscle Serum Smooth muscle Pulmanary Cell Line pBluescript Treated, Norm artery S3014 Smooth muscle, serum Smooth muscle Pulmanary Cell Line pBluescript induced, re-exc artery S6022 H. Adipose Tissue Human Adipose Tissue Uni-ZAP XR S6024 Alzheimers, spongy Alzheimer''s/Spongy Brain disease Uni-ZAP XR change change S6026 Frontal Lobe, Dementia Frontal Lobe Brain Uni-ZAP XR dementia/Alzheimer''s S6028 Human Manic Human Manic depression Brain disease Uni-ZAP XR Depression Tissue tissue T0002 Activated T-cells Activated T-Cell, PBL Blood Cell Line pBluescript SK− fraction T0003 Human Fetal Lung Human Fetal Lung pBluescript SK− T0004 Human White Fat Human White Fat pBluescript SK− T0006 Human Pineal Gland Human Pinneal Gland pBluescript SK− T0008 Colorectal Tumor Colorectal Tumor disease pBluescript SK− T0010 Human Infant Brain Human Infant Brain Other T0023 Human Pancreatic Human Pancreatic disease pBluescript SK− Carcinoma Carcinoma T0039 HSA 172 Cells Human HSA172 cell line pBluescript SK− T0040 HSC172 cells SA172 Cells pBluescript SK− T0041 Jurkat T-cell G1 phase Jurkat T-cell pBluescript SK− T0042 Jurkat T-Cell, S phase Jurkat T-Cell Line pBluescript SK− T0048 Human Aortic Human Aortic Endothilium pBluescript SK− Endothelium T0049 Aorta endothelial cells + TNF-a Aorta endothelial cells pBluescript SK− T0060 Human White Adipose Human White Fat pBluescript SK− T0067 Human Thyroid Human Thyroid pBluescript SK− T0069 Human Uterus, normal Human Uterus, normal pBluescript SK− T0078 Human Liver, normal Human Liver, normal Adult pBluescript SK− adult T0082 Human Adult Retina Human Adult Retina pBluescript SK− T0104 HCC cell line metastisis pBluescript SK− to liver T0109 Human (HCC) cell line pBluescript SK− liver (mouse) metastasis, remake T0110 Human colon carcinoma pBluescript SK− (HCC) cell line, remake T0112 Human (Caco-2) cell pBluescript SK− line, adenocarcinoma, colon T0114 Human (Caco-2) cell pBluescript SK− line, adenocarcinoma, colon, remake T0115 Human Colon pBluescript SK− Carcinoma (HCC) cell line L0002 Atrium cDNA library Human heart L0004 ClonTech HL 1065a L0005 Clontech human aorta polyA+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult lung 3″ directed MboI cDNA L0041 Human epidermal keratinocyte L0055 Human promyelocyte L0065 Liver HepG2 cell line. L0105 Human aorta polyA+ aorta L0142 Human placenta cDNA placenta (TFujiwara) L0143 Human placenta polyA+ placenta (TFujiwara) L0151 Human testis (C. De testis Smet) L0157 Human fetal brain brain (TFujiwara) L0158 Human fetal brain brain QBoqin L0163 Human heart cDNA heart (YNakamura) L0194 Human pancreatic pancreatic cancer Patu 8988t cancer cell line Patu 8988t L0351 Infant brain, Bento BA, M13-derived Soares L0352 Normalized infant brain, BA, M13-derived Bento Soares L0355 P, Human foetal Brain Bluescript Whole tissue L0361 Stratagene ovary ovary Bluescript SK (#937217) L0362 Stratagene ovarian Bluescript SK− cancer (#937219) L0363 NCI_CGAP_GC2 germ cell tumor Bluescript SK− L0364 NCI_CGAP_GC5 germ cell tumor Bluescript SK− L0366 Stratagene schizo brain schizophrenic brain S-11 Bluescript SK− S11 frontal lobe L0367 NCI_CGAP_Sch1 Schwannoma tumor Bluescript SK− L0368 NCI_CGAP_SS1 synovial sarcoma Bluescript SK− L0369 NCI_CGAP_AA1 adrenal adenoma adrenal gland Bluescript SK− L0371 NCI_CGAP_Br3 breast tumor breast Bluescript SK− L0372 NCI_CGAP_Co12 colon tumor colon Bluescript SK− L0373 NCI_CGAP_Co11 tumor colon Bluescript SK− L0374 NCI_CGAP_Co2 tumor colon Bluescript SK− L0375 NCI_CGAP_Kid6 kidney tumor kidney Bluescript SK− L0376 NCI_CGAP_Lar1 larynx larynx Bluescript SK− L0378 NCI_CGAP_Lu1 lung tumor lung Bluescript SK− L0381 NCI_CGAP_HN4 squamous cell carcinoma pharynx Bluescript SK− L0382 NCI_CGAP_Pr25 epithelium (cell line) prostate Bluescript SK− L0383 NCI_CGAP_Pr24 invasive tumor (cell line) prostate Bluescript SK− L0384 NCI_CGAP_Pr23 prostate tumor prostate Bluescript SK− L0386 NCI_CGAP_HN3 squamous cell carcinoma tongue Bluescript SK− from base of tongue L0387 NCI_CGAP_GCB0 germinal center B-cells tonsil Bluescript SK− L0388 NCI_CGAP_HN6 normal gingiva (cell line Bluescript SK− from immortalized kerati L0393 B, Human Liver tissue gt11 L0411 1-NIB Lafmid BA L0415 b4HB3MA Cot8-HAP- Lafmid BA Ft L0435 Infant brain, LLNL lafmid BA array of Dr. M. Soares 1NIB L0438 normalized infant brain total brain brain lafmid BA cDNA L0439 Soares infant brain whole brain Lafmid BA 1NIB L0442 4HB3MK Lafmid BK L0448 3HFLSK20 Lafmid K L0455 Human retina cDNA retina eye lambda gt10 randomly primed sublibrary Tsp509I-cleaved sublibrary L0459 Adult heart, Clontech Lambda gt11 L0462 WATM1 lambda gt11 L0470 BL29 Burkitt''s lambda ZAP 2 lymphoma, Pascalis Sideras L0471 Human fetal heart, Lambda ZAP Express Lambda ZAP Express L0480 Stratagene cat#937212 Lambda ZAP, (1992) pBluescript SK(−) L0481 CD34+DIRECTIONAL Lambda ZAPII L0483 Human pancreatic islet Lambda ZAPII L0485 STRATAGENE Human skeletal muscle leg muscle Lambda ZAPII skeletal muscle cDNA library, cat. #936215. L0497 NCI_CGAP_HSC4 CD34+, CD38− from bone marrow pAMP1 normal bone marrow donor L0498 NCI_CGAP_HSC3 CD34+, T negative, patient bone marrow pAMP1 with chronic myelogenou L0503 NCI_CGAP_Br17 adenocarcinoma breast pAMP1 L0506 NCI_CGAP_Br16 lobullar carcinoma in situ breast pAMP1 L0511 NCI_CGAP_Ov34 borderline ovarian ovary pAMP1 carcinoma L0512 NCI_CGAP_Ov36 borderline ovarian ovary pAMP1 carcinoma L0515 NCI_CGAP_Ov32 papillary serous carcinoma ovary pAMP1 L0517 NCI_CGAP_Pr1 pAMP10 L0518 NCI_CGAP_Pr2 pAMP10 L0519 NCI_CGAP_Pr3 pAMP10 L0520 NCI_CGAP_Alv1 alveolar pAMP10 rhabdomyosarcoma L0522 NCI_CGAP_Kid1 kidney pAMP10 L0523 NCI_CGAP_Lip2 liposarcoma pAMP10 L0525 NCI_CGAP_Li2 liver pAMP10 L0526 NCI_CGAP_Pr12 metastatic prostate bone pAMP10 lesion L0527 NCI_CGAP_Ov2 ovary pAMP10 L0529 NCI_CGAP_Pr6 prostate pAMP10 L0532 NCI_CGAP_Thy1 thyroid pAMP10 L0534 Chromosome 7 Fetal brain brain pAMP10 Brain cDNA Library L0540 NCI_CGAP_Pr10 invasive prostate tumor prostate pAMP10 L0542 NCI_CGAP_Pr11 normal prostatic epithelial prostate pAMP10 cells L0543 NCI_CGAP_Pr9 normal prostatic epithelial prostate pAMP10 cells L0544 NCI_CGAP_Pr4 prostatic intraepithelial prostate pAMP10 neoplasia - high grade L0545 NCI_CGAP_Pr4.1 prostatic intraepithelial prostate pAMP10 neoplasia - high grade L0549 NCI_CGAP_HN10 carcinoma in situ from pAMP10 retromolar trigone L0551 NCI_CCAP_HN7 normal squamous pAMP10 epithelium, floor of mouth L0555 NCI_CGAP_Lu34 large cell carcinoma lung pAMP10 L0561 NCI_CGAP_HN11 normal squamous tongue pAMP10 epithelium L0562 Chromosome 7 HeLa HeLa cell line; pAMP10 cDNA Library ATCC L0563 Human Bone Marrow bone marrow pBluescript Stromal Fibroblast L0564 Jia bone marrow stroma bone marrow stroma pBluescript L0565 Normal Human Bone Hip pBluescript L0581 Stratagene liver liver pBluescript SK (#937224) L0584 Stratagene cDNA pBluescript SK(+) library Human heart, cat#936208 L0586 HTCDL1 pBluescript SK(−) L0588 Stratagene endothelial pBluescript SK− cell 937223 L0589 Stratagene fetal retina pBluescript SK− 937202 L0590 Stratagene fibroblast pBluescript SK− (#937212) L0591 Stratagene HeLa cell s3 pBluescript SK− 937216 L0592 Stratagene hNT neuron pBluescript SK− (#937233) L0593 Stratagene pBluescript SK− neuroepithelium (#937231) L0594 Stratagene pBluescript SK− neuroepithelium NT2RAMI 937234 L0595 Stratagene NT2 neuroepithelial cells brain pBluescript SK− neuronal precursor 937230 L0596 Stratagene colon colon pBluescript SK− (#937204) L0597 Stratagene corneal cornea pBluescript SK− stroma (#937222) L0598 Morton Fetal Cochlea cochlea ear pBluescript SK− L0599 Stratagene lung lung pBluescript SK− (#937210) Epithelium L0601 Stratagene pancreas pancreas pBluescript SK− (#937208) L0602 Pancreatic Islet pancreatic islet pancreas pBluescript SK− L0603 Stratagene placenta placenta pBluescript SK− (#937225) L0604 Stratagene muscle muscle skeletal muscle pBluescript SK− 937209 L0605 Stratagene fetal spleen fetal spleen spleen pBluescript SK− (#937205) L0606 NCI_CGAP_Lym5 follicular lymphoma lymph node pBluescript SK− L0607 NCI_CGAP_Lym6 mantle cell lymphoma lymph node pBluescript SK− L0608 Stratagene lung lung carcinoma lung NCI-H69 pBluescript SK− carcinoma 937218 L0611 Schiller meningioma meningioma brain pBluescript SK− (Stratagene) L0612 Schiller oligodendroglioma brain pBluescript SK− oligodendroglioma (Stratagene) L0615 22 week old human fetal pBluescriptII SK(−) liver cDNA library L0617 Chromosome 22 exon pBluescriptIIKS+ L0622 HM1 pcDNAII (Invitrogen) L0623 HM3 pectoral muscle (after pcDNAII (Invitrogen) mastectomy) L0625 NCI_CGAP_AR1 bulk alveolar tumor pCMV-SPORT2 L0626 NCI_CGAP_GC1 bulk germ cell seminoma pCMV-SPORT2 L0627 NCI_CGAP_Co1 bulk tumor colon pCMV-SPORT2 L0629 NCI_CGAP_Mel3 metastatic melanoma to bowel (skin pCMV-SPORT4 bowel primary) L0631 NCI_CGAP_Br7 breast pCMV-SPORT4 L0634 NCI_CGAP_Ov8 serous adenocarcinoma ovary pCMV-SPORT4 L0635 NCI_CGAP_PNS1 dorsal root ganglion peripheral pCMV-SPORT4 L0636 NCI_CGAP_Pit1 four pooled pituitary brain pCMV-SPORT6 adenomas L0637 NCI_CGAP_Brn53 three pooled meningiomas brain pCMV-SPORT6 L0638 NCI_CGAP_Brn35 tumor, 5 pooled (see brain pCMV-SPORT6 description) L0639 NCI_CGAP_Brn52 tumor, 5 pooled (see brain pCMV-SPORT6 description) L0640 NCI_CGAP_Br18 four pooled high-grade breast pCMV-SPORT6 tumors, including two prima L0641 NCI_CGAP_Co17 juvenile granulosa tumor colon pCMV-SPORT6 L0642 NCI_CGAP_Co18 moderately differentiated colon pCMV-SPORT6 adenocarcinoma L0643 NCI_CGAP_Co19 moderately differentiated colon pCMV-SPORT6 adenocarcinoma L0644 NCI_CGAP_Co20 moderately differentiated colon pCMV-SPORT6 adenocarcinoma L0645 NCI_CGAP_Co21 moderately differentiated colon pCMV-SPORT6 adenocarcinoma L0646 NCI_CGAP_Co14 moderately-differentiated colon pCMV-SPORT6 adenocarcinoma L0647 NCI_CGAP_Sar4 five pooled sarcomas, connective pCMV-SPORT6 including myxoid tissue liposarcoma L0648 NCI_CGAP_Eso2 squamous cell carcinoma esophagus pCMV-SPORT6 L0649 NCI_CGAP_GU1 2 pooled high-grade genitourinary pCMV-SPORT6 transitional cell tumors tract L0650 NCI_CGAP_Kid13 2 pooled Wilms'' tumors, kidney pCMV-SPORT6 one primary and one metast L0651 NCI_CGAP_Kid8 renal cell tumor kidney pCMV-SPORT6 L0652 NCI_CGAP_Lu27 four pooled poorly- lung pCMV-SPORT6 differentiated L0653 NCI_CGAP_Lu28 two pooled squamous cell lung pCMV-SPORT6 carcinomas L0654 NCI_CGAP_Lu31 lung, cell line pCMV-SPORT6 L0655 NCI_CGAP_Lym12 lymphoma, follicular mixed lymph node pCMV-SPORT6 small and large cell L0656 NCI_CGAP_Ov38 normal epithelium ovary pCMV-SPORT6 L0657 NCI_CGAP_Ov23 tumor, 5 pooled (see ovary pCMV-SPORT6 description) L0658 NCI_CGAP_Ov35 tumor, 5 pooled (see ovary pCMV-SPORT6 description) L0659 NCI_CGAP_Pan1 adenocarcinoma pancreas pCMV-SPORT6 L0661 NCI_CGAP_Mel15 malignant melanoma, skin pCMV-SPORT6 metastatic to lymph node L0662 NCI_CGAP_Gas4 poorly differentiated stomach pCMV-SPORT6 adenocarcinoma with signet r L0663 NCI_CGAP_Ut2 moderately-differentiated uterus pCMV-SPORT6 endometrial adenocarcino L0664 NCI_CGAP_Ut3 poorly-differentiated uterus pCMV-SPORT6 endometrial adenocarcinoma, L0665 NCI_CGAP_Ut4 serous papillary carcinoma, uterus pCMV-SPORT6 high grade, 2 pooled t L0666 NCI_CGAP_Ut1 well-differentiated uterus pCMV-SPORT6 endometrial adenocarcinoma, 7 L0667 NCI_CGAP_CML1 myeloid cells, 18 pooled whole blood pCMV-SPORT6 CML cases, BCR/ABL rearra L0686 Stanley Frontal SN pool 2 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0698 Testis 2 PGEM 5 zf(+) hncDNA library L0709 NIH_MGC_21 choriocarcinoma placenta pOTB7 L0710 NIH_MGC_7 small cell carcinoma lung MGC3 pOTB7 L0717 Gessler Wilms tumor pSPORT1 L0718 Testis 5 pSPORT1 L0731 Soares_pregnant_uterus — uterus pT7T3-Pac NbHPU L0738 Human colorectal pT7T3D cancer L0740 Soares melanocyte melanocyte pT7T3D (Pharmacia) 2NbHM with a modified polylinker L0741 Soares adult brain brain pT7T3D (Pharmacia) N2b4HB55Y with a modified polylinker L0742 Soares adult brain brain pT7T3D (Pharmacia) N2b5HB55Y with a modified polylinker L0743 Soares breast 2NbHBst breast pT7T3D (Pharmacia) with a modified polylinker L0744 Soares breast 3NbHBst breast pT7T3D (Pharmacia) with a modified polylinker L0745 Soares retina N2b4HR retina eye pT7T3D (Pharmacia) with a modified polylinker L0746 Soares retina N2b5HR retina eye pT7T3D (Pharmacia) with a modified polylinker L0747 Soares_fetal_heart_NbHH19W heart pT7T3D (Pharmacia) with a modified L0748 Soares fetal liver spleen Liver and pT7T3D (Pharmacia) 1NFLS Spleen with a modified polylinker L0749 Soares_fetal_liver_spleen — Liver and pT7T3D (Pharmacia) 1NFLS_S1 Spleen with a modified polylinker L0750 Soares_fetal_lung_NbHL19W lung pT7T3D (Pharmacia) with a modified polylinker L0751 Soares ovary tumor ovarian tumor ovary pT7T3D (Pharmacia) NbHOT with a modified polylinker L0752 Soares_parathyroid_tumor — parathyroid tumor parathyroid pT7T3D (Pharmacia) NbHPA gland with a modified polylinker L0753 Soares_pineal_gland_N3HPG pineal gland pT7T3D (Pharmacia) with a modified polylinker L0754 Soares placenta Nb2HP placenta pT7T3D (Pharmacia) with a modified polylinker L0755 Soares_placenta_8to9weeks — placenta pT7T3D (Pharmacia) 2NbHP8to9W with a modified polylinker L0756 Soares_multiple_sclerosis — multiple sclerosis lesions pT7T3D (Pharmacia) 2NbHMSP with a modified polylinker V_TYPE L0757 Soares_senescent_fibroblasts — senescent fibroblast pT7T3D (Pharmacia) NbHSF with a modified polylinker V_TYPE L0758 Soares_testis_NHT pT7T3D-Pac (Pharmacia) with a L0759 Soares_total_fetus_Nb2HF8 — pT7T3D-Pac 9w (Pharmacia) with a modified polylinker L0760 Barstead aorta HPLRB3 aorta pT7T3D-Pac (Pharmacia) with a modified polylinker L0761 NCI_CGAP_CLL1 B-cell, chronic lymphotic pT7T3D-Pac leukemia (Pharmacia) with a modified polylinker L0762 NCI_CGAP_Br1.1 breast pT7T3D-Pac (Pharmacia) with a modified polylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia) with a modified polylinker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0765 NCI_CGAP_Co4 colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0766 NCI_CGAP_GCB1 germinal center B cell pT7T3D-Pac (Pharmacia) with a modified polylinker L0767 NCI_CGAP_GC3 pooled germ cell tumors pT7T3D-Pac (Pharmacia) with a modified polylinker L0768 NCI_CGAP_GC4 pooled germ cell tumors pT7T3D-Pac (Pharmacia) with a modified polylinker L0769 NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac oligodendroglioma (Pharmacia) with a L0770 NCI_CGAP_Brn23 glioblastoma (pooled) brain pT7T3D-Pac (Pharmacia) with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinoma colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0772 NCI_CGAP_Co10 colon tumor RER+ colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0773 NCI_CGAP_Co9 colon tumor RER+ colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0774 NCI_CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0775 NCI_CGAP_Kid5 2 pooled tumors (clear cell kidney pT7T3D-Pac type) (Pharmacia) with a modified polylinker L0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified polylinker L0777 Soares_NhHMPu_S1 Pooled human melanocyte, mixed (see pT7T3D-Pac fetal heart, and pregnant below) (Pharmacia) with a modified polylinker L0779 Soares_NFL_T_GBC_S1 pooled pT7T3D-Pac (Pharmacia) with a modified polylinker L0780 Soares_NSF_F8_9W_O pooled pT7T3D-Pac T_PA_P_S1 (Pharmacia) with a modified polylinker L0782 NCI_CGAP_Pr21 normal prostate prostate pT7T3D-Pac (Pharmacia) with a L0783 NCI_CGAP_Pr22 normal prostate prostate pT7T3D-Pac (Pharmacia) with a modified polylinker L0784 NCI_CGAP_Lei2 leiomyosarcoma soft tissue pT7T3D-Pac (Pharmacia) with a modified polylinker L0785 Barstead spleen spleen pT7T3D-Pac HPLRB2 (Pharmacia) with a modified polylinker L0786 Soares_NbHFB whole brain pT7T3D-Pac (Pharmacia) with a modified polylinker L0787 NCI_CGAP_Sub1 pT7T3D-Pac (Pharmacia) with a modified polylinker L0788 NCI_CGAP_Sub2 pT7T3D-Pac (Pharmacia) with a modified polylinker L0789 NCI_CGAP_Sub3 pT7T3D-Pac (Pharmacia) with a modified polylinker L0790 NCI_CGAP_Sub4 pT7T3D-Pac (Pharmacia) with a modified polylinker L0791 NCI_CGAP_Sub5 pT7T3D-Pac (Pharmacia) with a modified polylinker L0792 NCI_CGAP_Sub6 pT7T3D-Pac (Pharmacia) with a modified polylinker L0793 NCI_CGAP_Sub7 pT7T3D-Pac (Pharmacia) with a L0794 NCI_CGAP_GC6 pooled germ cell tumors pT7T3D-Pac (Pharmacia) with a modified polylinker L0796 NCI_CGAP_Brn50 medulloblastoma brain pT7T3D-Pac (Pharmacia) with a modified polylinker L0800 NCI_CGAP_Co16 colon tumor, RER+ colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0803 NCI_CGAP_Kid11 kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0804 NCI_CGAP_Kid12 2 pooled tumors (clear cell kidney pT7T3D-Pac type) (Pharmacia) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified polylinker L0806 NCI_CGAP_Lu19 squamous cell carcinoma, lung pT7T3D-Pac poorly differentiated (4 (Pharmacia) with a modified polylinker L0807 NCI_CGAP_Ov18 fibrotheoma ovary pT7T3D-Pac (Pharmacia) with a modified polylinker L0808 Barstead prostate BPH prostate pT7T3D-Pac HPLRB4 1 (Pharmacia) with a modified polylinker L0809 NCI_CGAP_Pr28 prostate pT7T3D-Pac (Pharmacia) with a modified polylinker L1430 CT0225 colon puc18 L1562 CN0027 colon_normal puc18 L2255 GLC corresponding non pBluescript sk(−) cancerous liver tissue L2257 NIH_MGC_65 adenocarcinoma colon pCMV-SPORT6 L2258 NIH_MGC_67 retinoblastoma eye pCMV-SPORT6 L2259 NIH_MGC_68 large cell carcinoma lung pCMV-SPORT6 L2260 NIH_MGC_69 large cell carcinoma, lung pCMV-SPORT6 undifferentiated L2261 NIH_MGC_70 epithelioid carcinoma pancreas pCMV-SPORT6 L2262 NIH_MGC_72 melanotic melanoma skin pCMV-SPORT6 L2263 NIH_MGC_66 adenocarcinoma ovary pCMV-SPORT6 L2264 NIH_MGC_71 leiomyosarcoma uterus pCMV-SPORT6 L2270 Lupski_dorsal_root_ganglion dorsal root ganglia pCMV-SPORT6 (Life Technologies) L2285 BT0723 breast puc18 L2293 BT0762 breast puc18 L2300 BT0789 breast puc18 L2323 CT0406 colon puc18 L2359 UT0023 uterus_tumor puc18 L2368 UT0041 uterus_tumor puc18 L2402 NN0118 nervous_normal puc18 L2482 HT0497 head_neck puc18 L2486 HT0527 head_neck puc18 L2491 HT0559 head_neck puc18 L2497 HT0618 head_neck puc18 L2499 HT0622 head_neck puc18 L2528 HT0713 head_neck puc18 L2630 HT0865 head_neck puc18 L2647 HT0894 head_neck puc18 L2652 NIH_MGC_57 glioblastoma brain pDNR-LIB (Clontech) L2653 NIH_MGC_58 hypernephroma kidney pDNR-LIB (Clontech) L2654 NIH_MGC_9 adenocarcinoma cell line ovary pOTB7 leukemia L2657 NIH_MGC_54 from chronic myelogenous bone marrow pDNR-LIB (Clontech) leukemia L2670 NT0023 nervous_tumor puc18 L2744 FT0004 prostate_tumor puc18 L2759 FT0028 prostate_tumor puc18 L2777 FT0056 prostate_tumor puc18 L2800 FT0097 prostate_tumor puc18 L2879 AN0032 amnion_normal puc18 L2903 BN0039 breast_normal puc18 L2906 BN0047 breast_normal puc18 L2909 BN0067 breast_normal puc18 L2910 BN0070 breast_normal puc18 L3012 BN0296 breast_normal puc18 L3109 ET0046 lung_tumor puc18 13180 MT0101 marrow puc18 L3181 MT0107 marrow puc18 L3215 OT0083 ovary puc18 L3271 FN0094 prostate_normal puc18 L3278 FN0104 prostate_normal puc18 L3311 FN0180 prostate_normal puc18 L3387 GKB hepatocellular carcinoma pBluescript sk(−) L3388 GKC hepatocellular carcinoma pBluescript sk(−) L3391 NIH_MGC_53 carcinoma, cell line bladder pDNR-LIB (Clontech) L3485 GN0070 placenta_normal puc18 L3499 HT0617 head_neck puc18 L3503 HT0870 head_neck puc18 L3576 TN0086 testis_normal puc18 L3643 ADB Adrenal gland pBluescript sk(−) L3644 ADC Adrenal gland pBluescript sk(−) L3645 Cu adrenal cortico adenoma for pBluescript sk(−) Cushing''s syndrome L3646 DCA pTriplEx2 L3647 Human HO-1 melanoma cells L3649 DCB pTriplEx2 L3652 FHTB hypothalamus pTriplEx2 L3653 HTB Hypothalamus pBluescript sk(−) L3655 HTC Hypothalamus pBluescript sk(−) L3657 HTF Hypothalamus pBluescript sk(−) L3658 cdA pheochromocytoma pTriplEx2 L3659 CB cord blood pBluescript L3663 NIH_MGC_60 adenocarcinoma prostate pDNR-LIB (Clontech) L3665 NIH_MGC_75 kidney pDNR-LIB (Clontech) L3709 CT0515 colon puc18 L3726 GN0038 placenta_normal puc18 L3796 UT0042 uterus_tumor puc18 L3811 NPC pituitary pBluescript sk(−) L3814 BM Bone marrow pTriplEx2 L3815 MDS Bone marrow pTriplEx2 L3816 HEMBA1 whole embryo, mainly head pME18SFL3 L3817 HEMBB1 whole embryo, mainly body pME18SFL3 L3818 MAMMA1 mammary gland pME18SFL3 L3821 NIH_MGC_48 primary B-cells from B-cells pOTB7 tonsils (cell line) L3822 NIH_MGC_59 mucoepidermoid carcinoma lung pDNR-LIB (Clontech) L3824 NT2RM2 NT2 pME18SFL3 L3825 NT2RM4 NT2 pME18SFL3 L3826 NT2RP1 NT2 pUC19FL3 L3827 NT2RP2 NT2 pME18SFL3 L3828 NT2RP3 NT2 pME18SFL3 L3829 NT2RP4 NT2 pME18SFL3 L3831 OVARC1 ovary, tumor tissue pME18SFL3 L3832 PLACE1 placenta pME18SFL3 L3833 PLACE2 placenta pME18SFL3 L3834 PLACE3 placenta pME18SFL3 L3837 THYRO1 thyroid gland pME18SFL3 L3872 NCI_CGAP_Skn1 skin, normal, 4 pCMV-SPORT6 pooled sa L3904 NCI_CGAP_Brn64 glioblastoma with EGFR brain pCMV-SPORT6 amplification L3905 NCI_CGAP_Brn67 anaplastic brain pCMV-SPORT6 oligodendroglioma with 1p/19q loss L4497 NCI_CGAP_Br22 invasive ductal carcinoma, breast pCMV-SPORT6 3 pooled samples L4501 NCI_CGAP_Sub8 pT7T3D-Pac (Pharmacia) with a modified polylinker L4508 NCI_CGAP_Thy8 normal epithelium thyroid pAMP10 L4556 NCI_CGAP_HN13 squamous cell carcinoma tongue pCMV-SPORT6 L4558 NCI_CGAP_Pan3 pancreas pCMV-SPORT6 L4559 NCI_CGAP_Thy3 follicular carcinoma thyroid pCMV-SPORT6 L4747 NCI_CGAP_Brn41 oligodendroglioma brain pT7T3D-Pac (Pharmacia) with a modified polylinker L5564 NCI_CGAP_HN20 normal pAMP1 head/neck tissue L5565 NCI_CGAP_Brn66 glioblastoma with probably brain pCMV-SPORT6 TP53 mutation and witho L5566 NCI_CGAP_Brn70 anaplastic brain pCMV-SPORT6.ccdb oligodendroglioma L5572 NCI_CGAP_Co27 adenocarcinoma (mucinous colon pAMP1 component) L5574 NCI_CGAP_HN19 normal epithelium nasopharynx pAMP10 L5575 NCI_CGAP_Brn65 glioblastoma without EGFR brain pCMV-SPORT6 amplification L5622 NCI_CGAP_Skn3 skin pCMV-SPORT6 L5623 NCI_CGAP_Skn4 squamous cell carcinoma skin pCMV-SPORT6

Description of Table 5

Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000. Web URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases with the cytologic band disclosed in Table 1B.1, column 8, as determined using the database.
TABLE 5 OMIM Reference Description 100690 Myasthenic syndrome, slow-channel congenital, 601462 100710 Myasthenic syndrome, slow-channel congenital, 601462 100730 Myasthenia gravis, neonatal transient 102200 Somatotrophinonia 102700 Severe combined immunodeficiency due to ADA deficiency 102700 Hemolytic anemia due to ADA excess 103581 Albright hereditary osteodystrophy-2 104770 Amyloidosis, secondary, susceptibility to 106100 Angioedema, hereditary 106150 Hypertension, essential, susceptibility to 106150 Preeclampsia, susceptibility to 106165 Hypertension, essential, 145500 107250 Anterior segment mesenchymal dysgenesis 107300 Antithrombin III deficiency 107670 Apolipoprotein A-II deficiency 108725 Atherosclerosis, susceptibility to 108985 Atrophia areata 109270 Renal tubular acidosis, distal, 179800 109270 Spherocytosis, hereditary 109270 [Acanthocytosis, one form] 109270 [Elliptocytosis, Malaysian-Melanesian type] 109270 Hemolytic anemia due to band 3 defect 110100 Blepharophimosis, epicanthus inversus, and ptosis, type 1 110700 Vivax malaria, susceptibility to 112410 Hypertension with brachydactyly 116806 Colorectal cancer 116860 Cavernous angiomatous malformations 117700 [Hypoceruloplasminemia, hereditary] 117700 Hemosiderosis, systemic, due to aceruloplasminemia 118485 Polycystic ovary syndrome with hyperandrogenemia 118800 Choreoathetosis, familial paroxysmal 120070 Alport syndrome, autosomal recessive, 203780 120120 Epidermolysis bullosa dystrophica, dominant, 131750 120120 Epidermolysis bullosa dystrophica, recessive, 226600 120120 Epidermolysis bullosa, pretibial, 131850 120131 Alport syndrome, autosomal recessive, 203780 120131 Hematuria, familial benign 120150 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 166220 120150 Osteoporosis, idiopathic, 166710 120150 Ehlers-Danlos syndrome, type VIIA1, 130060 120436 Muir-Torre family cancer syndrome, 158320 120436 Turcot syndrome with glioblastoma, 276300 120436 Colorectal cancer, hereditary nonpolyposis, type 2 120550 C1q deficiency, type A 120570 C1q deficiency, type B 120575 C1q deficiency, type C 120700 C3 deficiency 123000 Craniometaphyseal dysplasia 123620 Cataract, cerulean, type 2, 601547 123660 Cataract, Coppock-like 129900 EEC syndrome-1 130500 Elliptocytosis-1 131100 Multiple endocrine neoplasia I 131100 Prolactinoma, hyperparathyroidism, carcinoid syndrome 131100 Carcinoid tumor of lung 131210 Atherosclerosis, susceptibility to 133171 [Erythrocytosis, familial], 133100 133200 Erythrokeratodermia variabilis 133450 Neuroepithelioma 133450 Ewing sarcoma 133701 Exostoses, multiple, type 2 133780 Vitreoretinopathy, exudative, familial 134790 Hyperferritinemia-cataract syndrome, 600886 134820 Dysfibrinogenemia, alpha type, causing bleeding diathesis 134820 Dysfibrinogenemia, alpha type, causing recurrent thrombosis 134820 Amyloidosis, hereditary renal, 105200 134830 Dysfibrinogenemia, beta type 134850 Dysfibrinogenemia, gamma type 134850 Hypofibrinogenemia, gamma type 135600 Ehlers-Danlos syndrome, type X 135700 Fibrosis of extraocular muscles, congenital, 1 135940 Ichthyosis vulgaris, 146700 136132 [Fish-odor syndrome], 602079 136836 Fucosyltransferase-6 deficiency 138030 [Hyperproglucagonemia] 138140 Glucose transport defect, blood-brain barrier 138190 Diabetes mellitus, noninsulin-dependent 138300 Hemolytic anemia due to glutathione reductase deficiency 138320 Hemolytic anemia due to glutathione peroxidase deficiency 141750 Alpha-thalassemia/mental retardation syndrome, type 1 141800 Methemoglobinemias, alpha- 141800 Thalassemias, alpha- 141800 Erythremias, alpha- 141800 Heinz body anemias, alpha- 141850 Thalassemia, alpha- 141850 Erythrocytosis 141850 Heinz body anemia 141850 Hemoglobin H disease 141850 Hypochromic microcytic anemia 142989 Synpolydactyly, type II, 186000 145001 Hyperparathyroidism-jaw tumor syndrome i45260 Pseudohypoaldosteronism, type II 145981 Hypocalciuric hypercalcemia, type II 146150 Hypomelanosis of Ito 146790 Lupus nephritis, susceptibility to 147050 Atopy 147141 Leukemia, acute lymphoblastic 147200 [Kappa light chain deficiency] 148065 White sponge nevus, 193900 148080 Epidermolytic hyperkeratosis, 113800 148370 Keratolytic winter erythema 150210 Lactoferrin-deficient neutrophils, 245480 151410 Leukemia, chronic myeloid 152445 Vohwinkel syndrome, 124500 152445 Erythrokeratoderma, progressive symmetric, 602036 152760 Hypogonadotropic hypogonadism due to GNRH deficiency, 227200 153454 Ehiers-Danlos syndrome, type VI, 225400 153700 Macular dystrophy, vitelliform type 154275 Malignant hyperthermia susceptibility 2 156232 Mesomelic dysplasia, Kantaputra type 156850 Cataract, congenital, with microphthalmia 157147 Abetalipoproteinemia, 200100 157640 PEO with mitochondrial DNA deletions, type 1 157655 Lactic acidosis due to defect in iron-sulfur cluster of complex I 159001 Muscular dystrophy, limb-girdle, type 1B 161015 Mitochondrial complex I deficiency, 252010 164009 Leukemia, acute promyelocytic, NUMA/RARA type 164953 Liposarcoma 168360 Paraneoplastic sensory neuropathy 168461 Multiple myeloma, 254250 168461 Parathyroid adenomatosis 1 168461 Centrocytic lymphoma 168468 Metaphyseal chondrodysplasia, Murk Jansen type, 156400 168470 Humoral hypercalcemia of malignancy 168500 Parietal foramina 169600 Hailey-Hailey disease 171190 Hypertension, essential, 145500 171650 Lysosomal acid phosphatase deficiency 171760 Hypophosphatasia, adult, 146300 171760 Hypophosphatasia, infantile, 241500 173610 Platelet alpha/delta storage pooi deficiency 173870 Xeroderma pigmentosum 173870 Fanconi anemia 174000 Medullary cystic kidney disease, AD 174900 Polyposis, juvenile intestinal 176100 Porphyria cutanea tarda 176100 Porphyria, hepatoerythropoietic 176830 Obesity, adrenal insufficiency, and red hair 176830 ACTH deficiency 176930 Dysprothrombinemia 176930 Hypoprothrombinemia 176943 Apert syndrome, 101200 176943 Pfeiffer syndrome, 101600 176943 Beare-Stevenson cutis gyrata syndrome, 123790 176943 Crouzon craniofacial dysostosis, 123500 176943 Jackson-Weiss syndrome, 123150 177070 Spherocytosis, hereditary, Japanese type 177070 Hermansky-Pudlak syndrome, 203300 178300 Ptosis, hereditary congenital, 1 178600 Pulmonary hypertension, familial primary 178640 Pulmonary alveolar proteinosis, congenital, 265120 179755 Renal cell carcinoma, papillary, 1 180100 Retinitis pigmentosa-1 180380 Night blindness, congenital stationery, rhodopsin-related 180380 Retinitis pigmentosa, autosomal recessive 180380 Retinitis pigmentosa-4, autosomal dominant 180721 Retinitis pigmentosa, digenic 180840 Susceptibility to IDDM 181405 Scapuloperoneal spinal muscular atrophy, New England type 181430 Scapuloperoneal syndrome, myopathic type 181600 Sclerotylosis 182138 Anxiety-related personality traits 182279 Prader-Willi syndrome 182280 Small-cell cancer of lung 182500 Cataract, congenital 182601 Spastic paraplegia-4 182860 Pyropoikilocytosis 182860 Spherocytosis, recessive 182860 Elliptocytosis-2 185430 Atherosclerosis, susceptibility to 185800 Symphalangism, proximal 186580 Arthrocutaneouveal granulomatosis 186860 Leukemia/lymphoma, T-cell 186921 Leukemia, T-cell acute lymphoblastic 188070 Bleeding disorder due to defective thromboxane A2 receptor 188450 Goiter, adolescent multinodular 188450 Goiter, nonendemic, simple 188450 Hypothyroidism, hereditary congenital 188826 Sorsby fundus dystrophy, 136900 189800 Preeclampsia/eclampsia 191092 Tuberous sclerosis-2 191170 Colorectal cancer, 114500 191170 Li-Fraumeni syndrome 191181 Cervical carcinoma 191315 Insensitivity to pain, congenital, with anhidrosis, 256800 192340 Diabetes insipidus, neurohypophyseal, 125700 193235 Vitreoretinopathy, neovascular inflammatory 200990 Acrocallosal syndrome 201460 Acyl-CoA dehydrogenase, long chain, deficiency of 203100 Waardenburg syndrome/ocular albinism, digenic, 103470 203100 Albinism, oculocutaneous, type IA 203200 Albinism, ocular, autosomal recessive 203200 Albinism, oculocutaneous, type II 203500 Alkaptonuria 203800 Alstrom syndrome 205100 Amyotrophic lateral sclerosis, juvenile 209901 Bardet-Biedl syndrome 1 214500 Chediak-Higashi syndrome 216900 Achromatopsia 218000 Andermann syndrome 221820 Gliosis, familial progressive subcortical 227220 [Eye color, brown] 227646 Fanconi anemia, type D 229800 [Fructosuria] 230000 Fucosidosis 230800 Gaucher disease 230800 Gaucher disease with cardiovascular calcification 231680 Glutaricaciduria, type IIA 232050 Propionicacidemia, type II or pccB type 232600 McArdle disease 233700 Chronic granulomatous disease due to deficiency of NCF-1 234200 Neurodegeneration with brain iron accumulation 236730 Urofacial syndrome 238600 Chylomicronemia syndrome, familial 238600 Combined hyperlipemia, familial 238600 Hyperlipoproteinemia I 238600 Lipoprotein lipase deficiency 240400 Scurvy 243500 Isovalericacidemia 245000 Papillon-Lefevre syndrome 248510 Mannosidosis, beta- 248611 Maple syrup urine disease, type Ib 249000 Meckel syndrome 249270 Thiamine-responsive megaloblastic anemia 253250 Mulibrey nanism 254210 Myasthenia gravis, familial infantile 255800 Schwartz-Jampel syndrome 256700 Neuroblastoma 258870 Gyrate atrophy of choroid and retina with omithinemia, B6 responsive or unresponsive 259700 Osteopetrosis, recessive 259770 Osteoporosis-pseudoglioma syndrome 259900 Hyperoxaluria, primary, type 1 261510 Pseudo-Zellweger syndrome 262000 Bjornstad syndrome 263700 Porphyria, congenital erythropoietic 266150 Pyruvate carboxylase deficiency 266200 Anemia, hemolytic, due to PK deficiency 266300 [Hair color, red] 271900 Canavan disease 272800 Tay-Sachs disease 272800 [Hex A pseudodeficiency] 272800 GM2-gangliosidosis, juvenile, adult 276700 Tyrosinemia, type I 276902 Usher syndrome, type 3 276903 Usher syndrome, type 1B 276903 Deafness, autosomal dominant 11, neurosensory, 601317 276903 Deafness, autosomal recessive 2, neurosensory, 600060 278250 Wrinkly skin syndrome 600040 Colorectal cancer 600045 Xeroderma pigmentosum, group E, subtype 2 600079 Colon cancer 600119 Muscular dystrophy, Duchenne-like, type 2 600119 Adhalinopathy, primary 600140 Rubenstein-Taybi syndrome, 180849 600143 Epilepsy, progressive, with mental retardation 600163 Long QT syndrome-3 600179 Leber congenital amaurosis, type I, 204000 600194 Ichthyosis bullosa of Siemens, 146800 600231 Palmoplantar keratoderma, Bothnia type 600258 Colorectal cancer, hereditary nonpolyposis, type 3 600273 Polycystic kidney disease, infantile severe, with tuberous sclerosis 600281 Non-insulin-dependent diabetes mellitus, 125853 600281 MODY, type 1, 125850 600319 Diabetes mellitus, insulin-dependent, 4 600512 Epilepsy, partial 600525 Trichodontoosseous syndrome, 190320 600528 CPT deficiency, hepatic, type I, 255120 600623 Prostate cancer, 176807 600698 Salivary adenoma 600698 Uterine leiomyoma 600698 Lipoma 600698 Lipomatosis, mutiple, 151900 600759 Alzheimer disease-4 600808 Enuresis, nocturnal, 2 600811 Xeroderma pigmentosum, group E, DDB-negative subtype, 278740 600839 Bartter syndrome, 241200 600850 Schizophrenia disorder-4 600881 Cataract, congenital, zonular, with sutural opacities 600882 Charcot-Marie-Tooth neuropathy-2B 600897 Cataract, zonular pulverulent-1, 116200 600957 Persistent Mullerian duct syndrome, type I, 261550 600958 Cardiomyopathy, familial hypertrophic, 4, 115197 600977 Cone dystrophy, progressive 600983 Pseudohypoaldosteronism type I, autosomal dominant, 177735 600996 Arrhythmogenic right ventricular dysplasia-2 601105 Pycnodysostosis, 265800 601154 Cardiomyopathy, dilated, 1E 601199 Neonatal hyperparathyroidism, 239200 601199 Hypocalcemia, autosomal dominant, 601198 601199 Hypocalciuric hypercalcemia, type I, 145980 601202 Cataract, anterior olar-2 601238 Cerebellar ataxia, Cayman type 601277 Ichthyosis, lamellar, type 2 601284 Hereditary hemorrhagic telangiectasia-2, 600376 601313 Polycystic kidney disease, adult type I, 173900 601318 Diabetes mellitus, insulin-dependent, 13 601385 Prostate cancer 601412 Deafness, autosomal dominant 7 601471 Moebius syndrome-2 601623 Angelman syndrome 601652 Glaucoma 1A, primary open angle, juvenile-onset, 137750 601669 Hirschsprung disease, one form 601682 Glaucoma 1C, primary open angle 601744 Systemic lupus erythematosus, susceptibility to, 1 601769 Osteoporosis, involutional 601769 Rickets, vitamin D-resistant, 277440 601777 Cone dystrophy, progressive 601785 Carbohydrate-deficient glycoprotein syndrome, type I, 212065 601800 [Hair color, brown] 601844 Pseudohypoaldosteronism type II 601846 Muscular dystrophy with rimmed vacuoles 601884 [High bone mass] 601889 Lymphoma, diffuse large cell 601954 Muscular dystrophy, limb-girdle, type 2G 601969 Medulloblastoma, 155255 601969 Glioblastoma multiforme, 137800 601975 Ectodermal dysplasia/skin fragility syndrome 602025 Obesity/hyperinsulinism, susceptibility to 602084 Endometrial carcinoma 602092 Deafness, autosomal recessive 18 602116 Glioma 602117 Prader-Willi syndrome 602134 Tremor, familial essential, 2 602216 Peutz-Jeghers syndrome, 175200 602477 Febrile convulsions, familial, 2 602491 Hyperlipidemia, familial combined, 1 602568 Homocystinuria-megaloblastic anemia, cbl E type, 236270 602629 Dystonia-6, torsion 602685 Mental retardation, severe, with spasticity and tapetoretinal degeneration 602759 Prostate cancer, hereditary, 2, 176807

Mature Polypeptides

The present invention also encompasses mature forms of a polypeptide having the amino acid sequence of SEQ ID NO:Y and/or the amino acid sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional acitivities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:46834690 (1986) uses the information from the residues surrounding the cleavage site, typically residues —13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1A.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the predicted mature form of the polypeptide as delineated in columns 14 and 15 of Table 1A. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional acitivities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotides encoding proteins comprising, or consisting of, the predicted mature form of polypeptides of the invention (e.g., polynucleotides having the sequence of SEQ ID NO: X (Table 1A, column 4), the sequence delineated in columns 7 and 8 of Table 1A, and a sequence encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A (e.g., the sequence of SEQ ID NO:X encoding the mature polypeptide delineated in columns 14 and 15 of Table 1)) are also encompassed by the invention, as are fragments or variants of these polynucleotides (such as, fragments as described herein, polynucleotides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polyncueotides, and nucleic acids which hybridizes under stringent conditions to the complementary strand of the polynucleotide).
As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 15 residues of the predicted cleavage point (i.e., having 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, or 15 more or less contiguous residues of SEQ ID NO:Y at the N-termrinus when compared to the predicted mature form of the polypeptide (e.g., the mature polypeptide delineated in columns 14 and 15 of Table 1). Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
The present invention is also directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID NO:X that encodes the polypeptide sequence as defined in columns 13 and 14 of Table 1A, nucleotide sequences encoding the polypeptide sequence as defined in columns 13 and 14 of Table 1A, the nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B, nucleotide sequences encoding the polypeptide as defined in Table 1B, the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defined in column 6 of Table 1C, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1C, the cDNA sequence contained in ATCC Deposit No:Z, nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in ATCC Deposit No:Z, and/or nucleotide sequences encoding a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCC Deposit No:Z.
The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide as defined in columns 13 and 14 of Table 1A, the polypeptide sequence as defined in Table 1B, a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1C, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, the polypeptide sequence encoded by the cDNA sequence contained in ATCC Deposit No:Z and/or a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCC Deposit No:Z.
“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X or contained in the cDNA sequence of ATCC Deposit No:Z; (b) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC Deposit No:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC Deposit No:Z which encodes a mature polypeptide (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)); (d) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC Deposit No:Z, which encodes a biologically active fragment of a polypeptide; (e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC Deposit No:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID NO:Y (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)) or a mature polypeptide of the amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or () above, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA contained in ATCC Deposit No:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in Table 1B or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (b) the amino acid sequence of a mature (secreted) form of a polypeptide having the amino acid sequence of SEQ ID NO:Y (e.g., as delineated in columns 14 and 15 of Table 1A) or a mature form of the amino acid sequence encoded by the cDNA in ATCC Deposit No:Z mature; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No:Z.
The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in ATCC Deposit No:Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C, the amino acid sequence as defined in Table 1B, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Table 1B or 2 as the ORF (open reading frame), or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence delineated in columns 14 and 15) or a fragment thereof, Table 1B.1 (e.g., the amino acid sequence identified in column 6) or a fragment thereof, Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ D NO:B as defined in column 6 of Table 1C or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in ATCC Deposit No:Z, or a fragment thereof, the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC Deposit No:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N— or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N— and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N— and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N— and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N— and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N— and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N— and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N— or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N— and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli ).
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N— or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants which show a biological or functional activity of the polypeptides of the invention (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus). Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein and/or a mature (secreted) protein of the invention. Such functional activities include, but are not limited to, biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
The functional activity of the polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrate(s) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in ATCC Deposit No:Z, the nucleic acid sequence referred to in Table 1B (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 1A (e.g., the nucleic acid sequence delineated in columns 7 and 8), the nucleic acid sequence disclosed in Table 2 (e.g., the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides “having functional activity.” In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which, for example, comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, the amino acid sequence of the mature (e.g., secreted) polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X, an amino acid sequence encoded by cDNA contained in ATCC Deposit No:Z, and/or the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC Deposit No:Z, or a fragment thereof, which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in ATCC Deposit No:Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotide and Polypeptide Fragments
The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a “polynucleotide fragment” refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in ATCC Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the mature (secreted) polypeptide encoded by the cDNA contained in ATCC Deposit No:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the mature amino acid sequence as defined in columns 14 and 15 of Table 1A or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto.
The polynucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in ATCC Deposit No:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351400, 401450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351400, 401450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in ATCC Deposit No:Z, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1C column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1C. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C which correspond to the same ATCC Deposit No:Z (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of the amino acid sequence contained in SEQ ID NO:Y, is a portion of the mature form of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, is a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:X, is a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, is a portion of the amino acid sequence of a mature (secreted) polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or is a portion of an amino acid sequence encoded by the cDNA contained in ATCC Deposit No:Z. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 2140, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ D NO:Y, a polypeptide as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ D NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC Deposit No:Z). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ED NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC Deposit No:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue in a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above described N— or C-terminal deletions can be combined to produce a N— and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in ATCC Deposit No:Z, and/or the complement thereof, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N— and C-terminal deletions. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in ATCC Deposit No:Z, or the polynucleotide sequence as defined in column 6 of Table 1C, may be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the cDNA contained in ATCC Deposit No:Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, Wis. 53715 USA; http:Hlwww.dnastar.com/).
Polypeptide regions that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above.
Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g. biological activity such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diabetes mellitus; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a “functional activity” is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereto; the polypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in ATCC Deposit No:Z under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Imununospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Non-limiting examples of epitopes of polypeptides that can be used to generate antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y specified in Table 1B. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in the DNAStar suite of computer programs. By “comprise” it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y shown in Table 1B, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein. In particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in Table 1B.
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N— or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N and/or C-terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention. Polynucleotides encoding these proteins are also encompassed by the invention.
Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polypeptides of the Invention
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 . Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHEL-D2, pH[L-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., New York, and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBf 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidinibiotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine ( 121 I, 123 I, 125 I, 131 I, carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113m In, 115m In), technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 75Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, and 97 Ru.
In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177 Lu, 90 Y, 166 Ho, and 153 Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 111 In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 90 Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycolipropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 46, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in ATCC Deposit No:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a, specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in ATCC Deposit No:Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCr application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in Table 1B, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10 −2 M, 10 −2 M,5×10 −3 M, 10 −3 M, 5×10 −4 M, 10 −4 M, 5×10 −5 M, 10 −5 M, 5×10 −6 M, 10 −6 M, 5×10 −7 M, 10 7 M, 5×10 −8 M, 10 −8 M, 5×10 −9 M, 10 −9 M, 5×10 −10 M, 10 −10 M, 5×10 −11 M, 10 −1 M, 5×10 −12 M, 10 −12 M, 5×10 −13 M, 10 −13 M, 5×10 −14 M, 10 −14 M, 5×10 −15 M, or 10 −15 M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 11l(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by reference herein in its entirety.
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N— or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, New York, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCr Publication WO 86/05807; PCI Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, New York (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entirities by reference herein.
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCI publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidintbiotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamnide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:73749 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the arL For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
Therapeutic Uses
Table 1D also provides information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1D also provides information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA ATCC Deposit No:Z”) provides the unique clone identifier for each clone as previously described and indicated in Table 1A, Table 1B, and Table 1C. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Table 1A, Table 1B, and Table 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity.
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, diabetes mellitus. The treatment and/or prevention of diabetes mellitus associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with diabetes mellitus. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating diabetes mellitus. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in Table 1B; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, diagnose, prevent, treat, prognosticate, and/or ameliorate diabetes mellitus or conditions associated with aberrant expression and/or activity of a polypeptide of the invention. The treatment and/or prevention of diabetes mellitus or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diabetes mellitus related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10 −2 M, 10 −2 M, 5×10 −3 M, 10 −3 M, 5×10 −4 M, 10 −4 M, 5×10 −5 M, 10 −5 M, 5×10 −6 M, 10 −6 M, 5×10 −7 M, 10 −7 M, 5×10 −8 M, 10 −8 M, 5×10 −9 M, 10 −9 M, 5×10 −10 M, 10 −10 M, 5×10 −11 M, 10 −11 M, 5×10 −12 M, 10 −12 M, 5×10 −13 M, 10 −13 M, 5×10 −14 M, 10 −14 M, 5×10 −15 M, and 10 −15 M.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a diabetes mellitus associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990).
In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijistra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92106180; WO 92122635; WO92120316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94112649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, prognosticate, or monitor diabetes mellitus and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of 4 polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing diabetes mellitus, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of diabetes mellitus. With respect to insulin resistance, the presence of a relatively high or low amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of insulin resistance into diabetes mellitus.
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1B.1, column 8 provides the chromosome location of some of the polynucleotides of the invention.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 bp (e.g., 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO:X will yield an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456459 (1998) which is hereby incorporated by reference in its entirety).
Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1B and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
The polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping. For a review of these techniques and others known in the art, see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.
Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Column 9 of Table 1B.1 provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in column 8 of Table 1B.1, as determined using techniques described herein and by reference to Table 5. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled “Antibodies”, “Diagnostic Assays”, and “Methods for Detecting Diseases”).
Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).
In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.
Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
By “measuring the expression level of polynucleotides of the invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
The method(s) provided above may preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.
The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
The compounds of the present invention have uses which include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)
For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580). However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.
In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular, for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled “Antisense and Ribozyme (Antagonists)”).
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy Methods”, and Examples 16, 17 and 18).
The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1B. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
The polynucleotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues (e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in Table 1B, and/or cancerous and/or wounded tissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
Thus, the invention provides a diagnostic method of a disorder, which involves: (a) assaying gene expression level in cells or body fluid of an individual; (b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
Polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry assays).
Antibodies can be used to assay levels of polypeptides encoded by polynucleotides of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 131 I, 125 i, 123 i, 121 j, carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 33 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 pm, 140 La, 175 yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131 I, 112 In, 99m Tc, ( 131 I, 125 I, 123 I, 121 I, carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F, 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer , S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.
By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213 Bi, or other radioisotopes such as, for example, 103 Pd, 133 Xe, 133 I, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, 90 Yttrium, 117 Tin, 186 Rhenium, 166 Holmium, and 188 rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90 Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 111 In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131 I.
Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
Diagnostic Assays
The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those related to biological activities described in Table 1D and, also as described herein under the section heading “Biological Activities”.
For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2. column 5 (Tissue Distribution Library Code).
By “assaying the expression level of the gene encoding the polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.
Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include inmmunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I, carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in Table 1B) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
By “solid phase support or carrier” is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.
Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al, Nature 314:268 (1985).
Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131 I, 112 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer , S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
With respect to antibodies, one of the ways in which an antibody of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Methods for Detecting Diseases
In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of a binding agent(s) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.
The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
Gene Therapy Methods
Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85:207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotide of the present invention.
Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15 EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca 2+ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Nat. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E−86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
The polynucleotide encoding a polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a proteinoated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to treat the associated disease.
Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.
In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, treatment, and/or amelioration of diseases and/or disorders relating to diabetes mellitus and/or a condition associated with diabetes mellitus (e.g., hyperglycemia, obesity, diabetic refinopathy, mononeuropathy, polyneuropathy, atherosclerosis, ulcers, heart disease, anemia, stroke, gangrene (e.g., of the feet and hands), impotence, infection, cataract, poor kidney function, malfunctioning of the autonomic nervous system, impaired white blood cell function, Carpal tunnel syndrome, Dupuytren's contracture, diabetic ketoacidosis, and as described in “Renal Disorders”, “Wound Healing and Epithelial Cell Proliferation”, “Endocrine Disorders”, “Reproductive System Disorders”, and “Gastrointestinal Disorders” sections below).
In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection, prevention, prognistication, and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.
More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, treatment and/or amelioration of diseases and/or disorders associated with the following system or systems.
Renal Disorders
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders of the renal system. Renal disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
Kidney diseases which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)
In addition, compositions of the invention can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).
Compositions of the invention can also be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypematremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermiic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.
It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and H diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Endocrine Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).
Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
Reproductive System Disorders
The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoosperrnia, oligospermia, and teratozoospermia).
Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
Additionally, the compositions of the invention may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chiamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.
Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
Further, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of congenital uterine abnormalities, such as bicomuate uterus, septate uterus, simple unicornuate uterus, unicomuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicomuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.
Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and obstruction of the intestine.
Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
Other disorders and/or diseases of the female reproductive system that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Gastrointestinal Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate gastrointestinal diseases and disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).
Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms ( Ascariasis lumbricoides ), Hookworms ( Ancylostoma duodenale ), Threadworns ( Enterobius vermicularis ), Tapeworms ( Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T. solium ).
Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (ejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
Chemotaxis
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could be used as an inhibitor of chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli . Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
Additionally, the receptor to which the polypeptide of the present invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the polypeptide of the present invention thereby effectively generating agonists and antagonists of the polypeptide of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptide of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP4, BMP-5, BMP6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the polypeptide of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3 [H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3 [H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3 [H{] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues.
Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.
Targeted Delivery
In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenylacetamide derivatives of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
Antisense And Ribozyme (Antagonists)
In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to cDNA sequences contained in cDNA ATCC Deposit No:Z identified for example, in Table 1A and/or 1B. In one embodiment, antisense sequence is generated internally, by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the nonymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).
For example, the 5′ coding portion of a polynucleotide that encodes the polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding the polypeptide of the present invention or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of the present invention. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary to either the 5′ - or 3′-non-translated, non-coding regions of polynucleotide sequences described herein could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′-or coding region of mRNA of the present invention, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
While antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
The antagonist/agonist may also be employed to treat the diseases described herein.
Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:
contacting polypeptides of the invention with a plurality of molecules; and identifying a molecule that binds the polypeptides of the invention.

The step of contacting the polypeptides of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptides on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptides of the invention. The molecules having a selective affinity for the polypeptides can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptides to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the polypeptides of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptides and the individual clone. Prior to contacting the polypeptides with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for polypeptides of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptides of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of the polypeptides of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the polypeptides of the invention or the plurality of polypeptides are bound to a solid support.
The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind polypeptides of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been pernethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.
In a specific embodiment, screening to identify a molecule that binds polypeptides of the invention can be carried out by contacting the library members with polypeptides of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptides of the invention. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to polypeptides of the invention.
Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.
Other Activities
A polypeptide, polynucleotide, agonist, or antagonist of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No:Z.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in column 5, “ORF (From-To)”, in Table 1B.1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in columns 8 and 9, “NT From” and “NT To” respectively, in Table 2.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No:Z.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No:Z.
A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in column 5, “ORF (From-To)”, in Table 1B.1.
A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in columns 8 and 9, “NT From” and “NT To”, respectively, in Table 2.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table lB.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No:Z, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which comprises the cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides of the cDNA sequence contained in ATCC Deposit No:Z.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of an open reading frame sequence encoded by cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC Deposit No:Z.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC Deposit No:Z.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by cDNA contained in ATCC Deposit No:Z.
A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC Deposit No:Z; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of the cDNA contained in ATCC Deposit No:Z.
The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; or the cDNA contained in ATCC Deposit No:Z which encodes a protein, wherein the method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of cDNA contained in ATCC Deposit No:Z.
The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC Deposit No:Z. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a DNA microarray or “chip” of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 500, 1000, 2000, 3000, or 4000 nucleotide sequences, wherein at least one sequence in said DNA nicroarray or “chip” is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1A and/or 1B; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA “Clone ID” in Table 1A and/or 1B.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a polypeptide encoded by contained in ATCC Deposit No:Z
Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a portion of said polypeptide encoded by cDNA contained in ATCC Deposit No:Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of a polypeptide encoded by cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleic acid sequence identified in Table 1A, 1B or Table 2 encoding a polypeptide, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
Also preferred is a polypeptide molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.
Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a human protein comprising an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z. The isolated polypeptide produced by this method is also preferred.
Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to increase the level of said protein activity in said individual.
Also preferred is a method of treatment of an individual in need of a decreased level of a protein activity, which method comprised administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to decrease the level of said protein activity in said individual.
Also preferred is a method of treatment of an individual in need of a specific delivery of toxic compositions to diseased cells (e.g., tumors, leukemias or lymphomas), which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide of the invention, including, but not limited to a binding agent, or antibody of the claimed invention that are associated with toxin or cytotoxic prodrugs.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Description of Table 6
Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application. These deposits were made in addition to those described in the Table 1A.
TABLE 6 ATCC Deposits Deposit Date ATCC Designation Number LP01, LP02, LP03, LP04, May 20, 1997 209059, 209060, 209061, LP05, LP06, LP07, LP08, 209062, 209063, 209064, LP09, LP10, LP11, 209065, 209066, 209067, 209068, 209069 LP12 Jan. 12, 1998 209579 LP13 Jan. 12, 1998 209578 LP14 Jul. 16, 1998 203067 LP15 Jul. 16, 1998 203068 LP16 Feb. 1, 1999 203609 LP17 Feb. 1, 1999 203610 LP20 Nov. 17, 1998 203485 LP21 Jun. 18, 1999 PTA-252 LP22 Jun. 18, 1999 PTA-253 LP23 Dec. 22, 1999 PTA-1081

EXAMPLES
Example 1
Isolation of a Selected cDNA Clone From the Deposited Sample
Each ATCC Deposit No:Z is contained in a plasmid vector. Table 7 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The following correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 7 as being isolated in the vector “Lambda Zap,” the corresponding deposited clone is in “pBluescript.”
Vector Used to Corresponding Construct Library Deposited Plasmid Lambda Zap pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:5861 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for Sacl and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 ori generates sense strand DNA and in the other, antisense.
Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993)). Vector lafmid BA (Bento Soares, Columbia University, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: 1991)). Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 7, as well as the corresponding plasmid vector sequences designated above.
The deposited material in the sample assigned the ATCC Deposit Number cited by reference to Table 1A, Table 2, Table 6 and Table 7 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each ATCC Deposit No:Z.
TABLE 7 ATCC Libraries owned by Catalog Catalog Description Vector Deposit HUKA HUKB HUKC HUKD Human Uterine Cancer Lambda ZAP II LP01 HUKE HUKF HUKG HCNA HCNB Human Colon Lambda Zap II LP01 HFFA Human Fetal Brain, Lambda Zap II LP01 random primed HTWA Resting T-Cell Lambda ZAP II LP01 HBQA Early Stage Human Lambda ZAP II LP01 Brain, random primed HLMB HLMF HLMG HLMH breast lymph node Lambda ZAP II LP01 HLMI HLMJ HLMM HLMN CDNA library HCQA HCQB human colon cancer Lamda ZAP II LP01 HMEA HMEC HMED HMEE Human Microvascular Lambda ZAP II LP01 HMEF HMEG HMEI HMEJ Endothelial Cells, HMEK HMEL fract. A HUSA HUSC Human Umbilical Vein Lambda ZAP II LP01 Endothelial Cells, fract. A HLQA HLQB Hepatocellular Tumor Lambda ZAP II LP01 HHGA HHGB HHGC HHGD Hemangiopericytoma Lambda ZAP II LP01 HSDM Human Striatum Depression, Lambda ZAP II LP01 re-rescue HUSH H Umbilical Vein Lambda ZAP II LP01 Endothelial Cells, frac A, re-excision HSGS Salivary gland, subtracted Lambda ZAP II LP01 HFXA HFXB HFXC HFXD Brain frontal cortex Lambda ZAP II LP01 HFXE HFXF HFXG HFXH HPQA HPQB HPQC PERM TF274 Lambda ZAP II LP01 HFXJ HFXK Brain Frontal Cortex, Lambda ZAP II LP01 re-excision HCWA HCWB HCWC CD34 positive cells ZAP Express LP02 HCWD HCWE HCWF (Cord Blood) HCWG HCWH HCWI HCWJ HCWK HCUA HCUB HCUC CD34 depleted Buffy ZAP Express LP02 Coat (Cord Blood) HRSM A-14 cell line ZAP Express LP02 HRSA A1-CELL LINE ZAP Express LP02 HCUD HCUE HCUF HCUG CD34 depleted Buffy ZAP Express LP02 HCUH HCUI Coat (Cord Blood), re-excision HBXE HBXF HBXG H. Whole Brain #2, ZAP Express LP02 re-excision HRLM L8 cell line ZAP Express LP02 HBXA HBXB HBXC HBXD Human Whole Brain #2 - ZAP Express LP02 Oligo dT > 1.5 Kb HUDA HUDB HUDC Testes ZAP Express LP02 HHTM HHTN HHTO H. hypothalamus, frac A; ZAP Express LP02 re-excision HHTL H. hypothalamus, frac A ZAP Express LP02 HASA HASD Human Adult Spleen Uni-ZAP XR LP03 HFKC HFKD HEKE HFKF Human Fetal Kidney Uni-ZAP XR LP03 HFKG HE8A HE8B HE8C HE8D Human 8 Week Whole Uni-ZAP XR LP03 HE8E HE8F HE8M HE8N Embryo HGBA HGBD HGBE HGBF Human Gall Bladder Uni-ZAP XR LP03 HGBG HGBH HGBI HLHA HLHB HLHC HLHD Human Fetal Lung III Uni-ZAP XR LP03 HLHE HLHF HLHG HLHH HLHQ HPMA HPMB HPMC HPMD Human Placenta Uni-ZAP XR LP03 HPME HPMF HPMG HPMH HPRA HPRB HPRC HPRD Human Prostate Uni-ZAP XR LP03 HSIA HSIC HSID HSIE Human Adult Small Uni-ZAP XR LP03 Intestine HTEA HTEB HTEC HTED Human Testes Uni-ZAP XR LP03 HTEE HTEF HTEG HTEH UTEI HTEJ HTEK HTPA HTPB HTPC HTPD Human Pancreas Tumor Uni-ZAP XR LP03 HTPE HTTA HTTB HTTC HTTD Human Testes Tumor Uni-ZAP XR LP03 HTTE HTTF HAPA HAPB HAPC HAPM Human Adult Pulmonary Uni-ZAP XR LP03 HETA HETB HETC HETD Human Endometrial Tumor Uni-ZAP XR LP03 HETE HETF HETG HETH HETI HHFB HHFC HHFD HHFE Human Fetal Heart Uni-ZAP XR LP03 HHFF HHFG HHFH HHFI HHPB HHPC HHPD HHPE Human Hippocampus Uni-ZAP XR LP03 HHPF HHPG HHPH HCE1 HCE2 HCE3 HCE4 Human Cerebellum Uni-ZAP XR LP03 HCE5 HCEB HCEC HCED HCEE HCEF HCEG HUVB HUVC HUVD HUVE Human Umbilical Vein, Uni-ZAP XR LP03 Endo. remake HSTA HSTB HSTC HSTD Human Skin Tumor Uni-ZAP XR LP03 HTAA HTAB HTAC HTAD Human Activated T-Cells Uni-ZAP XR LP03 HTAE HFEA HEEB HFEC Human Fetal Epithelium Uni-ZAP XR LP03 (Skin) HJPA HJPB HJPC HJPD HUMAN JURKAT Uni-ZAP XR LP03 MEMBRANE BOUND POLYSOMES HESA Human epithelioid sarcoma Uni-Zap XR LP03 HLTA HLTB HLTC HLTD Human T-Cell Lymphoma Uni-ZAP XR LP03 HLTE HLTF HFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XR LP03 HRDA HRDB HRDC HRDD Human Rhabdomyosarcoma Uni-ZAP XR LP03 HRDE HRDF HCAA HCAB HCAC Cem cells cyclohexamide Uni-ZAP XR LP03 treated HRGA HRGB HRGC HRGD Raji Cells, cyclohexamide Uni-ZAP XR LP03 treated HSUA HSUB HSUC HSUM Supt Cells, cyclohexamide Uni-ZAP XR LP03 treated HT4A HT4C HT4D Activated T-Cells, 12 hrs. Uni-ZAP XR LP03 HE9A HE9B HE9C HE9D Nine Week Old Early Stage Uni-ZAP XR LP03 HE9E HE9F HE9G HE9H Human HE9M HE9N HATA HATB HATC HATD Human Adrenal Gland Tumor Uni-ZAP XR LP03 HATE HT5A Activated T-Cells, 24 hrs. Uni-ZAP XR LP03 HFGA HFGM Human Fetal Brain Uni-ZAP XR LP03 HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XR LP03 HNEE HBGB HBGD Human Primary Breast Uni-ZAP XR LP03 Cancer HBNA HBNB Human Normal Breast Uni-ZAP XR LP03 HCAS Cem Cells, cyclohexamide Uni-ZAP XR LP03 treated, subtra HHPS Human Hippocampus, pBS LP03 subtracted HKCS HKCU Human Colon Cancer, pBS LP03 subtracted HRGS Raji cells, cyclohexamide pBS LP03 treated, subtracted HSUT Supt cells, cyclohexamide pBS LP03 treated, differentially expressed HT4S Activated T-Cells, 12 hrs, Uni-ZAP XR LP03 subtracted HCDA HCDB HCDC HCDD Human Chondrosarcoma Uni-ZAP XR LP03 HCDE HOAA HOAB HOAC Human Osteosarcoma Uni-ZAP XR LP03 HTLA HTLB HTLC HTLD Human adult testis, Uni-ZAP XR LP03 HTLE HTLF large inserts HLMA HLMC HLMD Breast Lymph node cDNA Uni-ZAP XR LP03 library H6EA H6EB H6EC HL-60, PMA 4H Uni-ZAP XR LP03 HTXA HTXB HTXC HTXD Activated T-Cell Uni-ZAP XR LP03 HTXE HTXF HTXG HTXH (12hs)/Thiouridine labelledEco HNFA HNFB HNFC HNFD Human Neutrophil, Uni-ZAP XR LP03 HNFE HNFF HNFG HNFH Activated HNFJ HTOB HTOC HUMAN TONSILS, Uni-ZAP XR LP03 FRACTION 2 HMGB Human OB MG63 control Uni-ZAP XR LP03 fraction I HOPB Human OB HOS control Uni-ZAP XR LP03 fraction HORB Human OB HOS treated Uni-ZAP XR LP03 (10 nM E2) fraction I HSVA HSVB HSVC Human Chronic Synovitis Uni-ZAP XR LP03 HROA HUMAN STOMACH Uni-ZAP XR LP03 HBJA HBJB HBJC HBJD HUMAN B CELL LYMPHOMA Uni-ZAP XR LP03 HBJE HBJF HBJG HBJH HBJI HBJJ HBJK HCRA HCRB HCRC human corpus colosum Uni-ZAP XR LP03 HODA HODB HODC HODD human ovarian cancer Uni-ZAP XR LP03 HDSA Dermatofibrosarcoma Uni-ZAP XR LP03 Protuberance HMWA HMWB HMWC Bone Marrow Cell Line Uni-ZAP XR LP03 HMWD HMWE HMWF (Rs4;11) HMWG HMWH HMWI HMWJ HSOA stomach cancer (human) Uni-ZAP XR LP03 HERA SKIN Uni-ZAP XR LP03 HMDA Brain-medulloblastoma Uni-ZAP XR LP03 UGLA HGLB HGLD Glioblastoma Uni-ZAP XR LP03 HEAA H. Atrophic Endometrium Uni-ZAP XR LP03 HBCA HBCB H. Lymph node breast Cancer Uni-ZAP XR LP03 HPWT Human Prostate BPH, Uni-ZAP XR LP03 re-excision HFVG HFVH HFVI Fetal Liver, subtraction II pBS LP03 HNFI Human Neutrophils, pBS LP03 Activated, re-excision HBMB HBMC HBMD Human Bone Marrow, re- pBS LP03 excision HKML HKMM HKMN H. Kidney Medulla, re- pBS LP03 excision HKIX HKIY H. Kidney Cortex, subtracted pBS LP03 HADT H. Amygdala Depression, pBS LP03 subtracted H6AS H1-60, untreated, Uni-ZAP XR LP03 subtracted H6ES HL-60, PMA 4H, Uni-ZAP XR LP03 subtracted H6BS HL-60, RA 4h, Subtracted Uni-ZAP XR LP03 H6CS HL-60, PMA 1d, subtracted Uni-ZAP XR LP03 HTXJ HTXK Activated T- Uni-ZAP XR LP03 cell(12h)/Thiouridine- re-excision HMSA HMSB HMSC HMSD Monocyte activated Uni-ZAP XR LP03 HMSE HMSF HMSG HMSH HMSI HMSJ HMSK HAGA HAGB HAGC HAGD Human Amygdala UnI-ZAP XR LP03 HAGE HAGF HSRA HSRB HSRE STROMAL- Uni-ZAP XR LP03 OSTEOCLASTOMA HSRD HSRF HSRG HSRH Human Osteoclastoma Uni-ZAP XR LP03 Stromal Cells-unamplified HSQA HSQB HSQC HSQD StroMal cell TF274 Uni-ZAP XR LP03 HSQE HSQF HSQG HSKA HSKB HSKC HSKD Smooth muscle, serum Uni-ZAP XR LP03 HSKE HSKF HSKZ treated HSLA HSLB HSLC HSLD Smooth muscle,control Uni-ZAP XR LP03 HSLE HSLF HSLG HSDA HSDD HSDE HSDF Spinal cord Uni-ZAP XR LP03 HSDG HSDH HPWS Prostate-BPH subtracted II pBS LP03 HSKW HSKX HSKY Smooth Muscle- HASTE pBS LP03 normalized HFPB HFPC HFPD H. Frontal cortex,epileptic; Uni-ZALP XR LP03 re-excision HSDI HSDJ HSDK Spinal Cord, re-excision Uni-ZAP XR LP03 HSKN HSKO Smooth Muscle Serum pBS LP03 Treated, Norm HSKG HSKH HSKI Smooth muscle, serum pBS LP03 induced,re-exc HFCA HFCB HFCC HFCD Human Fetal Brain Uni-ZAP XR LP04 HFCE HFCF HPTA HPTB HPTD Human Pituitary Uni-ZAP XR LP04 HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP04 HB6B HE6C HE6D HE6E Human Whole Six Week Old Uni-ZAP XR LP04 HE6F HE6G HE6S Embryo HSSA HSSB HSSC HSSD Human Synovial Sarcoma Uni-ZAP XR LP04 HSSE HSSF HSSG HSSH HSSI HSSJ HSSK HE7T 7 Week Old Early Stage Uni-ZAP XR LP04 Human, subtracted HEPA HEPB HEPC Human Epididymus Uni-ZAF XR LP04 HSNA HSNB HSNC HSNM Human Synovium Uni-ZAP XR LP04 HSNN HPFB HPFC HPFD HPFE Human Prostate Cancer, Uni-ZAP XR LP04 Stage C fraction HE2A HE2D HE2E HE2H 12 Week Old Early Stage Uni-ZAP XR LP04 HE2I HE2M HE2N HE2O Human HE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP04 HE2P HE2Q Human, II HPTS HPTT HPTU Human Pituitary, subtracted Uni-ZAP XR LP04 HAUA HAUB HAUC Amniotic Cells-TNF induced Uni-ZAP XR LP04 HAQA HAQB HAQC HAQD Amniotic Cells-Primary Culture Uni-ZAP XR LP04 HWTA HWTB HWTC wilm's tumor Uni-ZAP XR LP04 HBSD Bone Cancer, re-excision Uni-ZAP XR LP04 HSGB Salivary gland, re-excision Uni-ZAP XR LP04 HSJA HSJB HSJC Smooth muscle-ILb induced Uni-ZAP XR LP04 HSXA HSXB HSXC HSXD Human Substantia Nigra Uni-ZAP XR LP04 HSHA HSHB HSHC Smooth muscle, ILb induced Uni-ZAP XR LP04 HOUA HOUB HOUC HOUD Adipocytes Uni-ZAP XR LP04 HOUE HPWA HPWB HPWC HPWD Prostate BPH Uni-ZAP XR LP04 HPWE HELA HELB HELC HELD Endothelial cells-control Uni-ZAP XR LP04 HELE HELF HELG HELH HEMA HEMB HEMC HEMD Endothelial-induced Uni-ZAP XR LP04 HEME HEMF HEMG HEMH HBIA HBIB HBIC Human Brain, Striatum Uni-ZAP XR LP04 HHSA HHSB HHSC HHSD Human Uni-ZAP XR LP04 HHSE Hypothalmus, Schizophrenia HNGA HNGB HNGC HNGD neutrophils control Uni-ZAP XR LP04 HNGE HNGF HNGG HNGH HNGI HNGJ HNHA HNHB HNHC HNHD Neutrophils IL-1 and LPS Uni-ZAP XR LP04 HNHE HNHF HNHG HNHH induced HNHI HNHJ HSDB HSDC STRIATUM DEPRESSION Uni-ZAP XR LP04 HHPT Hypothalamus Uni-ZAP XR LP04 HSAT HSAU HSAV HSAW Anergic T-cell Uni-ZAP XR LP04 HSAX HSAY HSAZ HEMS HBMT HBMU HBMV Bone marrow Uni-ZAP XR LP04 HBMW HBMX HOEA HOEB HOEC HOED Osteoblasts Uni-ZAP XR LP04 HOEE HOEF HOEJ HAIA HAIB HAIC HAID Epithelial-TNFa and INF Uni-ZAP XR LP04 HAIE HAIF induced HTGA HTGB HTGC HTGD Apoptotic T-cell Uni-ZAP XR LP04 HMCA HMCB HMCC Macrophage-oxLDL Uni-ZAP XR LP04 HMCD HMCE HMAA HMAB HMAC Macrophage (GM-CSF Uni-ZAP XR LP04 HMAD HMAE HMAF treated) HMAG HPHA Normal Prostate Uni-ZAP XR LP04 HPIA HPIB HPIC LNCAP prostate cell line Uni-ZAP XR LP04 HPJA HPJB HPJC PC3 Prostate cell line Uni-ZAP XR LP04 HOSE HOSF HOSG Human Osteoclastoma, re- Uni-ZAP XR LP04 excision HTGE HTGF Apoptotic T-cell, Uni-ZAP XR LP04 re-excision HMAJ HMAK H Macrophage (GM-CSF Uni-ZAP XR LP04 treated), re-excision HACB HACC HACD Human Adipose Tissue, Uni-ZAP XR LP04 re-excision HEPA H. Frontal Cortex, Uni-ZAP XR LP04 Epileptic HFAA HFAB HFAC HFAD Alzheimer's, spongy Uni-ZAP XR LP04 HFAE change HFAM Frontal Lobe, Dementia Uni-ZAP XR LP04 HMIA HMIB HMIC Human Manic Depression Uni-ZAP XR LP04 Tissue HTSA HTSE HTSF HTSG Human Thymus pBS LP05 HTSH HPBA HPBB HPBC HPBD Human Pineal Gland pBS LP05 HPBE HSAA HSAB HSAC HSA 172 Cells pBS LP05 HSBA HSBB HSBC HSBM HSC172 cells pBS LP05 HJAA HJAB HJAC HJAD Jurkat T-cell G1 phase pBS LP05 HJBA HJBB HJBC HJBD Jurkat T-Cell, S phase pBS LP05 HAFA HAFB Aorta endothelial pBS LP05 cells +TNF-a HAWA HAWB HAWC Human White Adipose pBS LP05 HTNA HTNB Human Thyroid pBS LP05 HONA Normal Ovary, Premenopausal pBS LP05 HARA HARB Human Adult Retina pBS LP05 HLJA HLJB Human Lung pCMVSport 1 LP06 HOFM HOFN HOFO H. Ovarian Tumor, II, pCMVSport 2.0 LP07 OV5232 HOGA HOGB HOGC OV 10-3-95 pCMVSport 2.0 LP07 HCGL CD34+cells, II pCMVSport 2.0 LP07 HDLA Hodgkin's Lymphoma I pCMVSport 2.0 LP07 HDTA HDTB HDTC HDTD Hodgkin's Lymphoma II pCMVSport 2.0 LP07 HDTE HKAA HKAB HXAC HXAD Keratinocyte pCMVSport 2.0 LP07 HKAE HKAF HKAG HKAH HCIM CAPFINDER, Crohn's pCMVSport 2.0 LP07 Disease, lib 2 HKAL Keratinocyte, lib 2 pCMVSport 2.0 LP07 HKAT Keratinocyte, lib 3 pCMVSport 2.0 LP07 HNDA Nasal polyps pCMVSport 2.0 LP07 HDRA H. Primary Dendritic pCMVSport 2.0 LP07 Cells, lib 3 HOHA HOHB HOHC Human Osteoblasts II pCMVSport 2.0 LP07 HLDA HLDB HLDC Liver, Hepatoma pCMVSport 3.0 LP08 HLDN HLDO HLDP Human Liver, normal pCMVSport 3.0 LP08 HMTA pBMC stimulated w/poly pCMVSport 3.0 LP08 I/C HNTA NTERA2, control pCMVSport 3.0 LP08 HDPA HDPB HDPC HDPD Primary Dendritic Cells, pCMVSport 3.0 LP08 HDPF HDPG HDPH HDPI lib 1 HDPJ HDPK HDPM HDPN HDPO HDPP Primary Dendritic cells, pCMVSport 3.0 LP08 frac 2 HMUA HMUB HMUC Myoloid Progenitor Cell Line pCMVSport 3.0 LP08 HHEA HHEB HHEC HHED T Cell helper I pCMVSport 3.0 LP08 HHEM HHEN HHEO HHEP T cell helper II pCMVSport 3.0 LP08 HEQA HEQB HEQC Human endometrial stromal pCMVSport 3.0 LP08 cells HJMA HJMB Human endometrial stromal pCMVSport 3.0 LP08 cells-treated with progesterone HSWA HSWB HSWC Human endometrial stromal pCMVSport 3.0 LP08 cells-treated with estradiol HSYA HSYB HSYC Human Thymus Stromal Cells pCMVSport 3.0 LP08 HLWA HLWB HLWC Human Placenta pCMVSport 3.0 LP08 HRAA HRAB HRAC Rejected Kidney, lib 4 pCMVSport 3.0 LP08 HMTM PCR, pBMC I/C treated PCRII LP09 HMJA H. Meniingima, M6 pSport 1 LP10 HMKA HMKB HMKC H. Meningima, M1 pSport 1 LP10 HMKD HMKE HUSG HUSI Human umbilical vein pSport 1 LP10 endothelial cells, IL-4 induced HUSX HUSY Human Umbilical Vein pSport 1 LP10 Endothelial Cells, uninduced HOFA Ovarian Tumor I, OV5232 pSport 1 LP10 HCFA HCFB HCFC HCFD T-Cell PHA 16 hrs pSport 1 LP10 HCFL HCFM HCFN HCFO T-Cell PHA 24 hrs pSport 1 LP10 HADA HADC HADD HADE Human Adipose pSport 1 LP10 HADF HADG HOVA HOVB HOVC Human Ovary pSport 1 LP10 HTWB HTWC HTWD HTWE Resting T-Cell Library, II pSport 1 LP10 HTWF HMMA Spleen metastic melanoma pSport 1 LP10 HLYA HLYB HLYC HLYD Spleen, Chronic lymphocytic pSport 1 LP10 HLYE leukemia HCGA CD34+ cell, I pSport 1 LP10 HEOM HEON Human Eosinophils pSport 1 LP10 HTDA Human Tonsil, Lib 3 pSport 1 LP10 HSPA Salivary Gland, Lib 2 pSport 1 LP10 HCHA HCHB HCHC Breast Cancer cell line, pSport 1 LP10 MDA 36 HCHM HCHN Breast Cancer Cell line, pSport 1 LP10 angiogenic HCIA Crohn's Disease pSport 1 LP10 HDAA HDAB HDAC HEL cell line pSport 1 LP10 HABA Human Astrocyte pSport 1 LP10 HUFA HUFB HUFC Ulcerative Colitis pSport 1 LP10 HNTM NTERA2 + retinoic acid, pSport 1 LP10 14 days HDQA Primary Dendritic pSport 1 LP10 cells, CapFinder2, frac 1 HDQM Primary Dendritic Cells, pSport 1 LP10 CapFinder, frac 2 HLDX Human Liver, normal, pSport 1 LP10 CapFinder HULA HULB HULC Human Dermal Endothelial pSport 1 LP10 Cells, untreated HUMA Human Dermal Endothelial pSport 1 LP10 cells, treated HCJA Human Stromal Endometrial pSport 1 LP10 fibroblasts, untreated HCJM Human Stromal endometrial pSport 1 LP10 fibroblasts, treated w/estradiol HEDA Human Stromal endometrial pSport 1 LP10 fibroblasts, treated with progesterone HFNA Human ovary tumor cell pSport 1 LP10 OV350721 HKGA HKGB HXGC HKGD Merkel Cells pSport 1 LP10 HISA HISB HISC Pancreas Islet Cell Tumor pSport 1 LP10 HLSA Skin, burned pSport 1 LP10 HBZA Prostate, BPH, Lib 2 pSport 1 LP10 HBZS Prostate BPH, Lib 2, subtracted PSport 1 LP10 HFIA HFIB HFIC Synovial Fibroblasts (control) pSport 1 LP10 HFIH HFII HFIJ Synovial hypoxia pSport 1 LP10 HFIT HFIU HFIV Synovial IL-1/TNF stimulated pSport 1 LP10 HGCA Messangial cell, frac 1 pSport 1 LP10 HMVA HMVB HMVC Bone Marrow Stromal Cell, pSport 1 LP10 untreated HFIX HFIY HFIZ Synovial Fibroblasts pSport 1 LP10 (II1/TNF), subt HFOX HFOY HFOZ Synovial hypoxia-RSF pSport 1 LP10 subtracted HMQA HMQB HMQC Human Activated Monocytes Uni-ZAP XR LP11 HMQD HLIA HLIB HLIC Human Liver pCMVSport 1 LP012 HHBA HHBB HHBC HHBD Human Heart pCMVSport 1 LP012 HHBE HBBA HBBB Human Brain pCMVSport 1 LP012 HLJA HLJB HLJG HLJD Human Lung pCMVSport 1 LP012 HLJE HOGA HOGB HOGC Ovarian Tumor pCMVSport 2.0 LP012 HTJM Human Tonsils, Lib 2 pCMVSport 2.0 LP012 HAMF HAMG KMH2 pCMVSport 3.0 LP012 HAJA HAJB HAJC L428 pCMVSport 3.0 LP012 HWBA HWBB HWBC Dendritic cells, pCMVSport 3.0 LP012 HWBD HWBE pooled HWAA HWAB HWAC Human Bone Marrow, pCMVSport 3.0 LP012 HWAD HWAE treated HYAA HYAB HYAC B Cell lymphoma pCMVSport 3.0 LP012 HWHG HWHH HWHI Healing groin wound, pCMVSport 3.0 LP012 6.5 hours post incision HWHP HWHQ HWHR Healing groin wound; pCMVSport 3.0 LP012 7.5 hours post incision HARM Healing groin wound- pCMVSport 3.0 LP012 zero hr post-incision (control) HBIM Olfactory epithelium; pCMVSport 3.0 LP012 nasalcavity HWDA Healing Abdomen wound; pCMVSport 3.0 LP012 70&90 min post incision HWEA Healing Abdomen Wound;15 pCMVSport 3.0 LP012 days post incision HWJA Healing Abdomen Wound; pCMVSport 3.0 LP012 21&29 days HNAL Human Tongue, frac 2 pSport 1 LP012 HMJA H. Meniingima, M6 pSport 1 LP012 HMKA HMKB HMKC H. Meningima, M1 pSport 1 LP012 HMKD HMKE HOFA Ovarian Tumor I, OV5232 pSport 1 LP012 HCFA HCFB HCFC HCFD T-Cell PHA 16 hrs pSport 1 LP012 HCFL HCFM HCFN HCFO T-Cell PHA 24 hrs pSport 1 LP012 HMMA HMMB HMMC Spleen metastic melanoma pSport 1 LP012 HTDA Human Tonsil, Lib 3 pSport 1 LP012 HDBA Human Fetal Thymus pSport 1 LP012 HDUA Pericardium pSport 1 LP012 HBZA Prostate,BPH, Lib 2 pSport 1 LP012 HWCA Larynx tumor pSport 1 LP012 HWKA Normal lung pSport 1 LP012 HSMB Bone marrow stroma, pSport 1 LP012 treated HBHM Normal trachea pSport 1 LP012 HLFC Human Larynx pSport 1 LP012 HLRB Siebben Polyposis pSport 1 LP012 HNIA Mammary Gland pSport 1 LP012 HNJB Palate carcinoma pSport 1 LP012 HNKA Palate normal pSport 1 LP012 HMZA Pharynx carcinoma pSport 1 LP012 HABG Cheek Carcinoma pSport 1 LP012 HMZM Pharynx Carcinoma pSport 1 LP012 HDRM Larynx Carcinoma pSport 1 LP012 HVAA Pancreas normal PCA4 No pSport 1 LP012 HICA Tongue carcinoma pSport 1 LP012 HUKA HUKB HUKC HUKD Human Uterine Cancer Lambda ZAP II LP013 HUKE HFFA Human Fetal Brain, Lambda ZAP II LP013 random primed HTUA Activated T-ceIl labeled with 4- Lambda ZAP II LP013 thioluri HBQA Early Stage Human Brain, Lambda ZAP II LP013 random primed HMEB Human microvascular Lambda ZAP II LP013 Endothelial cells, fract. B HUSH Human Umbilical Vein Lambda ZAP II LP013 Endothelial cells, fract. A, re-excision HLQC HLQD Hepatocellular tumor, Lambda ZAP II LP013 re-excision HTWJ HTWK HTWL Resting T-ceIl, re-excision Lambda ZAP II LP013 HF6S Human Whole 6 week Old pBluescript LP013 Embryo (II), subt HHPS Human Hippocampus, pBluescript LP013 subtracted HL1S LNCAP, differential pBluescnpt LP013 expression HLHS HLHT Early Stage Human Lung, pBluescript LP013 Subtracted HSUS Supt cells, cyclohexamide pBluescript LP013 treated, subtracted HSUT Supt cells, cyclohexamide pBluescript LP013 treated, differentially expressed HSDS H. Striatum Depression, pBluescript LP013 subtracted HPTZ Human Pituitary, Subtracted Bluescript LP013 VII HSDX H. Striatum Depression, pBluescript LP013 subt II HSDZ H. Striatum Depression, pBluescript LP013 subt HPBA HPBB HPBC HPBD Human Pineal Gland pBluescript SK- LP013 HPBE HRTA Colorectal Tumor pBluescript SK- LP013 HSBA HSBB HSBC HSBM H5C172 cells pBluescropt SK- LP013 HJAA HJAB HJAC HJAD Jurkat T-cell G1 phase pBluescript SK- LP013 HJBA HJBB HJBC HJBD Jurkat T-cell, S1 phase pBluescript SK- LP013 HTNA HTNB Human Thyroid pBluescript SK- LP013 HAHA HAHB Human Adult Heart Uni-ZAP XR LP013 HE6A Whole 6 week Old Embryo Uni-ZAP XR LP013 HFCA HFCB HFCC HFCD Human Fetal Brain Uni-ZAP XR LP013 HFCE HFKC HFKD HFKE HFKF Human Fetal Kidney Uni-ZAP XR LP013 HFKG HGBA HGBD HGBE HGBF Human Gall Bladder Uni-ZAP XR LP013 HGBG HPRA HPRB HPRC HPRD Human Prostate Uni-ZAP XR LP013 HTEA HTEB HTEC HTED Human Testes Uni-ZAP XR LP013 HTEE HTTA HTTB HTTC HTTD Human Testes Tumor Uni-ZAP XR LP013 HTTE HYBA HYBB Human Fetal Bone Uni-ZAP XR LP013 HFLA Human Fetal Liver Uni-ZAP XR LP013 HHFB HHFC HHFD HHFE Human Fetal Heart Uni-ZAP XR LP013 HHFF HUVB HUVC HUVD HUVE Human Umbilical Vein, Uni-ZAP XR LP013 End. remake HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP013 HSTA HSTB HSTC HSTD Human Skin Tumor Uni-ZAP XR LP013 HTAA HTAB HTAC HTAD Human Activated T-cells Uni-ZAP XR LP013 HTAE HFEA HFEB HFEC Human Fetal Epithelium Uni-ZAP XR LP013 (skin) HJPA HJPB HJPC HJPD Human Jurkat Membrane Uni-ZAP XR LP013 Bound Polysomes HESA Human Epithelioid Sarcoma Uni-ZAF XR LP013 HALS Human Adult Liver, Uni-ZAP XR LP013 Subtracted HFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XR LP013 HCAA HCAB HCAC Cem cells, cyclohexamide Uni-ZAP XR LP013 treated HRGA HRGB HRGC HRGD Raji Cells, cyclohexamide Uni-ZAP XR LP013 treated HE9A HE9B HE9C HE9D Nine Week Old Early Stage Uni-ZAP XR LP013 HE9E Human HSFA Human Fibrosarcoma Uni-ZAP XR LP013 HATA HATB HATC HATD Human Adrenal Gland Uni-ZAP XR LP013 HATE Tumor HTRA Human Trachea Tumor Uni-ZAP XR LP013 HE2A HE2D HE2E HE2H 12 Week Old Early Uni-ZAP XR LP013 HE2I Stage Human HE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP013 HE2P Human, II HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XR LP013 HNEE HBGA Human Primary Uni-ZAP XR LP013 Breast Cancer HPTS HPTT HPTU Human Pituitary, Uni-ZAP XR LP013 subtracted HMQA HMQB HMQC Human Activated Uni-ZAP XR LP013 HMQD Monocytes HOAA HOAB HOAC Human Osteosarcoma Uni-ZAP XR LP013 HTOA HTOD HTOE HTOF human tonsils Uni-ZAP XR LP013 HTOG HMGB Human OB MG63 control Uni-ZAP XR LP013 fraction I HOPB Human OB HOS control Uni-ZAP XR LP013 fraction HOQB Human OB HOS treated (1 nM Uni-ZAP XR LP013 E2) fraction I HAUA HAUB HAUC Amniotic Cells-TNF Uni-ZAP XR LP013 induced HAQA HAQB HAQC HAQD Amniotic Cells-Primary Uni-ZAP XR LP013 Culture HROA HROC HUMAN STOMACH Uni-ZAP XR LP013 HBJA HBJB HBJC HBJD HUMAN B CELL LYMPHOMA Uni-ZAP XR LP013 HBJE HODA HODB HODC HODD human ovarian cancer Uni-ZAP XR LP013 HCPA Corpus Callosum Urn-ZAP XR LP013 HSOA stomach cancer (human) Uni-ZAP XR LP013 HERA SKIN Uni-ZAP XR LP013 HMDA Brain-medulloblastoma Uni-ZAP XR LP013 HGLA HGLB HGLD Glioblastoma Uni-ZAP XR LP013 HWTA HWTB HWTC wilm's tumor Urn-ZAP XR LP013 HEAA H. Atrophic Endometrium Uni-ZAP XR LP013 HAPN HAPO HAPP HAPQ Human Adult Pulmonary; Uni-ZAP XR LP013 HAPR re-excision HLTG HLTH Human T-cell lymphoma; Uni-ZAP XR LP013 re-excision HAHC HAHD HAHE Human Adult Heart; Uni-ZAP XR LP013 re-excision HAGA HAGB HAGC HAGD Human Amygdala Uni-ZAP XR LP013 HAGE HSJA HSJB HSJC Smooth muscle-ILb Uni-ZAP XR LP013 induced HSHA HSHB HSHC Smooth muscle, IL1b Uni-ZAP XR LP013 induced HPWA HPWB HPWC HPWD Prostate BPH Uni-ZAP XR LP013 HPWE HPIA HPIB HPIC LNCAP prostate cell Uni-ZAP XR LP013 line HPJA HPJB HPJC PC3 Prostate cell line Uni-ZAP XR LP013 HBTA Bone Marrow Stroma, Uni-ZAP XR LP013 TNF&LPS ind HMCF HMCG HMCH HMCI Macrophage-oxLDL; Uni-ZAP XR LP013 HMCJ re-excision HAGG HAGH HAGI Human Amygdala;re-excision Uni-ZAP XR LP013 HACA H. Adipose Tissue Uni-ZAP XR LP013 HKFB K562 + PMA (36 hrs), ZAP Express LP013 re-excision HCWT HCWU HCWV CD34 positive cells (cord ZAP Express LP013 blood),re-ex HBWA Whole brain ZAP Express LP013 HBXA HBXB HBXC HBXD Human Whole Brain #2- ZAP Express LP013 Oligo dT > 1.5 Kb HAVM Temporal cortex-Alzheizmer pT-Adv LP014 HAVT Hippocampus, Alzheimer pT-Adv LP014 Subtracted HHAS CHME Cell Line Uni-ZAP XR LP014 HAJR Larynx normal pSport 1 LP014 HWLE HWLF HWLG HWLH Colon Normal pSport 1 LP014 HCRM HCRN HCRO Colon Carcinoma pSport 1 LP014 HWLI HWLJ HWLK Colon Normal pSport 1 LP014 HWLQ HWLR HWLS HWLT Colon Tumor pSport 1 LP014 HBFM Gastrocnemius Muscle pSport 1 LP014 HBOD HBOE Quadriceps Muscle pSport 1 LP014 HBKD HBKE Soleus Muscle pSport 1 LP014 HCCM Pancreatic Langerhans pSport 1 LP014 HWGA Larynx carcinoma pSport 1 LP014 HWGM HWGN Larynx carcinoma pSport 1 LP014 HWLA HWLB HWLC Normal colon pSport 1 LP014 HWLM HWLN Colon Tumor pSport 1 LP014 HVAM HVAN HVAO Pancreas Tumor pSport 1 LP014 HWGQ Larynx carcinoma nSport 1 LP014 HAQM HAQN Salivary Gland pSport 1 LP014 HASM Stomach; normal pSport 1 LP014 HBCM Uterus; normal pSport 1 LP014 HCDM Testis; normal pSport 1 LP014 HDJM Brain; normal pSport 1 LP014 HEFM Adrenal Gland, normal pSport 1 LP014 HBAA Rectum normal pSport 1 LP014 HFDM Rectum tumour pSport 1 LP014 HGAM Colon, normal pSport 1 LP014 HHMM Colon, tumour pSport 1 LP014 HCLB HCLC Human Lung Cancer Lambda Zap II LP015 HRLA L1 Cell line ZAP Express LP015 HHAM Hypothalamus, Alzheimer's pCMVSport 3.0 LP015 HKBA Ku 812F Basophils Line pSport 1 LP015 HS2S Saos2, Dexamethosome Treated pSport 1 LP016 HA5A Lung Carcinoma A549 TNFalpha pSport 1 LP016 activated HTFM TF-1 Cell Line GM-CSF Treated pSport 1 LP016 HYAS Thyroid Tumour pSport 1 LP016 HUTS Larynx Normal pSport 1 LP016 HXOA Larynx Tumor pSport 1 LP016 HEAH Ea.hy.926 cell line pSport 1 LP016 HINA Adenocarcinoma Human pSport 1 LP016 HRMA Lung Mesothelium pSport 1 LP016 HLCL Human Pre-Differentiated Uni-Zap XR LP017 Adipocytes HS2A Saos2 Cells pSport 1 LP020 HS2I Saos2 Cells; Vitamin pSport 1 LP020 D3 Treated HUCM CHME Cell Line, untreated pSport 1 LP020 HEPN Aryepiglottis Normal pSport 1 LP020 HPSN Sinus Piniformis Tumour pSport 1 LP020 HNSA Stomach Normal pSport 1 LP020 HNSM Stomach Tumour pSport 1 LP020 HNLA Liver Normal Met5No pSport 1 LP020 HUTA Liver Tumour Met 5 Tu pSport 1 LP020 HOCN Colon Normal pSport 1 LP020 HOCT Colon Tumor pSport 1 LP020 HTNT Tongue Tumour pSport 1 LP020 HLXN Larynx Normal pSport 1 LP020 HLXT Larynx Tumour pSport 1 LP020 HTYN Thymus pSport 1 LP020 HPLN Placenta pSport 1 LP020 HTNG Tongue Normal pSport 1 LP020 HZAA Thyroid Normal pSport 1 LP020 (SDCA2 No) HWES Thyroid Thyroiditis pSport 1 LP020 HFHD Ficolled Human Stromal pTrip1Ex2 LP021 Cells, 5Fu treated HFHM, HFHN Ficolled Human Stromal pTrip1Ex2 LP021 Cells, Untreated HPCI Hep G2 Cells, lambda library lambda Zap- LP021 CMV XR HBCA, HBCB, HBCC H. Lymph node breast Cancer Uni-ZAP XR LP021 HCOK Chondrocytes pSPORT1 LP022 HDCA, HDCB, HDCC Dendritic Cells From pSPORT1 LP022 CD34 Cells HDMA, HDMB CD40 activated monocyte pSPORT1 LP022 dendritic cells HDDM, HDDN, UDDO LPS activated derived pSPORT1 LP022 dendritic cells HPCR Hep G2 Cells, PCR library lambda Zap- LP022 CMV XR HAAA, HAAB, HAAC Lung, Cancer (4005313A3): pSPORT1 LP022 Invasive Poorly Differentiated Lung Adenocarcinoma HIPA, HIPB, HIPC Lung, Cancer (4005163 B7): pSPORT1 LP022 Invasive, Poorly Diff. Adenocarcinoma, Metastatic HOOH, HOOI Ovary, Cancer: (4004562 B6) pSPORT1 LP022 Papillary Serous Cystic Neoplasm, Low Malignant Pot HIDA Lung, Normal: (4005313 B1) pSPORT1 LP022 HUJA, HUJB, HUJC, B-Cells pCMVSport 3.0 LP022 HUJD, HUJE HNOA, HNOB, HNOC, Ovary, Normal: (9805C040R) pSPORT1 LP022 HNOD HNLM Lung, Normal: (4005313 B1) pSPORT1 LP022 HSCL Stromal Cells pSPORT1 LP022 HAAX Lung, Cancer: (4005313 A3) pSPORT1 LP022 Invasive Poorly-differentiated Metastatic lung adenocarcinoma HUUA, HUUB, HUUC, B-cells (unstimulated) pTrip1Ex2 LP022 HUUD HWWA, HWWB, HWWC, B-cells (stimulated) pSPORT1 LP022 HWWD, HWWE, HWWF, HWWG HCCC Colon, Cancer: (9808C064R) pCMVSport 3.0 LP023 HPDO HPDP HPDQ HPDR Ovary, Cancer (9809C332): pSport 1 LP023 HPD Poorly differentiated adenocarcinoma HPCO HPCP HPCQ HPCT Ovary, Cancer (15395A1F): pSport 1 LP023 Grade II Papillary Carcinoma HOCM HOCO HOCP HOCQ Ovary, Cancer: (15799A1F) pSport 1 LP023 Poorly differentiated carcinoma HCBM HCBN HCBO Breast, Cancer: (4004943 A5) pSport 1 LP023 HNBT HNBU HNBV Breast, Normal: (4005522B2) pSport 1 LP023 HBCP HBCQ Breast, Cancer: (4005522 A2) pSport 1 LP023 HBCJ Breast, Cancer: (9806C012R) pSport 1 LP023 HSAM HSAN Stromal cells 3.88 pSport 1 LP023 HVCA HVCB HVCC HVCD Ovary, Cancer: (4004332 A2) pSport 1 LP023 HSCK HSEN HSEO Stromal cells (HBM3.18) pSport 1 LP023 HSCP HSCQ stromal cell clone 2.5 pSport 1 LP023 HUXA Breast Cancer: (4005385 A2) pSport 1 LP023 HCOM HCON HCOO HCOP Ovary, Cancer (4004650 A3): pSport 1 LP023 HCOQ Well-Differentiated Micropapillary Serous Carcinoma HBNM Breast, Cancer: (9802C020E) pSport 1 LP023 HVVA HVVB HVVC HVVD Human Bone Manow, treated pSport 1 LP023 HVVE

Two nonlimiting examples are provided below for isolating a particular clone from the deposited sample of plasmid cDNAs cited for that clone in Table 7. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to the nucleotide sequence of SEQ ID NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32 P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the nucleotide sequence of SEQ ID NO:X are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl 2 , 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.
Example 2
Isolation of Genomic Clones Corresponding to a Polynucleotide
A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the sequence corresponding to SEQ ID NO:X according to the method described in Example 1. (See also, Sambrook.)
Example 3
Tissue Specific Expression Analysis
The Human Genome Sciences, Inc. (HGS) database is derived from sequencing tissue and/or disease specific cDNA libraries. Libraries generated from a particular tissue are selected and the specific tissue expression pattern of EST groups or assembled contigs within these libraries is determined by comparison of the expression patterns of those groups or contigs within the entire database. ESTs and assembled contigs which show tissue specific expression are selected.
The original clone from which the specific EST sequence was generated, or in the case of an assembled contig, the clone from which the 5′ most EST sequence was generated, is obtained from the catalogued library of clones and the insert amplified by PCR using methods known in the art. The PCR product is denatured and then transferred in 96 or 384 well format to a nylon membrane (Schleicher and Scheull) generating an array filter of tissue specific clones. Housekeeping genes, maize genes, and known tissue specific genes are included on the filters. These targets can be used in signal normalization and to validate assay sensitivity. Additional targets are included to monitor probe length and specificity of hybridization.
Radioactively labeled hybridization probes are generated by first strand cDNA synthesis per the manufacturer's instructions (Life Technologies) from mRNA/RNA samples prepared from the specific tissue being analyzed (e.g., prostate, prostate cancer, ovarian, ovarian cancer, etc.). The hybridization probes are purified by gel exclusion chromatography, quantitated, and hybridized with the array filters in hybridization bottles at 65° C. overnight. The filters are washed under stringent conditions and signals are captured using a Fuji phosphorimager.
Data is extracted using AIS software and following background subtraction, signal normalization is performed. This includes a normalization of filter-wide expression levels between different experimental runs. Genes that are differentially expressed in the tissue of interest are identified.
Example 4
Chromosomal Mapping of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95° C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.
Example 5
Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/nl) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4° C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni—NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni—NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.
In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.
DNA can be inserted into the pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 6
Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10° C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10° C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 MM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 ×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4° C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4° C. without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 μm membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A 280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7
Cloning and Expression of a Polypeptide in a Baculovirus Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon, is amplified using the PCR protocol described in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five μg of a plasmid containing the polynucleotide is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27° C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 μCi of 35 S-methionine and 5 μCi 35 S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Example 8
Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli BB 101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9
Protein Fusions
The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
For example, if pC4 (ATCC Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
If the naturally occurring signal sequence is used to produce the polypeptide of the present invention, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)
Human IgG Fc region: (SEQ ID NO: 1) GGAATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10
Production of an Antibody from a Polypeptide
a) Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of a polypeptide of the present invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for a polypeptide of the present invention are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and TCell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with a polypeptide of the present invention or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Eagle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide of the present invention.
Alternatively, additional antibodies capable of binding to a polypeptide of the present invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide-specific antibody can be blocked by said polypeptide. Such antibodies comprise anti-idiotypic antibodies to the polypeptide-specific antibody and are used to immunize an animal to induce formation of further polypeptide-specific antibodies.
For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).
b) Isolation Of Antibody Fragments Directed Against a Polypeptide of the Present Invention From A Library Of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against a polypeptide of the present invention to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and 100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2xTY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene m, see International Publication No. WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m for 10 min. and the pellet resuspended in 2 liters of 2xTY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in International Publication No. WO 92/01047.
M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene m particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene RI protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 10 13 transducing units/rnl (ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 10 13 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCI, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG 1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., International Publication No. WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Example 11
Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
RNA isolated from entire families or individual patients presenting with diabetes mellitus is isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X; and/or the nucleotide sequence of the cDNA contained in ATCC Deposit No:Z. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).
PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase (Epicentre Technologies). The intron-exon boundaries of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing.
PCR products are cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991)). Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.
Example 12
Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample
A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 μg/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.
Example 13
Formulation
The invention also provides methods of preventing, treating and/or ameliorating diabetes mellitus by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).
The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Therapeutics can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials. (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).
In a preferred embodiment, polypeptide, polynucleotide, and antibody compositions of the invention are formulated in a biodegradable, polymeric drug delivery system, for example as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and 5,487,897 and in International Publication Numbers WO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated by reference in their entirety. In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
Examples of biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions, include but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The preferred polymers are those that have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Preferred materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility. In specific preferred embodiments, the biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired polypeptide, polynucleotide, or antibody release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is hereby incorporated by reference in its entirety).
It is also preferred that the solvent for the biodegradable polymer be non-toxic, water miscible, and otherwise biocompatible. Examples of such solvents include, but are not limited to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, and tetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such as dimethylformamide, dimethylacetamide and caprolactam; C3 to C20 ethers such as tetrahydrofuran, or solketal; tweens, triacetin, propylene carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl alchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate because of the solvating ability and their compatibility.
Additionally, formulations comprising polypeptide, polynucleotide, and antibody compositions and a biodegradable polymer may also include release-rate modification agents and/or pore-forming agents. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other like hydrophobic compounds, organic solvents, plasticizing compounds and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols, 2-ethoxyethanol. The release rate modification agent may be used singly or in combination with other such agents. Suitable combinations of release rate modification agents include, but are not limited to, glycerin/propylene glycol, sorbitolglycerine, ethylene oxidelpropylene oxide, butylene glycolladipic acid, and the like. Preferred release rate modification agents include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore-forming agents that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a defined pore size, such as salt or sugar, are preferred.
In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the BEMA™ BioErodible Mucoadhesive System, MCA™ MucoCutaneous Absorption System, SMP™ Solvent MicroParticle System, or BCP™ BioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 mi of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.
The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., TBERACYS®), MPL and nonviable prepartions of Corynebacterium parvum. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, μluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the Therapeutics of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.
In another embodiment, the Therapeutics of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKMASE™), antiresplace (e.g., EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICAR™). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
In another embodiment, the Therapeutics of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).
In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ iraine), and SUSTIVA™ (eravirenz). Protease inhibitors that may be administered in nation with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ avir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In ific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non- side reverse transcriptase inhibitors, and/or protease inhibitors may be used in any nation with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV ion.
Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; sle/Abbott; COVIRACIL™ (emtricitabine/FFC; structurally related to lamivudine (3TC) but - to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally to larnivudine but retains activity against a substantial proportion of lamivudine-resistant s; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead es); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is -pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active olite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC- nt virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)- g prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).
Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of EPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a next generation I with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 50-fold greater activity than its predecessor delavirdine and is active against K103N its; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of enz, designed to be active against viruses with the K103N mutation; DuPont); GW420867X 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo ome); CALANOLIDE A (naturally occurring agent from the latex tree; active against s containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).
Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott atories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™ (PNU- 0, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic ropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an vir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a lornimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX- W-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 ); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).
Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX404C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (968), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1, MIP-1,, etc., may also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).
In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIIMTHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.
In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.
In other embodiments, the Therapeutics of the invention are administered in combination with immunestimulants. Immunostimulants that may be administered in combination with the Therapeutics of the invention include, but are not limited to, levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).
In other embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDINI), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
In certain embodiments, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, dL-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChlMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J. Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not limited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/ Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).
In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as Remicader™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.) In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophospharnide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophospharnide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
In another specific embodiment, the compositions of the invention are administered in combination Zevalin™. In a further embodiment, compositions of the invention are administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Particularly preferred isotopes are 90 Y and 111 In.
In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, lL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.
In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNP, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in Gernan Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.
In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-I 1, FGF-12, FGF-13, FGF-14, and FGF-15.
In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostirn, LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.
In certain embodiments, Therapeutics of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
In another embodiment, the Therapeutics of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).
In another embodiment, the Therapeutics of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenarnide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na + -K + -2Cl − symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).
In one embodiment, the Therapeutics of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, 127 I, radioactive isotopes of iodine such as 131 I and 123 I; recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin); growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such as PARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionic gonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTREL™ and LUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRH™ and THYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T 4 ™, SYNTHROID™ and LEVOTHROID™ (levothyroxine sodium), L-T 3 ™, CYTOMEL™ and TRIOSTAT™ (liothyroine sodium), and THYROLAR™ (liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca 2+ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodium ipodate).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACE™ (estradiol), ESTNYL™ (ethinyl estradiol), PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™ (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen), SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™ (hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™ (medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™ (megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ and AYGESTIN™ (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins such as RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™ (norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device that releases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™, NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinyl estradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ and TREPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™ (ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodiol diacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™ (norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinyl estradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinyl estradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), and OVRETTE™ (norgestrel).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™ (oxandrolone); testosterone transdermal systems such as TESTODERM™; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropic hormone preparations such as CORTROSYN™ (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™ (amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolone pivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEF ACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™ (flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone), MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™ (methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™ (methylprednisolone sodium succinate), ELOCON™ (mometasone furoate), HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone), ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodium phosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™ (prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™ (triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™ (triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADREN™ (aminoglutethirnide), NIZORAL™ (ketoconazole), MODRASTANE™ (trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™, DIDBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide); and diazoxides such as PROGLYCEM™ (diazoxide).
In an additional embodiment, the Therapeutics of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-iron complex (e.g., NEFEREX™), iron dextran injection (e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B 12 , cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.
In another embodiment, Therapeutics of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.
In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for gastrointestinal disorders. Treatments for gastrointestinal disorders that may be administered with the Therapeutic of the invention include, but are not limited to, H 2 histamine receptor antagonists (e.g., TAGAMET™ (cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™ (nizatidine)); inhibitors of H + , K + ATPase (e.g., PREVACID™ (lansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™ (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate), MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide), antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Example 14
Method of Treating Decreased Levels of the Polypeptide
The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of polypeptides (including agonists thereto), and/or antibodies of the invention. Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a polypeptide of the present invention in an individual may be treated by administering agonists of said polypeptide. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the agonist (including polypeptides and antibodies of the present invention) to increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the agonist for six consecutive days. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 13.
Example 15
Method of Treating Increased Levels of the Polypeptide
The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).
In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, due to a variety of etiologies, such as cancer.
For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The antisense polynucleotides of the present invention can be formulated using techniques and formulations described herein (e.g. see Example 13), or otherwise known in the art.
Example 16
Method of Treatment Using Gene Therapy-Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a subconfluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 17
Gene Therapy Using Endogenous Genes Corresponding To Polynucleotides of the Invention
Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.
The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.
In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.
Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×10 6 cells/ml. Electroporation should be performed immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×10 6 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.
Example 18
Method of Treatment Using Gene Therapy—In Vivo
Another aspect of the present invention is using in vivo gene therapy methods to prevent, treat, and/or ameliorate diabetes mellitus. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to (i.e., associated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).
The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 19
Transgenic Animals
The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 20
Knock-Out Animals
Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 21
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
Diabetic db+/db+Mouse Model.
To demonstrate that an agonist or antagonist of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have-a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 16 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BLKsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
An agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm 2 , the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an agonist or antagonist of the invention. This assessment included verification of the presence of cell accumulation, inflanmmatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.
Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
To demonstrate that an agonist or antagonist of the invention can accelerate the healing process, the effects of multiple topical applications of the agonist or antagonist on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
The agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm 2 , the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an agonist or antagonist of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 22
Production Of Polypeptide of the Invention For High-Throughput Screening Assays
The following protocol produces a supernatant containing polypeptide of the present invention to be tested. This supernatant can then be used in the Screening Assays described in Examples 24-27.
First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2×10 5 cells/well in 0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/1× Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multihannel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8-10, into an appropriately labeled 96-well round bottom plate. With a multihannel pipetter, add 50 ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multihannel pipetter to add 150 ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C. for 6 hours.
While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO 4 -5H 2 O; 0.050 mg/L of Fe(NO 3 ) 3 -9H 2 O; 0.417 mg/L of FeSO 4 -7H 2 O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl 2 ; 48.84 mg/L of MgSO 4 ; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO 3 ; 62.50 mg/L of NaH 2 PO 4 -H 2 O; 71.02 mg/L of Na 2 HPO4; 0.4320 mg/L of ZnSO 4 -7H 2 O; 0.002 mg/L of Arachidonic Acid ; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Olcic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H 2 O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H 2 O; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H 2 O; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H 2 O; and 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B 12 ; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamine and 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15 ml polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37 degree C. for 45 or 72 hours depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 24-27.
It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide of the present invention directly (e.g., as a secreted protein) or by polypeptide of the present invention inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.
Example 23
Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.
JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) Il-11 (Pleiotropic) ? + ? ? 1, 3 OnM (Pleiotropic) ? + + ? 1, 3 LIF (Pleiotropic) ? + + ? 1, 3 CNTF (Pleiotropic) −/+ + + ? 1, 3 G-CSF (Pleiotropic) ? + ? ? 1, 3 IL-12 (Pleiotropic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS (B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRFI promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is:
(SEQ ID NO: 3) 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGAT TTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4) PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:
(SEQ ID NO: 5) 5′: CTCGAG ATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCC CCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTT TTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGA GGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA AAGCTT :3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding.
Other constructs can be made using the above description and replacing GAS with a different promoter sequence, for example, with EGR and NF-KB promoter sequences. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB3, Il-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as BELA (epithelial), HUVEC (endothelial), Reh (Bcell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 24
Assay for SEAP Acdivity
SEAP activity is assayed using the Tropix Phospholight Kit (Cat. BP400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of 2.5× dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 25
High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO 2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO 2 incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×10 6 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10 6 cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 ul, followed by an aspiration step to 100 ul final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4 . The supernatant is added to the well, and a change in fluorescence is detected.
To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event caused by the a molecule, either polypeptide of the present invention or a molecule induced by polypeptide of the present invention, which has resulted in an increase in the intracellular Ca ++ concentration.
Example 26
High-Throughput Screening Assay Identifying Tyrosine Kinase Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, Ick, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether polypeptide of the present invention or a molecule induced by polypeptide of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford,Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,Mass.) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example 22, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C. at 16,000×g.
Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/Mg 2+ (5mM ATP/50 mM MgCl 2 ), then 10 ul of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl 2 , 5 mM MnCl 2 , 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate(1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degree C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C. for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degree C. for one hour. Wash the well as above.
Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 27
High-Throughput Screening Assay Identifying Phosphorylation Activity
As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 26, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C. until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 22 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Waflac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by polypeptide of the present invention or a molecule induced by polypeptide of the present invention.
Example 28
Detection of Inhibition of a Mixed Lymphocyte Reaction
This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.
Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.
Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×10 6 cells/ml in RPMI-1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is adjusted to 2×10 5 cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO 2 , and 1 μC of [ 3 H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.
Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
Example 29
Assays for Protease Activity
The following assay may be used to assess protease activity of the polypeptides of the invention.
Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in 2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37° C. 5 to 16 hours. After staining in amido black areas of proteolysis apear as clear areas agains the blue-black background. Trypsin (Sigma T8642) is used as a positive control.
Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500. Reactions are set up in (25 mMNaPO 4 ,1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in adsorbance at 260nm is monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.
Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as adsorbance at 280 nm or colorimetrically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).
Example 30
Identifying Serine Protease Substrate Specificity
Methods known in the art or described herein may be used to determine the substrate specificity of the polypeptides of the present invention having serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB2 324 529 (incorporated herein in its entirety).
Example 31
Ligand Binding Assays
The following assay may be used to assess ligand binding activity of the polypeptides of the invention.
Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for a polypeptide is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its polypeptide. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
Example 32
Functional Assay in Xenopus Oocytes
Capped RNA transcripts from linearized plasmid templates encoding the polypeptides of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus ooctyes in response polypeptides and polypeptide agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 33
Microphysiometric Assays
Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation of polypeptide which is coupled to an energy utilizing intracellular signaling pathway.
Example 34
Extract/Cell Supernatant Screening
A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the polypeptides of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify its natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
Example 35
Calcium and cAMP Functional Assays
Seven transmembrane receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day >150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
Example 36
ATP-binding Assay
The following assay may be used to assess ATP-binding activity of polypeptides of the invention.
ATP-binding activity of the polypeptides of the invention may be detected using the ATP-binding assay described in U.S. Pat. No. 5,858,719, which is herein incorporated by reference in its entirety. Briefly, ATP-binding to polypeptides of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg/ml of the ABC transport protein of the present invention are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP ( 32 P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2 mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the particular polypeptides of the invention are excised, and the radioactivity quantified. A decrease in radioactivity with increasing ATP or adenly-5′-imidodiphosphate provides a measure of ATP affinity to the polypeptides.
Example 37
Small Molecule Screening
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and polypeptide of the invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the invention. These methods comprise contacting such an agent with a polypeptide of the invention or fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is herein incorporated by reference in its entirety. Briefly stated, large numbers of different small molecule test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with polypeptides of the invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and inmmobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
Example 38
Phosphorylation Assay
In order to assay for phosphorylation activity of the polypeptides of the invention, a phosphorylation assay as described in U.S. Pat. No. 5,958,405 (which is herein incorporated by reference) is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled 32 P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The polypeptides of the invention are incubated with the protein substrate, 32 P-ATP, and a kinase buffer. The 32 P incorporated into the substrate is then separated from free 32 P-ATP by electrophoresis, and the incorporated 32 P is counted and compared to a negative control. Radioactivity counts above the negative control are indicative of phosphorylation activity of the polypeptides of the invention.
Example 39
Detection of Phosphorylation Activity (Activation) of the Polypeptides of the Invention in the Presence of Polypeptide Ligands
Methods known in the art or described herein may be used to determine the phosphorylation activity of the polypeptides of the invention. A preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in U.S. Pat. No. 5,817,471 (incorporated herein by reference).
Example 40
Identification Of Signal Transduction Proteins That Interact With Polypeptides Of The Present Invention
The purified polypeptides of the invention are research tools for the identification, characterization and purification of additional signal transduction pathway proteins or receptor proteins. Briefly, labeled polypeptides of the invention are useful as reagents for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptides of the invention are covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 41
Assay for Phosphatase Activity
The following assay may be used to assess serine/threonine phosphatase (PTPase) activity of the polypeptides of the invention.
In order to assay for serine/threonine phosphatase (PIPase) activity, assays can be utilized which are widely known to those skilled in the art. For example, the serine/threonine phosphatase (PSPase) activity is measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [ 32 P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.
Example 42
Interaction of Serine/Threonine Phosphatases with other Proteins
The polypeptides of the invention with serine/threonine phosphatase activity as determined in Example 41 are research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, labeled polypeptide(s) of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptide of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The polypeptides of the invention-complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 43
Assaying for Heparanase Activity
In order to assay for heparanase activity of the polypeptides of the invention, the heparanase assay described by Vlodavsky et al is utilized (Vlodavsky, I., et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media or intact cells (1×10 6 cells per 35-mm dish) are incubated for 18 hrs at 37° C., pH 6.2-6.6, with 35 S-labeled ECM or soluble ECM derived peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharose 6B at 0.5<K av <0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to “peak II,” as described by Vlodavsky et al., is indicative of the activity of the polypeptides of the invention in cleaving heparan sulfate.
Example 44
Immobilization of Biomolecules
This example provides a method for the stabilization of polypeptides of the invention in non-host cell lipid bilayer constucts (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), hereby incorporated by reference in its entirety herein) which can be adapted for the study of polypeptides of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to the extracellular domain of the polypeptides of the invention, thus allowing uniform orientation upon immobilization. A 50 uM solution of polypeptides of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150 ul). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford Ill.) at 4C first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 45
TAQMAN
Quantitative PCR (QPCR). Total RNA from cells in culture are extracted by Trizol separation as recommended by the supplier (LifeTechnologies). (Total RNA is treated with DNase I (Life Technologies) to remove any contaminating genomic DNA before reverse transcription.) Total RNA (50 ng) is used in a one-step, 50 ul, RT-QPCR, consisting of Taqman Buffer A (Perkin-Elmer; 50 mM KCl/10 mM Tris, pH 8.3), 5.5 mM MgCl 2 , 240 μM each dNTP, 0.4 units RNase inhibitor(Promega), 8% glycerol, 0.012% Tween-20, 0.05% gelatin, 0.3 uM primers, 0.1 uM probe, 0.025 units Amplitaq Gold (Perkin-Elmer) and 2.5 units Superscript II reverse transcriptase (Life Technologies). As a control for genomnic contamination, parallel reactions are setup without reverse transcriptase. The relative abundance of (unknown) and 18S RNAs are assessed by using the Applied Biosystems Prism 7700 Sequence Detection System (Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K. (1995) PCR Methods Appl. 4, 357-362). Reactions are carried out at 48° C. for 30 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s, 60° C. for 1 min. Reactions are performed in triplicate.
Primers (f & r) and FRET probes sets are designed using Primer Express Software (Perkin-Elmer). Probes are labeled at the 5′-end with the reporter dye 6-FAM and on the 3′-end with the quencher dye TAMRA (Biosource International, Camarillo, Calif. or Perkin-Elmer).
Example 46
Assays for Metalloproteinase Activity
Metalloproteinases (EC 3.4.24.-) are peptide hydrolases which use metal ions, such as Zn 2+ , as the catalytic mechanism. Metalloproteinase activity of polypeptides of the present invention can be assayed according to the following methods.
Proteolysis of alpha-2-macroglobulin
To confirm protease activity, purified polypeptides of the invention are mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; Boehringer Mannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl 2 , 25 μM ZnCl 2 and 0.05% Brij-35) and incubated at 37° C. for 1-5 days. Trypsin is used as positive control. Negative controls contain only alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining. Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.
Inhibition of alpha-2-macroglobulin proteolysis by inhibitors of metalloproteinases Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND HgCl 2 ), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) are used to characterize the proteolytic activity of polypeptides of the invention. The three synthetic MMP inhibitors used are: MMP inhibitor I, [IC 50 =1.0 μM against MMP-1 and MMP-8; IC 50 =30 μM against MMP-9; IC 50 =150 μM against MMP-3]; MMP-3 (stromelysin-inhibitor I [IC 50 =5 μM against MMP-3], and MMP-3 inhibitor II [K i =130 nM against MMP-3]inhibitors available through Calbiochem, catalog #444250, 444218, and 444225, respectively). Briefly, different concentrations of the small molecule MMP inhibitors are mixed with purified polypeptides of the invention (50 μg/ml) in 22.9 μl of 1× HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl 2 , 25 μM ZnCl 2 and 0.05% Brij-35) and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl of substrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for 20-hr. The reactions are stopped by adding 4× sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.
Synthetic Fluorogenic Peptide Substrates Cleavage Assay
The substrate specificity for polypeptides of the invention with demonstrated metalloproteinase activity can be determined using synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE). All the substrates are prepared in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 μM. Fluorescent assays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nnm Briefly, the assay is carried out by incubating 176 μl 1× HEPES buffer (0.2 M NaCl, 10 MM CaCl 2 , 0.05% Brij-35 and 50 mM BEPES, pH 7.5) with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, and then adding 20 μl of a purified polypeptide of the invention into the assay cuvett. The final concentration of substrate is 1 μM. Initial hydrolysis rates are monitored for 30-min.
Example 47
Characterization of the cDNA contained in a Deposited Plasmid
The size of the cDNA insert contained in a deposited plasmid may be routinely determined using techniques known in the art, such as PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the cDNA sequence. For example, two primers of 17-30 nucleotides derived from each end of the cDNA (i.e., hybridizable to the absolute 5′ nucleotide or the 3′ nucleotide end of the sequence of SEQ ID NO:X, respectively) are synthesized and used to amplify the cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl 2 , 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
Incorporation by Reference
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. In addition, the sequence listing submitted herewith is incorporated herein by reference in its entirety. The specification and sequence listing of each of the following U.S. and PCT applications are herein incorporated by reference in their entirety (filing dates shown in format “year-month-day” (yyyy-mm-dd)): Application No. 60/278,650 filed on 2001 Mar. 27, application Ser. No. 09/950,082 filed on 2001 Sep. 12, application Ser. No. 09/950,083 filed on 2001 Sep. 12, application Ser. No. 60/306,171 filed on 19 Jul. 2001, application Ser. No. 09/833,245 filed on 2001 Apr. 12, Application No. PCT/US01/11988 filed on 2001 Apr. 12, Application No. 60/331,287 filed on 2001 Nov. 13, Application No. 60/277,340 filed on 2001 Mar. 21, Application No. PCT/US00/06043 filed on 2000 Mar. 9, Application No. PCT/US00/06012 filed on 2000 Mar. 9, Application No. PCT/US00/06058 filed on 2000 Mar. 9, Application No. PCT/US00/06044 filed on 2000 Mar. 9, Application No. PCT/US00/06059 filed on 2000 Mar. 9, Application No. PCT/US00/06042 filed on 2000 Mar. 9, Application No. PCT/US00/06014 filed on 2000 Mar. 9, Application No. PCT/US00/06013 filed on 2000 Mar. 9, Application No. PCT/US00/06049 filed on 2000 Mar. 9, Application No. PCT/US00/06057 filed on 2000 Mar. 9, Application No. PCT/US00/06824 filed on 2000 Mar. 16, Application No. PCT/US00/06765 filed on 2000 Mar. 16, Application No. PCT/US00/06792 filed on 2000 Mar. 16, Application No. PCT/US00/06830 filed on 2000 Mar. 16, Application No. PCT/US00/06782 filed on 2000 Mar. 16, 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on 2000 Jan. 20, Application No. U.S. 60/128,699 filed on 1999 Apr. 9, Application No. U.S. 60/177,050 filed on 2000 Jan. 20, Application No. U.S. 60/128,701 filed on 1999 Apr. 9, Application No. U.S. 60/177,166 filed on 2000 Jan. 20, Application No. U.S. 60/128,700 filed on 1999 Apr. 9, Application No. U.S. 60/176,930 filed on 2000 Jan. 20, Application No. U.S. 60/128,694 filed on 1999 Apr. 9, Application No. U.S. 60/176,931 filed on 2000 Jan. 20, Application No. U.S. 60/128,702 filed on 1999 Apr. 9, Application No. U.S. 60/177,049 filed on 2000 Jan. 20, Application No. U.S. 60/138,629 filed on 1999 Jun. 11, Application No. U.S. 60/138,628 filed on 1999 Jun. 11, Application No. U.S. 60/138,631 filed on 1999 Jun. 11, Application No. U.S. 60/138,632 filed on 1999 Jun. 11, Application No. U.S. 60/138,599 filed on 1999 Jun. 11, Application No. U.S. 60/138,572 filed on 1999 Jun. 11, Application No. U.S. 60/138,625 filed on 1999 Jun. 11, Application No. U.S. 60/138,633 filed on 1999 Jun. 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