(en)Novel proteins and methods of treatment using same are disclosed.

1.ApplicationNumber: US-4101602-A
1.PublishNumber: US-2002165151-A1
2.Date Publish: 20021107
3.Inventor: JACOBS KENNETH
MCCOY JOHN M.
RACIE LISA A.
LAVALLIE EDWARD R.
TREACY MAURICE
EVANS CHERYL
AGOSTINO MICHAEL J.
LU ZHIJIAN
MERBERG DAVID
TASHIRO KEI
NAKAMURA TOMOYUKI
HONJO TASUKU

4.Inventor Harmonized: JACOBS KENNETH(US)
MCCOY JOHN M(US)
RACIE LISA A(US)
LAVALLIE EDWARD R(US)
TREACY MAURICE(US)
EVANS CHERYL(US)
AGOSTINO MICHAEL J(US)
LU ZHIJIAN(US)
MERBERG DAVID(US)
TASHIRO KEI(JP)
NAKAMURA TOMOYUKI(US)
HONJO TASUKU(JP)

5.Country: US
6.Claims:
(en)Novel proteins and methods of treatment using same are disclosed.

7.Description:
(en)[0001] This application is a continuation-in-part of application Ser. No. 08/885,610, filed Jun. 30, 1997, which was a continuation-in-part of application Ser. No. 08/634,325, filed Apr. 18, 1996.



FIELD OF THE INVENTION
[0002] The present invention provides novel proteins, along with therapeutic, diagnostic and research utilities for these proteins.
BACKGROUND OF THE INVENTION
[0003] Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins that the present invention is directed.
[0004] In modem medical research, cardiovascular biology is a field that attracts considerable attention because cardiovascular disease is the leading cause of mortality. Cardiovascular research has revealed important facts about neointimal formation and arterial remodeling, both of which are thought to contribute to plaque formation in atherosclerosis and blood vessel narrowing. For example, there are three aspects of the cellular process in hypercholesterolaemia induced blood vessel damage in animal models that mimic human development of atherosclerotic coronary disease. The three elements that form lesions on the artery wall are: a) proliferation of smooth muscle cells, macrophages and lymophocytes, b) formation of connective tissues (mainly elastic fiber proteins, collagen and proteoglycans made by smooth muscle cells in a process similar to scar formation), and c) the accumulation of lipid and cholesterol in the newly formed connective tissue matrices. The exact sequence of the three damaging elements are debatable, but it is clear that the abnormal dedifferentiation, redifferentiation and growth of smooth muscle cells contribute structurally to vessel damage.
[0005] Another significant pathological process that involves abnormal smooth muscle cell growth is restenosis after “percutaneous transluminal coronary angioplasty” (PTCA). The success rate of this procedure currently stands at 30% to 50%, largely due to vessel renarrowing and remodeling resulted from uncontrolled growth and migration of vessel intimal smooth muscle cells.
[0006] It is also known that in embryonic development there are different cellular signals that stimulate, direct and control cellular differentiation and growth. In such processes, both positive and negative signals play important roles. Some of these signals are temporally and spatially restricted to a specific developmental stage. However, their existence, signaling pathway and cellular effects signifies the possibility of using these molecules produced ex vivo to treating adult individuals for particular medical conditions. For example, BMP-2 plays a major morphogenetic role in bone and joint development during embryogenesis, but it can be used therapeutically in adults to induce bone growth. Similarly, it is reasonable to envision the use of molecules involved in the smooth muscle component of blood vessel formation during embryogenesis for the purpose of controlling abnormal SMC growth in the pathological processes in adults.
[0007] The signaling molecules in multicellular organisms that transmit accurate and precise spatial and temporal inter-cellular messages for cellular growth, movement, differentiation, dormancy or death are secreted proteins. When these proteins interact with their cognate cell surface receptors, signals are received and intra-cellular processes started. Most of these two groups of proteins have an identifiable structural motif—signal peptide which causes a protein to move to the cell surface or to be secreted from the cell.
[0008] It would, therefore, be desirable to identify secreted proteins involved in regulation of smooth muscle formation (including blood vessel tissues) for use as therapeutics and as targets for discovery of small molecule drugs.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
[0010] (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
[0011] (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 186 to nucleotide 1532:
[0012] (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 261 to nucleotide 1532;
[0013] (d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 255 to nucleotide 1532;
[0014] (e) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AK647 deposited under accession number ATCC 98026;
[0015] (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AK647 deposited under accession number ATCC 98026;
[0016] (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AK647 deposited under accession number ATCC 98026;
[0017] (h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AK647 deposited under accession number ATCC 98026;
[0018] (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
[0019] (j) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 24 to amino acid 448;
[0020] (k) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 26 to amino acid 448;
[0021] (l) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
[0022] (m) a polynucleotide which is an allelic variant of a polynucleotide of any of (a)-(h) above; and
[0023] (n) a polynucleotide which encodes a species homologue of the protein of any of (i)-(k) above.
[0024] Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:1 from nucleotide 186 to nucleotide 1532; the nucleotide sequence of SEQ ID NO:1 from nucleotide 261 to nucleotide 1532; the nucleotide sequence of SEQ ID NO:1 from nucleotide 255 to nucleotide 1532; the nucleotide sequence of the full-length protein coding sequence of clone AK647 deposited under accession number ATCC 98026; or the nucleotide sequence of the mature protein coding sequence of clone AK647 deposited under accession number ATCC 98026. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AK647 deposited under accession number ATCC 98026. In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 104. In further preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 93; or from amino acid 24-448 or 26-448.
[0025] In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
[0026] (a) the amino acid sequence of SEQ ID NO:2;
[0027] (b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 104;
[0028] (c) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 93;
[0029] (d) the amino acid sequence of SEQ ID NO:2 from amino acid 24 to amino acid 448;
[0030] (e) the amino acid sequence of SEQ ID NO:2 from amino acid 26 to amino acid 448;
[0031] (f) fragments of the amino acid sequence of SEQ ID NO:2; and
[0032] (g) the amino acid sequence encoded by the cDNA insert of clone AK647 deposited under accession number ATCC 98026;
[0033] the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2, or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 104, or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 93, or the amino acid sequence of SEQ ID NO:2 from amino acid 24 to amino acid 448, or the amino acid sequence of SEQ ID NO:2 from amino acid 26 to amino acid 448.
[0034] Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
[0035] Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF FIGURES
[0036]FIGS. 1A and 1B depict the pED6 and pNotS vectors used to deposit the clone encoding the proteins of the present invention.
[0037]FIG. 2 is an autoradiograph evidencing the expression of clone AK647 in COS cells.
[0038]FIG. 3: AK647 expression in COS-1 cells. 3 A: Metabolic labeling of transient expression in media containing different amount of bovine fetal serum. The conditioned media from mock, AK647 and CF50 (positive control) transfected COS cells are analyzed on a 12% SDS-PAGE and visualized by autoradiography. 3 B: SDS-PAGE analysis of conditioned media concentrate from COS cells transfected by AK647. The intensity of the Coomassie blue stained AK 647 band is the basis for estimating the amount of AK647 present in the conditioned media.
[0039]FIG. 4: Diagram of plasmid constructs for making CHO cell lines expressing HEK-AK647-QA and HEK-AK647-QC.
[0040]FIG. 5: Bacterial expression of AK647. 5 A: Diagram of plasmid construct for expressing GroHEK2/AK647-QA in E. coli strain GI934. 5 B: SDS-PAGE analysis of E. coli cells express GroHEK2/AK647-QA. The protein exists in the bacterial cells as inclusion bodies.
[0041]FIG. 6: Morphology of CRL 1444 rat aortic smooth muscle cells treated with and without AK647 conditioned medium. 6 A: Cells are incubated for 24 hours in serum free medium containing 10% AK647 COS conditioned medium. 6 B: Cells are incubated for 24 hours in serum free media containing 10% mock transfected COS conditioned medium.
[0042]FIG. 7: Effect of AK647 on PDGF stimulated CRL 1444 rat aortic smooth muscle cells, as measured by MTT assay. Cells are exposed to the treatment condition for a period of 24 hours. The amount of formazan generated by mitochondrial dehydrogenase activity present in viable cells, as measured by the absorbance at A570, reflects the relative growth of cells under different conditions.
[0043]FIG. 8: Effect of AK647 on proliferation of PDGF stimulated CRL 1444 rat aortic smooth muscle cells, as measured by 3H-thymidine incorporation. Cells are exposed to the treatment condition for a period of 24 hours. After washing cells to remove unincorporated 3H-thymidine, the incorporated radioactivity in the cells are counted on a BetaPlate counter.
[0044]FIG. 9: Concentration dependence of AK647 on inhibition of proliferation in CRL 1444 rat aortic smooth muscle cells, as measured by 3H-thymidine incorporation. Cells are incubated for 24 hours in the treatment condition. The concentration of AK647 in the first well of the dilution series is 1 mg/ml-estimated by Coomassie Blue staining of SDS-PAGE of AK647 conditioned medium concentrate. Mock transfected conditioned medium is concentrated to the same extent as AK647 conditioned medium.
[0045]FIG. 10: Concentration dependence of AK647 on inhibition of proliferation of CRL 2018 rat aortic smooth muscle cells, as measured by 3H-thymidine incorporation. Cells are incubated in the treatment condition for 24 hours. Initial concentration of AK647 in the medium is 0.5 mg/ml estimated by Coomassie blue staining.
[0046]FIG. 11: Concentration dependence of AK647 on inhibition of proliferation of CRL 1476 rat aortic smooth muscle cells, as measured by 3H-thymidine incorporation. Cells are incubated in the treatment condition for 24 hours. Initial concentration of AK647 in the medium is 0.5 mg/ml estimated by Coomassie Blue staining.



DETAILED DESCRIPTION
[0047] Isolated Proteins
[0048] Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
[0049] As used herein a “secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
[0050] Protein “AK647”
[0051] One protein of the present invention has been identified as protein “AK647”. A partial cDNA clone encoding AK647 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank and GeneSeq databases using BLASTN/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST identified as H17726 (ym40a05.r1 Homo-sapiens cDNA clone 50483 5′), U03877 (Human extracellular protein (S1-5) mRNA, complete cds), N50529 (yy89c07.s1 Homo sapiens cDNA clone 280716 3′), and T21312 (Human gene signature HUMGS02672). The predicted amino acid sequence disclosed herein for AK647 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted AK647 protein demonstrated at least some identity with sequences identified as U13646 (homeotic region most like HMPB_DROME homeotic proboscipedia protein [ Caenorhabditis elegans ]), U03877 (extracellular protein [ Homo sapiens ]), R11150 (Fibulin C), and U03272, (HSU03272 — 1 fibrillin-2 [ Homo sapiens ]). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo., a distributor of the I.M.A.G.E. Consortium library. The clone received from the distributor was examined and determined to be a full-length clone, including a 5′ end and 3′ UTR (including a polyA tail). This full-length clone is also referred to herein as “AK647”.
[0052] Applicants' methods identified clone AK647 as encoding a secreted protein.
[0053] The nucleotide sequence of AK647 as presently determined is reported in SEQ ID NO:1. What applicants believe is the proper reading frame and the predicted amino acid sequence of the AK647 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Amino acids 13 to 25 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 26, or are a transmembrane domain.
[0054] The EcoRI/NotI restriction fragment obtainable from the deposit containing clone AK647 should be approximately 2383 bp.
[0055] Deposit of Clones
[0056] Clone AK647 was deposited on Apr. 17, 1996 with the American Type Culture Collection under accession number ATCC 98026, from which the clone comprising a particular polynucleotide is obtainable. This deposit is a mixture of clone AK647 with other unrelated clones. Each clone has been transfected into separate bacterial cells ( E. coli ) in this composite deposit.
[0057] Clone AK647 can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5′ site, EcoRI; 3′ site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNotS vector depicted in FIG. 1. In some instances, the deposited clone can become “flipped” (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5′ site and EcoRI will produce the 3′ site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
[0058] Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
[0059] An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences.
[0060] In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
[0061] The design of the oligonucleotide probe should preferably follow these parameters:
[0062] (a) It should be designed to an area of the sequence which has the fewest ambiguous bases (“N's”), if any;
[0063] (b) It should be designed to have a T m of approx. 80° C. (assuming 2° for each A or T and 4 degrees for each G or C).
[0064] The oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
[0065] The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37° C., and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37° C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
[0066] Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
[0067] The filter is then preferably incubated at 65° C. for 1 hour with gentle agitation in 6× SSC (20× stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to 1e+6 dpm/mL. The filter is then preferably incubated at 65° C. with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2× SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2× SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1× SSC/0.5% SDS at 65° C. for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
[0068] The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
[0069] Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through “linker” sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein-IgM fusion would generate a decavalent form of the protein of the invention.
[0070] The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
[0071] Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman. Methods in Enzymology identified in accordance with known techniques for determination of such domains from sequence information.
[0072] Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
[0073] Species homologs of the disclosed proteins are also provided by the present invention. 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 from the desired species.
[0074] The invention also encompasses allelic variants of the disclosed proteins; that is, naturally-occurring alternative forms of the isolated proteins which are identical, homologous or related to that encoded by the polynucleotides disclosed herein.
[0075] The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
[0076] The isolated polynucleotide endcoing the protein of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman. Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
[0077] A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A43 1 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
[0078] Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
[0079] The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.”
[0080] The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3 GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
[0081] Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“Flag”) is commercially available from Kodak (New Haven, Conn.).
[0082] Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.”
[0083] The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
[0084] The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
[0085] The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
[0086] Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
[0087] Uses and Biological Activity
[0088] The proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
[0089] Research Uses and Utilities
[0090] The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
[0091] Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
[0092] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.
[0093] Nutritional Uses
[0094] Proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein of the invention can be added to the medium in or on which the microorganism is cultured.
[0095] Cytokine and Cell Proliferation/Differentiation Activity
[0096] A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
[0097] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0098] Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli. et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152:1756-1761, 1994.
[0099] Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, R. D. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
[0100] Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al, Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
[0101] Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
[0102] Immune Stimulating or Suppressing Activity
[0103] A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
[0104] Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
[0105] Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or energy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
[0106] Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
[0107] The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
[0108] Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
[0109] Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
[0110] Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
[0111] In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
[0112] The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β 2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
[0113] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0114] Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowman et al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
[0115] Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e. a. Coligan eds. Vol. 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0116] Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
[0117] Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
[0118] Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
[0119] Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
[0120] Hematopoiesis Regulating Activity
[0121] A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g., in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and, paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
[0122] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0123] Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
[0124] Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
[0125] Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al eds. Vol. pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney. et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.
[0126] Tissue Growth Activity
[0127] A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of bums, incisions and ulcers.
[0128] A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
[0129] A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
[0130] Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
[0131] The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e., for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
[0132] Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
[0133] It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
[0134] A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
[0135] A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above. AK647 is particularly useful in inhibition of the growth, formation and development of smooth muscle tissue, including vascular tissue, as further described below.
[0136] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0137] Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).
[0138] Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H I and Rovee, D T, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest, Dermatol 71:382-84 (1978).
[0139] Activin/Inhibin Activity
[0140] A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
[0141] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0142] Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
[0143] Chemotactic/Chemokinetic Activity
[0144] A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
[0145] A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
[0146] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0147] Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al. Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
[0148] Hemostatic and Thrombolytic Activity
[0149] A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophiliac) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
[0150] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0151] Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
[0152] Receptor/Ligand Activity
[0153] A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selecting, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
[0154] The activity of a protein of the invention may, among other means, be measured by the following methods:
[0155] Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
[0156] Anti-Inflammatory Activity
[0157] Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
[0158] Cadherin/Tumor Invasion Suppressor Activity
[0159] Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
[0160] The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the super family have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
[0161] E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
[0162] Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
[0163] Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e., the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
[0164] Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
[0165] Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11:2547-2552. 1995; Ozawa et al. Cell 63:1033-1038, 1990.
[0166] Tumor Inhibition Activity
[0167] In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
[0168] Other Activities
[0169] A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive-disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
[0170] Further Characterization of AK647 and Examination of Activity AK647 in Inhibiting Growth of Smooth Muscle Tissue
[0171] Below we establish that AK647 is a molecule found in fetal kidney tissue and that its homologs in rodents and chicks are involved in aortic tissue development. We also demonstrate further recombinant DNA manipulation for expressing this molecule in E. coli, COS and CHO cells as well as procedures for making the purified protein. In addition, we describe the effect of AK647 on the growth and differentiation of cell lines derived from aortic tissues. We then further describe ways of using products derived from AK647 for treating medical indications such as atherosclerosis, restenosis, blood vessel remodeling and degeneration.
[0172] Materials and Methods
[0173] Signal Sequence Trap.
[0174] Signal sequences are highly conserved among secreted proteins, and even between diverse organisms. In order to use this sequence as a trap to isolate cDNA clones encoding secreted proteins, a cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with a strain of Saccharomyces cerivisiae deleted for its endogenous invertase gene. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as raffinose or sucrose. The methods used in the signal sequence trap are further described in U.S. Pat. Nos. 5,712,116 and 5,536,637.
[0175] Expression of AK647 in E. coli.
[0176] The DNA fragment encoding the mature AK647-QA (SEQ ID NO:2 beginning with amino acid 24) fused to the C-terminus of an expression/purification accessory sequence AAKDVKSGHHHHHHGDDDDK (GroHEK2) was subcloned into the E. coli expression vector pAL985 to an ATG which serves as the translation initiation codon. The resulting plasmid pAL985-GroHEK2-AK647-QA was used to transform the E. coli strain of GI934. For expression, a fresh overnight culture of GI934 harboring the construct was used to inoculate IMC/Amp medium (M9 media containing 0.2% casamino acids, 0.5% glucose, 1 mM MgSO 4 and 100 μg/ml ampicillin) to an OD 550 of 0.05. The culture was grown at 30° C. until the OD 550 reached 0.5, then L-tryptophan was added to a concentration of 100 μg/ml and the culture temperature shifted to 37° C. (LaVallie, 1993). Four hours following tryptophan addition the cells were harvested by centrifugation and stored at −80° C. until used.
[0177] Expression of AK647 in COS Cells.
[0178] The cDNA of novel secreted protein AK647 was subcloned into a pNot expression vector, and transiently expressed in COS cells using a lipofectamine protocol. pNot vector containing the cDNA of tissue factor inserted in reverse orientation was also used in COS transfection for obtaining conditioned media which can be used as a control in the cellular assay studies. The expression of the proteins in conditioned media was assessed by SDS-PAGE combined with Coomassie Blue staining, or in metabolic labeling experiments, by autoradiograph.
[0179] Generation of Purified AK647.
[0180] The nucleotide sequences encoding two potential forms (named AK647-QA (amino acids 24-448 of SEQ ID NO:2) and AK647-QC (amino acids 26-448 of SEQ ID NO:2)) of the mature AK647 were fused in-frame to an artificial gene that encodes a generic secretion signal peptide and a tag (HEK) removable by enterokinase (FIG. 5). The fusion genes were subcloned into pHTOP vector for transfecting CHO cells. Seven to 14 days after transfection, individual clones were selected and their level of secreted HEK-AK647-QA or HEK-AK647-QC were examined by metabolic labeling and SDS-PAGE/autoradiography.
[0181] Large-scale expression of the CHO cell lines in defined medium yielded enough conditioned media for purification of HEK-AK647-QA and HEK-AK647-QC on Ni-NTA chromatography. Following the IMAC purification, the tag HEK was removed by enterokinase, resulting in pure AK647-QA and AK647-QC.
[0182] Aortic Cell Lines.
[0183] Four aortic smooth muscle cell lines obtained from ATCC (CRL-1444, CRL-1476, CRL-1999 and CRL-2018) were used for in vitro cell assays. CRL 1999 is a cell line established by selective serial passage of cells isolated from human thoracic aortic smooth muscle. Cells were screened during the selection process for smooth muscle cell markers and the ability to propagate an action potential. CRL 1476 and 1444 are rat thoracic aortic smooth muscle cell lines established and were screened in the same manner as CRL1999. CRL 2018 is an SV-40 transformed rat abdominal aortic smooth muscle cell line. It was also screened to confirm the presence of smooth muscle cell markers, and the ability to propagate an action potential.
[0184] Cell Proliferation Measured by MTT Assay.
[0185] Cleavage and conversion of the soluble yellow dye MTT by active mitochondrial dehydrogenases into an insoluble purple formazan precipitate was used as a measure of cell viability and proliferation. The amount of the cleavage product which was generated was measured spectrophotometrically. To carry out the assays, cells were seeded onto 96-well plates at a concentration of 5×10 4 /ml in the media containing 10% BFS and allowed to attach overnight. Cells were then washed 3× in PBS, and incubated with treatment factor in the media containing a defined amount of serum or no serum for a period of 24 or 48 hours. After the incubation, freshly made MTT dye was added to the wells at 0.5 mg/ml final concentration, and allowed to incubate for a further 4 hrs at 37° C. Any precipitate which was generated during this time was then solubilized by addition of an isopropanol/acid mixture stop solution, and the absorbance at 570 nm for each well was measured on a Molecular Devices microplate reader.
[0186] Cell Proliferation Measured by 3H-Thymidine Incorporation.
[0187] In proliferation assays measured by 3H-thymidine incorporation, cells were seeded onto 96-well plates at a concentration of 3×104/ml in the media containing 10% BFS and allowed to attach overnight. Cells were then washed 3× in PBS, and incubated with treatment factor in media containing defined amount of serum or no serum for a period of 24 or 48 hours followed by addition of 3H-thymidine (New England Nuclear) to the wells at 0.5 mCi/well. Four to six hours after the addition of the isotope label, media were removed from the wells, and 2.5% Trypsin/EDTA was added to each well to detach the cells from the culture surface. Cells were then harvested on a TOMTEC cell harvester to a filter, and the radioactivity incorporated in the cells counted on a BetaPlate counter.
[0188] Results
[0189] The sequence of AK647.
[0190] The open reading frame of AK647, spanning nucleotides 194 to 1537 of SEQ ID NO:1, encodes a polypeptide of 448 amino acids (SEQ ID NO:2). The analysis of the polypeptide sequence reveals a signal peptide preceding the mature protein, which was consistent with its selection from the signal sequence trap. The mature protein consists of six EGF domains, five of which are contiguous. There is a region of nonhomologous sequence between the first and second EGF domains, and there is an extended nonhomologous region following the sixth EGF domain.
[0191] The protein sequence analysis algorithm “SigCleave” predicts signal peptide cleavage occurring at two different peptide bonds (i.e., between Ala23 and Gln24 and between Ala25 and Gln26) which indicates the potential existence of two forms of the mature protein. The protein has two N-linked glycosylation sites featured by Asn-Xxx-Ser/Thr, where Xxx could be any of the naturally occurring amino acids except proline. There are numerous serine and threonine residues in the protein that are potential O-linked glycosylation sites.
[0192] Transient Expression of AK647 in COS Cells.
[0193] COS-1 cells were transiently transfected with pNOT vector containing the entire AK647 cDNA according to standard lipofectamine protocol. Twenty-four hours before harvesting the conditioned medium, the transfected cells were washed once with DME and then cultured in DME medium containing 35 S labeled methionine/cysteine supplement and different amounts of bovine serum. The harvested conditioned media were analyzed by SDS-PAGE and autoradiography. As can be seen in FIG. 3A, AK647 appears as a 55 to 58 kDa band on the 12% SDS-PAGE, compared to a 47 kDa species predicted for the mature polypeptide. This indicates the molecule is modified post-translationally resulting in the increased molecular weight. It also can be noted in FIG. 3A that the amount of serum in the culture medium has a minimal effect on the level of AK647 expression. Conditioned media from large-scale AK647 or mock transfections were harvested 24 hours after changing to serum-free DME. They were then concentrated and their expression levels assessed on SDS-PAGE by Coomassie blue staining (FIG. 3B).
[0194] E. coli Expression of AK647.
[0195] The DNA segment of mature AK647-QA was fused in-frame to a linker sequence encoding the GroHEK2 chimera whose amino acid sequence is MAAKDVKSGHHHHHHGDDDDK. The DNA fusion was subcloned into expression vector pAL985 for tryptophan-induced expression in E. coli strain GI934 (FIG. 5A). The expressed fusion protein GroHEK2/AK647-QA, in the form of inclusion bodies, was recovered from the insoluble fractions of the bacterial lysate (FIG. 5B). The inclusion bodies were washed with PBS containing 1 M NaCl, solubilized with SDS gel sample buffer and then loaded on a preparative SDS-PAGE for purification. The band corresponding to GroHEK2/AK647-QA (50 kDa) was electro-eluted, and its N-terminal sequence confirmed by Edman sequencing on a ProciseHT (Applied Biosystems, Inc. CA). The purified protein was used for animal injection to raise polyclonal and monoclonal antibodies against AK647.
[0196] Morphological Changes/Viability.
[0197] Cells treated over a 24 hour period with serum-free medium containing 10% AK647 COS conditioned medium display a marked change in morphology. Detachment from the substrate, loss of cellular volume and “rounding up” of cells was prevalent. Those cells which were still attached to the substrate form tight clusters with one another (FIG. 6A), leaving large, open spaces on the culture plate. Cells at this stage retain the ability to exclude the vital dye, Trypan blue, to a similar extent as cells treated for 24 hours with serum free medium. In contrast, the same cells treated over a 24 hour period with serum free medium alone or serum-free medium containing 10% mock (vector) COS conditioned medium (FIG. 6B), display no observable changes in morphology.
[0198] Aortic Cell Proliferation Measured by Mitochondrial Dehydrogenase Activity.
[0199] The effects of AK647 conditioned medium, harvested from transiently transfected COS cells, on PDGF stimulated human and rat aortic smooth muscle cells are shown in FIG. 6. Following incubation for 24 hours in culture media of various composition, CRL 1444 cells exhibit different levels of proliferation as determined by mitochondrial dehydrogenase activity (absorbance at 570 nm of the chromophore released during the assay). The cells grown in DME show an activity at about 0.275 while for cells in the same medium supplemented with 10% FBS, the activity was about 0.43. Cells grown in serum-free medium containing increasing concentrations of PDGF show increasing activity, while the activity from the cells grown in DME containing 10% AK647 conditioned medium concentrate was suppressed to 0.246 even in the presence of 100 ng/ml of PDGF, as compared with an activity of 0.44 for DME containing 100 ng/ml of PDGF. The enzyme activity values for all the culturing conditions tested are the average of eight duplicate samples, and the difference between the results from DME containing 100 ng/ml PDGF with and without AK647 are highly significant (p<0.0001).
[0200] Over the course of the treatment period (24 hours), CRL 1444 cells generally proliferate by approximately 2 fold over their seeding density, while those incubated in serum free medium proliferate at a slower rate, approximately equal to 1.5 fold of the seeding density. Those cells incubated in DME augmented with PDGF proliferate at a greater rate than those in the serum free condition. At 100 ng/ml PDGF, the proliferation was approximately equal to that seen in normal growth medium. By contrast, those cells treated with 100 ng/ml PDGF in DME containing 10% AK647, showed little or no proliferation over their seeding density. These observations closely reflect the absorbance readings.
[0201] Aortic Cell Proliferation Measured by 3H-Thymidine Incorporation.
[0202] Proliferation of rat aortic vascular smooth muscle cells CRL1444 in response to treatment with PDGF-BB was significantly inhibited upon incubation with AK647 conditioned medium. Incubation of this cell type for a period of 24 hours in serum free medium containing 1 ng/ml PDGF gives a 3 H-Thymidine uptake value of 2.5×10 4 +/−1.0×10 4 cpm (FIG. 8), while incubation in medium containing both 1 ng/ml PDGF and 10% AK647 conditioned medium results in a reading of 3.0×10 3 +/−1.0×10 3 cpm. Treatment with serum free medium containing 1 ng/ml PDGF and 10% mock (vector) conditioned medium gives a result of 2.1×10 4 +/−1.0×10 4 cpm. The same experiment carried out in the presence of 10 ng/ml PDGF gives a 3 H-Thymidine uptake value of 3.5×10 4 +/−0.9×10 4 cpm, 4×10 3 +/−0.8×10 3 and 3.3×10 4 +/−0.9×10 4 , respectively. When the same experiment was performed in the presence of 50 ng/ml PDGF, the cpm values were 3.8×10 4 +/−1.0×10 4 , 5×10 3 +/−1.0×10 3 , and 3.7×10 4 +/−1.2×10 4 , respectively.
[0203] The inhibitory effect of AK647 was directly related to its concentration in the culture medium (FIG. 9). Incubation over 24 hours with the conditioned medium containing an estimated concentration of 1-2 ng/ml AK647, significantly inhibits the proliferation of CRL 1444 rat aortic smooth muscle cells, even in the presence of 10 ng/ml PDGF. This inhibition of smooth muscle cell growth was also observed, to a similar degree, with rat aortic smooth muscle cell lines CRL 2018 and CRL 1476 (FIGS. 10 and 11).
[0204] Smooth muscle cells cultured with mock conditioned medium also shows some inhibition of PDGF induced proliferation, but to a significantly lesser degree than that seen with AK647 conditioned media, and only at very high concentrations.
[0205] Discussion
[0206] The protein AK647 is highly conserved among mouse, rat, chicken and humans. Many growth/differentiation molecules are found to be highly conserved during evolution. The spatial and temporal distribution of the AK647 molecule indicates that it acts as a modulator of smooth muscle cells in vasculogenesis during embryonic development.
[0207] The primary structure of AK647 consists of multiple EGF domains. This type of domain organization has been found in many other growth factors. The existence of T16, a homolog of AK647 indicates the possibility that there is a family of proteins like AK647, each having a specific tissue expression pattern and target cell type.
[0208] The effect of AK647 to down-regulate the growth of smooth muscle cells is most apparent in a culture medium containing no bovine serum supplement. The simplest explanation for this phenomenon is that bovine serum contains a significant amount of PDGF which is released from platelets due to thrombosis during its preparation. PDGF is shown in our experiments, and those of others, to be a mitogen in vitro that stimulates smooth muscle cell growth. However, this is not a problem for the in vivo use of AK647 to suppress smooth muscle cell growth, because the normal serum content of PDGF is low.
[0209] When AK647 is added to aortic cell line cultures, the cell growth becomes inhibited and the attachment to the culture plate is weakened. This initial reaction from the cells resembles the effects of proteases that digest extracellular matrices. Longer incubation of AK647 induces cell morphological changes consistent with apoptosis or programmed cell death. There are many examples where apoptosis serves as a mechanism for morphogenesis. The exact mechanism of growth inhibition can be studied in a number of ways. For example, experiments designed to examine apoptosis such as TUNEL assays and DNA laddering assays can be used to address the potential apoptosis phenomenon; GeneChip technology can be used to examine the intracellular signal transduction pathways; and the known smooth muscle cell stimulating factors can be tested for their neutralizing effects on AK647.
[0210] A factor that inhibits vascular smooth muscle cell growth is very desirable in modem cardiology. The application of such a compound can reduce the formation of arterial lesions, reduce the rate of vessel wall thickening, and prevent restenosis after PTCA. The effects of AK647 on aortic cell line cultures demonstrate that it is capable of inhibiting smooth muscle cell growth, thus indicating its usefulness clinically for treating a number of cardiovascular conditions (including, for example, systemic delivery for atherosclerosis, local application in the balloon angioplasty, etc.)
[0211] Administration and Dosing
[0212] A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
[0213] A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
[0214] The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
[0215] The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are incorporated herein by reference.
[0216] As used herein, the term “therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
[0217] In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
[0218] Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
[0219] When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
[0220] When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
[0221] The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
[0222] The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
[0223] Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
[0224] For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.
[0225] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
[0226] Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
[0227] A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
[0228] In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).
[0229] The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
[0230] The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
[0231] Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucteotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
[0232] Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
[0233] Patent and literature references cited herein are incorporated by reference as if fully set forth.



1



2




2362 base pairs
nucleic acid
double
linear


cDNA

1

TAGCTTGGCA CGAGGGGACC CCGGCGCTCT CCCCGTGTCC TCTCCACGAC TCGCTCGGCC 60

CCTCTGGAAT AAAACACCCG CGAGCCCCGA GGGCCCAGAG GAGGCCGACG TGCCCGAGCT 120

CCTCCGGGGG TCCCGCCCGC GAGCTTTCTT CTCGCCTTCG CATCTCCTCC TCGCGCGTCT 180

TGGACATGCC AGGAATAAAA AGGATACTCA CTGTTACCAT TCTGGCTCTC TGTCTTCCAA 240

GCCCTGGGAA TGCACAGGCA CAGTGCACGA ATGGCTTTGA CCTGGATCGC CAGTCAGGAC 300

AGTGTTTAGA TATTGATGAA TGCCGAACCA TCCCCGAGGC CTGCCGAGGA GACATGATGT 360

GTGTTAACCA AAATGGCGGG TATTTATGCA TTCCCCGGAC AAACCCTGTG TATCGAGGGC 420

CCTACTCGAA CCCCTACTCG ACCCCCTACT CAGGTCCGTA CCCAGCAGCT GCCCCACCAC 480

TCTCAGCTCC AAACTATCCC ACGATCTCCA GGCCTCTTAT ATGCCGCTTT GGATACCAGA 540

TGGATGAAAG CAACCAATGT GTGGATGTGG ACGAGTGTGC AACAGATTCC CACCAGTGCA 600

ACCCCACCCA GATCTGCATC AATACTGAAG GCGGGTACAC CTGCTCCTGC ACCGACGGAT 660

ATTGGCTTCT GGAAGGCCAG TGCTTAGACA TTGATGAATG TCGCTATGGT TACTGCCAGC 720

AGCTCTGTGC GAATGTTCCT GGATCCTATT CTTGTACATG CAACCCTGGT TTTACCCTCA 780

ATGAGGATGG AAGGTCTTGC CAAGATGTGA ACGAGTGTGC CACCGAGAAC CCCTGCGTGC 840

AAACCTGCGT CAACACCTAC GGCTCTTTCA TCTGCCGCTG TGACCCAGGA TATGAACTTG 900

AGGAAGATGG CGTTCATTGC AGTGATATGG ACGAGTGCAG CTTCTCTGAG TTCCTCTGCC 960

AACATGAGTG TGTGAACCAG CCCGGCACAT ACTTCTGCTC CTGCCCTCCA GGCTACATCC 1020

TGCTGGATGA CAACCGAAGC TGCCAAGACA TCAACGAATG TGAGCACAGG AACCACACGT 1080

GCAACCTGCA GCAGACGTGC TACAATTTAC AAGGGGGCTT CAAATGCATC GACCCCATCC 1140

GCTGTGAGGA GCCTTATCTG AGGATCAGTG ATAACCGCTG TATGTGTCCT GCTGAGAACC 1200

CTGGCTGCAG AGACCAGCCC TTTACCATCT TGTACCGGGA CATGGACGTG GTGTCAGGAC 1260

GCTCCGTTCC CGCTGACATC TTCCAAATGC AAGCCACGAC CCGCTACCCT GGGGCCTATT 1320

ACATTTTCCA GATCAAATCT GGGAATGAGG GCAGAGAATT TTACATGCGG CAAACGGGCC 1380

CCATCAGTGC CACCCTGGTG ATGACACGCC CCATCAAAGG GCCCCGGGAA ATCCAGCTGG 1440

ACTTGGAAAT GATCACTGTC AACACTGTCA TCAACTTCAG AGGCAGCTCC GTGATCCGAC 1500

TGCGGATATA TGTGTCGCAG TACCCATTCT GAGCCTCGGG CTGGAGCCTC CGACGCTGCC 1560

TCTCATTGGC ACCAAGGGAC AGGAGAAGAG AGGAAATAAC AGAGAGAATG AGAGCGACAC 1620

AGACGTTAGG CATTTCCTGC TGAACGTTTC CCCGAAGAGT CAGCCCCGAC TTCCTGACTC 1680

TCACCTGTAC TATTGCAGAC CTGTCACCCT GCAGGACTTG CCACCCCCAG TTCCTATGAT 1740

ACAGTTATCA AAAAGTATTA TCATTGCTCC CCTGATAGAA GATTGTTGGT GAATTTTCAA 1800

GGCCTTCAGT TTATTTCCAC TATTTTCAAA GAAAATAGAT TAGGTTTGCG GGGGTCTGAG 1860

TCTATGTTCA AAGACTGTGA ACAGCTTGCT GTCACTTCTT CACCTCTTCC ACTCCTTCTC 1920

TCACTGTGTT ACTGCTTTGC AAAGACCCGG GAGCTGGCGG GGAACCCTGG GAGTAGCTAG 1980

TTTGCTTTTT GCGTACACAG AGAAGGCTAT GTAAACAAAC CACAGCAGGA TCGAAGGGTT 2040

TTTAGAGAAT GTGTTTCAAA ACCATGCCTG GTATTTTCAA CCATAAAAGA AGTTTCAGTT 2100

GTCCTTAAAT TTGTATAACG GTTTAATTCT GTCTTGTTCA TTTTGAGTAT TTTTAAAAAA 2160

TATGTCGTAG AATTCCTTCG AAAGGCCTTC AGACACATGC TATGTTCTGT CTTCCCAAAC 2220

CCAGTCTCCT CTCCATTTTA GCCCAGTGTT TTCTTTGAGG ACCCCTTAAT CTTGCTTTCT 2280

TTAGAATTTT TACCCAATTG GATTGGAATG CAGAGGTCTC CAAACTGATT AAATATTTGA 2340

AGAGAAAAAA AAAAAAAAAA AA 2362






448 amino acids
amino acid
Not Relevant
linear


protein

2

Met Pro Gly Ile Lys Arg Ile Leu Thr Val Thr Ile Leu Ala Leu Cys
1 5 10 15

Leu Pro Ser Pro Gly Asn Ala Gln Ala Gln Cys Thr Asn Gly Phe Asp
20 25 30

Leu Asp Arg Gln Ser Gly Gln Cys Leu Asp Ile Asp Glu Cys Arg Thr
35 40 45

Ile Pro Glu Ala Cys Arg Gly Asp Met Met Cys Val Asn Gln Asn Gly
50 55 60

Gly Tyr Leu Cys Ile Pro Arg Thr Asn Pro Val Tyr Arg Gly Pro Tyr
65 70 75 80

Ser Asn Pro Tyr Ser Thr Pro Tyr Ser Gly Pro Tyr Pro Ala Ala Ala
85 90 95

Pro Pro Leu Ser Ala Pro Asn Tyr Pro Thr Ile Ser Arg Pro Leu Ile
100 105 110

Cys Arg Phe Gly Tyr Gln Met Asp Glu Ser Asn Gln Cys Val Asp Val
115 120 125

Asp Glu Cys Ala Thr Asp Ser His Gln Cys Asn Pro Thr Gln Ile Cys
130 135 140

Ile Asn Thr Glu Gly Gly Tyr Thr Cys Ser Cys Thr Asp Gly Tyr Trp
145 150 155 160

Leu Leu Glu Gly Gln Cys Leu Asp Ile Asp Glu Cys Arg Tyr Gly Tyr
165 170 175

Cys Gln Gln Leu Cys Ala Asn Val Pro Gly Ser Tyr Ser Cys Thr Cys
180 185 190

Asn Pro Gly Phe Thr Leu Asn Glu Asp Gly Arg Ser Cys Gln Asp Val
195 200 205

Asn Glu Cys Ala Thr Glu Asn Pro Cys Val Gln Thr Cys Val Asn Thr
210 215 220

Tyr Gly Ser Phe Ile Cys Arg Cys Asp Pro Gly Tyr Glu Leu Glu Glu
225 230 235 240

Asp Gly Val His Cys Ser Asp Met Asp Glu Cys Ser Phe Ser Glu Phe
245 250 255

Leu Cys Gln His Glu Cys Val Asn Gln Pro Gly Thr Tyr Phe Cys Ser
260 265 270

Cys Pro Pro Gly Tyr Ile Leu Leu Asp Asp Asn Arg Ser Cys Gln Asp
275 280 285

Ile Asn Glu Cys Glu His Arg Asn His Thr Cys Asn Leu Gln Gln Thr
290 295 300

Cys Tyr Asn Leu Gln Gly Gly Phe Lys Cys Ile Asp Pro Ile Arg Cys
305 310 315 320

Glu Glu Pro Tyr Leu Arg Ile Ser Asp Asn Arg Cys Met Cys Pro Ala
325 330 335

Glu Asn Pro Gly Cys Arg Asp Gln Pro Phe Thr Ile Leu Tyr Arg Asp
340 345 350

Met Asp Val Val Ser Gly Arg Ser Val Pro Ala Asp Ile Phe Gln Met
355 360 365

Gln Ala Thr Thr Arg Tyr Pro Gly Ala Tyr Tyr Ile Phe Gln Ile Lys
370 375 380

Ser Gly Asn Glu Gly Arg Glu Phe Tyr Met Arg Gln Thr Gly Pro Ile
385 390 395 400

Ser Ala Thr Leu Val Met Thr Arg Pro Ile Lys Gly Pro Arg Glu Ile
405 410 415

Gln Leu Asp Leu Glu Met Ile Thr Val Asn Thr Val Ile Asn Phe Arg
420 425 430

Gly Ser Ser Val Ile Arg Leu Arg Ile Tyr Val Ser Gln Tyr Pro Phe
435 440 445