(en)The present invention relates to anti-IL-1 beta binding members and in particular to monovalent high potency IL-1 beta-binding antibody fragments being highly stable and soluble. Such binding members may be used in the treatment of inflammatory and other diseases as well as in diagnostics. Also provided are related nucleic acids, vectors, cells, and compositions.

1.ApplicationNumber: US-201615202982-A
1.PublishNumber: US-2017051054-A1
2.Date Publish: 20170223
3.Inventor: GRABULOVSKI STEFANIE
KRETZSCHMAR TITUS
SCHMITT SIMONE
SHAMSHIEV ABDIJAPAR
SCHAFER THORSTEN ALEXANDER

4.Inventor Harmonized: GRABULOVSKI STEFANIE(CH)
KRETZSCHMAR TITUS(CH)
SCHMITT SIMONE(CH)
SHAMSHIEV ABDIJAPAR(CH)
SCHAFER THORSTEN ALEXANDER(DE)

5.Country: US
6.Claims:
(en)The present invention relates to anti-IL-1 beta binding members and in particular to monovalent high potency IL-1 beta-binding antibody fragments being highly stable and soluble. Such binding members may be used in the treatment of inflammatory and other diseases as well as in diagnostics. Also provided are related nucleic acids, vectors, cells, and compositions.

7.Description:
(en)RELATED APPLICATIONS
This application is a divisional of application Ser. No. 14/072,165, filed on Nov. 5, 2013, which claims the benefit of U.S. Provisional Application No. 61/722,532, filed on Nov. 5, 2012. This application claims priority under 35 U.S.C, §119 or 365 to EP Application No. 12007503.1, filed Nov. 5, 2012. The entire teachings of the above applications are incorporated herein by reference.


The invention relates to humanized anti-IL-1 beta antibodies, in particular monovalent, highly potent anti-IL-1 beta antibody fragments. The invention also relates to nucleic acids encoding such antibodies, vectors, host cells containing such sequences, pharmaceutical and diagnostic compositions comprising the antibodies or nucleic acids, and uses thereof.
BACKGROUND OF THE INVENTION
Interleukin-1 beta (IL-1 beta) is a pro-inflammatory cytokine which is produced as a precursor by activated macrophages. Upon proteolytic cleavage, signal transduction is initiated by binding of the active form to the IL-1 receptor type I (IL-1R1) which in turn associates with the transmembrane IL-1 receptor accessory protein (IL-1RAP). The formed complex is competent of signal transduction. Being a key mediator in the inflammatory response, the cytokine affects a number of cellular activities such as cell proliferation, differentiation, and apoptosis. Therefore, IL-1 beta has been considered an important target for a variety of pharmaceuticals.
There is a need in the art for antibodies with high therapeutic potential against human IL-1 beta. For being therapeutically successful, it is important that such antibody displays desirable biophysical and biochemical characteristics. For example, since the target IL-1 beta is a highly efficient interleukin that is potent at very low concentrations and thus needs to be comprehensively blocked, such antibody needs to be highly potent as well as highly stable and soluble.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides a monovalent antibody fragment directed against IL-1 beta having a potency of lower than 50 picomolar (pM), as determined by the half-maximum inhibitory concentration IC 50 with regard to inhibiting the biological effect of human IL-1 beta.
Monovalent antibody fragments, whether being humanized or not, having potency values in the pM-range are particular and not routinely obtained. In addition and typically, an antibody loses affinity to its target upon humanization when compared to the parent non-human antibody. It is therefore a challenge to humanize an antibody such that the affinity parameters are close or equal to the parent antibody. This is particularly true for monovalent antibody fragments which comprise only one variable light and heavy chain, and therefore bind to the target less strongly than bivalent antibodies displaying two light and heavy chains.
Moreover, when converting a full-length antibody into a smaller fragment, its potency usually becomes diminished. This is not only due to the accompanying change of valency (for example, the antibody fragment might only be monovalent whereas a full-length immunoglobulin is bi- or multivalent) but may also be caused by steric reasons.
A potent antibody is particularly useful since it allows administering lower amounts of drug to the patient, thereby decreasing the overall costs of treatment. In addition, a more complete neutralization of the molecular target of the disease is rendered feasible.
Moreover, different application routes in animal models as well as in human therapy can be envisioned when applying highest potency antibodies. For example, as to topical drugs, although delivery may be limited due to the barrier function of the epithelial layer, efficacy of treatment is restored by the high potency of the limited quantity of drug molecules that passes this physiological barrier.
Often, the high amount of a less potent drug, which needs to be administered to achieve similar pharmacodynamic effects, translates into much higher intravenous or subcutaneous application volumes than with a more potent drug. Such higher application volumes are a disadvantage for use in animals and humans for two reasons; firstly, the impracticality of treating patients with a high volume of drug, and secondly, because antibodies are very expensive per unit of mass.
Therefore, lower quantities of antibody used for treatment translate into lower production costs of the drug. In particular, antibody fragments are suitable for production using, e.g., bacterial or yeast culture systems, which are of comparatively lower cost than mammalian expression systems typically used for the production of full-length immunoglobulins such as IgG. The combination of smaller quantities of drug to be administered and cheaper manufacturing processes opens the possibility of more cost-efficient medicines per patient. Thus, a larger number of patients may benefit from such drug.
Stability and solubility parameters are other factors crucial for providing a viable medicament. The more stable and soluble an antibody drug, the smaller the volume of administration and the longer the shelf half-life time. The antibodies provided herein are highly stable and soluble, i.e., they remain monomeric for prolonged periods of time and also at high concentrations.
In one aspect, an antibody is provided, in particular the monovalent antibody fragment above, comprising:
(a) at least one of the variable heavy chain (VH) complementarity determining region (CDR) sequences CDR-H1, CDR-H2 or CDR-H3 as set forth in SEQ ID Nos.: 1, 2 and 3, respectively, or variants thereof; and/or
(b) at least one of the variable light chain (VL) CDR sequences CDR-L1, CDR-L2 or CDR-L3 as set forth in SEQ ID Nos.: 4, 5, and 6, respectively, or variants thereof.
In another embodiment, the antibody, and in particular said monovalent antibody fragment, comprises:
(a) at least one of the variable heavy chain (VH) complementarity determining region (CDR) sequences CDR-H1, CDR-H2 or CDR-H3 as set forth in SEQ ID Nos.: 155, 156 and 157, respectively, or variants thereof; and/or
(b) at least one of the variable light chain (VL) CDR sequences CDR-L1, CDR-L2 or CDR-L3
(i) as set forth in SEQ ID Nos.: 158, 159 and 160, respectively, or variants thereof, or
(ii) as set forth in SEQ ID Nos.: 161, 162 and 163, respectively, or variants thereof.
In some embodiments, the antibody comprises:
(a) a VH having at least 85% identity to a sequence selected from the group consisting of SEQ ID No.: 7 and SEQ ID No.: 146; and/or
(b) a VL having at least 85% identity to a sequence selected from the group consisting of SEQ ID No.: 8, SEQ ID No.: 136 and SEQ ID No.: 145.
The antibody can comprise a linker sequence, being or derived from SEQ ID No.: 9. In some embodiments, such antibody is an antibody fragment having at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 10, SEQ ID No.: 73 and SEQ ID No.: 82.
In one aspect the invention provides binding members that bind to IL-1 beta and compete for binding with the antibodies described herein. Said binding member can be monovalent or multivalent. A preferred multivalent binding member is bivalent. A multivalent binding member can be bispecific.
In one aspect, the invention provides an isolated nucleic acid sequence encoding the antibody or the binding member disclosed herein.
In one aspect, a vector comprising said nucleic acid sequence is provided.
In one aspect, the invention provides a host cell comprising the nucleic acid sequence above or the vector above.
In one aspect, a composition comprising the antibody above, the binding member above, the nucleic acid sequence above, the vector above or the host cell above; and further a suitable carrier, diluent or excipient. The composition is preferably a pharmaceutical composition, comprising a pharmaceutically acceptable carrier, diluent or excipient. Such pharmaceutical composition is preferably in a form suitable for topical, intradermal, transdermal, intravenous, subcutaneous, intramuscular, parenteral, sublingual, buccal, oral, nasal, intranasal, rectal, local or ocular administration.
Further provided is a method of treating an IL-1 beta-mediated disease comprising administering to a subject in need thereof the pharmaceutical composition above.
Also provided is the antibody above, the binding member above, the nucleic acid sequence above, the vector above or the host cell disclosed herein:
(i) for use in the treatment of an IL-1 beta-mediated disease;
(ii) for use in diagnostics;
(iii) for use in cosmetics; and/or
(iv) for detection purposes.
In still another aspect, the invention provides a method of producing the antibody or the binding member described herein, either comprising: (i) the steps of cultivating the host cell above and recovering and purifying the antibody fragment or the binding member, respectively; or (ii) the use of a cell-free system. Additionally or alternatively, the method can comprise at least one step of chemical protein synthesis.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the results of an ELISA to determine binding of DLX2323 to recombinant human (rh) IL-1 beta at various concentrations.
FIG. 2 is a graph depicting the results of DLX2323 binding to natural human IL-11 beta in comparison to binding to rhIL-1 beta. Natural human IL-1 beta was derived from supernatant of activated THP-1 cells.
FIGS. 3A and 3B show comparisons of DLX2323 with several commercially available IL-1 beta inhibitors for neutralization of rhIL-1 beta in a human fibroblast assay from two independent experiments. FIG. 3A shows the results for DLX2323 and MAB201, and FIG. 3B summarizes data for DLX2323, rhIL-1 receptor antagonist (ra) and canakinumnab (ILARIS®).
FIG. 4 shows the in vive efficacy of DLX2323 in a human IL-1 beta-induced mouse inflammation model.
FIG. 5 illustrates the definition of CDR-H1 as used herein.
FIG. 6 illustrates the results of an ELISA assay wherein cleared cell lysates of DLX2323 variants expressed in E. coli cells bind bound to coated rhIL-1 beta.



DETAILED DESCRIPTION
So that the invention may be more readily understood, certain terms are first defined. Unless otherwise defined within the specification, all technical and scientific terms used herein have their art-recognized meaning. Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and not intended to be limiting.
Within the scope of the present invention, the term “antibody” refers to full-length immunoglobulins as well as to fragments thereof. Such full-length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies.
“Antibody fragments” comprise portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fe region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. HOLLIGER. P. and Hudson, J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p. 1126-1136). Examples of antibody fragments, without being limited to, include scFv, Fab, Fv, Fab′, F(ab′) 2 fragments, dAb, VHH, nanobodles, V(NAR) or minimal recognition units.
“Single chain variable fragments” or “single chain antibodies” or “scFv” are one type of antibody fragments. scFv are fusion proteins comprising the VH and VL of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full-length immunoglobulins, but retain the specificity of the original immunoglobulin.
A “binding member” as used herein refers to full-length immunoglobulins, antibody fragments, non-antibody scaffolds, and/or other binding compounds. Such binding member can be monovalent or multivalent, i.e. having one or more antigen binding sites. Non-limiting examples of monovalent binding members include scFv, Fab fragments, dAb, VHH, DARPins, affilins and nanobodies. A multivalent binding member can have two, three, four or more antigen binding sites whereby one or more different antigens can be recognized. Full-length immunoglobulins, F(ab′) 2 fragments, bis-scFv and diabodies are non-limiting examples of multivalent binding members; in said exemplary multivalent binding members, two binding sites are present, i.e. the binding member is bivalent.
In one embodiment, the multivalent binding member is bispecific, i.e. the binding member is directed against two different targets or two different target sites on one target molecule. Bispecific antibodies are, e.g., reviewed in MÜLLER, D. and Kontermann, R. E. Bispecific antibodies. Edited by DÜBEL, S. Weinheim: Wiley-VCH, 2007. ISBN 3527314539. p. 345-378. In another embodiment, the multivalent binding member comprises more than two, e.g., three Or four different binding sites for three or four, respectively, different antigens. Such binding member is multivalent and multispecific in particular tri- or tetra-specific, respectively.
“Non-antibody scaffolds” are antigen-binding polypeptides which are e.g. described in FIELDER, M. and Skerra, A. Non-antibody scaffolds. Edited by DÜBEL, S. Weinheim: Wiley-VCH, 2007. ISBN 3527314539. p. 467-500; or GILBRETH, R. N. and Koide, S. Structural insights for engineering binding proteins based on nonantibody scaffolds. Current Opinion in Structural Biology 2012, vol. 22, p. 413-420. Non-limiting examples include affibodies, affilin molecules, AdNectin, Anticalin, DARPins, Knottin, Kunitz-type domain, Avimer, Tetranectin and trans-body.
“Binding compounds” are chemical or biological molecules that bind to a target and that are not belonging to the class of full-length immunoglobulins, antibody fragments and non-antibody scaffolds as defined above. Examples of binding compounds, without being limited to, include macrolides (GUNDLURU, M. K. et al. Design, synthesis and initial biological evaluation of a novel pladienolide analog scaffold. Med chem comm. 2011, vol. 2, p. 904-908; PATERSON, L. et al. Total synthesis and biological evaluation of a series of macrocyclic hybrids and analogies of the antimitotic natural products dictyostatin, discodermolide and taxol. Chem Asian J. 2011, vol. 6, p. 459-473; MORITA, H. et al. Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase. PNAS 2011, vol. 108, p. 13504-13509), molecular imprinted polymers (HOSHINO, Y. et al. Recognition, neutralization and clearance of target peptides in the blood stream of living mice by molecular imprinted polymer nanoparticles: a plastic antibody. Journal of the American Chemical Society, 2010, vol. 19, p. 664-6645), aptamers (STREHLITZ, B., et al. Aptamers for pharmaceuticals and their application in environmental analytics. Bioanalytical reviews 2012, vol. 4, p. 1-30; YE, M. et al. Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine. International Journal of Molecular Sciences 2012, vol. 13, p. 3341-3353), Spiegelmers (see e.g., MAASCH, C. et al. Polyethylenimine-Polyplexes of Spiegelmer NOX-A50 directed against intracellular high mobility group protein A1 (HMGA1) reduce tumor growth in vivo. JBC 2010, vol. 285, p. 40012-40018), or peptides (cyclic or linear; see, e.g., GOULD, A. et al. Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery. Curr Pharm Des. 2011, vol. 17, p. 4294-4307).
The “IC 50 ” or “half-maximum inhibitory concentration” is a measure of antagonist drug potency and describes quantitatively the effectiveness of a compound to inhibit a biological or biochemical function. This measure indicates how much of the compound is needed to inhibit by 50% a certain biological or biochemical process. Although no direct indicator of affinity, both values are correlated and can be determined via the Cheng-Prusoff equation (CHENG Y. and Prusoff W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (150) of an enzymatic reaction. Biochemical Pharmacology 1973, vol. 22, p. 3099-3108; RAMMES, G., et al. Identification of a domain which affects kinetics and antagonistic potency of clozapine at 5-HT3 receptors. PLOS one 2009, vol. 4, p. 1-14; ZHEN, J., et al. Concentration of receptor and ligand revisited in a modified receptor binding protocol for high-affinity radioligands: [ 3 H] spiperone binding to D 2 and D 3 dopamine receptors. Journal of Neuroscience Methods 2010, vol. 188, p. 32-38).
The term “IL-1 beta specific binding” as used herein describes that a binding member binds to IL-1 beta with higher affinity than to a structurally different antigen which does not comprise the IL-1 beta epitope to which the anti-IL-1 beta binding member binds. Specific binding is reflected by a dissociation equilibrium constant (K D ) of lower than 1 micromolar. This constant can be determined, e.g. using Quartz Crystal Microbalance (QCM) in an Attana instrument, or Surface Plasmon Resonance (SPR) technology in a BIACORE instrument.
As used herein, “IL-1 beta” refers to the molecule as described in, e.g., Dinarello C. A., Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nature reviews 2012, vol. 11, p. 633-652. “hIL-1 beta” as used herein refers to human IL-1 beta. “rIL-1 beta” refers to recombinant IL-1 beta. Recombinant IL-1 beta may or may not have an amino terminal methionine residue, depending upon the method by which it is prepared. “rhIL-” beta refers to recombinant human IL-1 beta. rhIL-1 beta may, e.g., be obtained from Peprotech, USA, cat. no. 200-01B. IL-1 beta may also be obtained by isolation from biological samples of human or non-human origin.
“Humanized” antibodies refer to antibodies comprising one or more, typically all six CDR regions of a non-human parent antibody or variants thereof, and of which the framework is, e.g., (i) a human framework, potentially comprising one or more framework residues of the non-human parent antibody, or (ii) a framework from a non-human antibody modified to increase similarity to naturally produced human frameworks. Methods of humanizing antibodies are known in the art, see e.g. LEGER, O. and Saldanha, J. Antibody Drug Discovery. Edited by WOOD, C. London: Imperial College Press, 2011. ISBN 1848166281. p. 1-23.
“Framework” (PR) refers to the scaffold of the variable immunoglobulin domain, either the variable light chain (VL) or variable heavy chain (VH), embedding the respective CDRs. A VL and/or VH framework typically comprises four framework sections, FR1, FR2, FR3 and FR4, flanking the CDR regions. Thus, as known in the art, a VL has the general structure: (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), whereas a VH has the general structure: (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H3)-(FR-H4).
“CDR” refers to the hypervariable regions of the antibody which mainly contribute to antigen binding. Typically, an antigen binding site comprises six CDRs, embedded into a framework scaffold. Herein, the CDRs of the VL are referred to as CDR-L1, CDR-L2 and CDR-L3 whereas the CDRs of the VH are referred to as CDR-H1. CDR-H2 and CDR-H3. These can be identified as described in KABAT, E. A., et al. Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991, p. 91-3242. CDR-H1 as used herein, however, differs from the Kabat definition in that it starts with position 27 and ends prior to position 36 (see FIG. 5 for illustration).
As used herein, the numbering system to identify amino acid residue positions in the VH and VL of the antibody corresponds to the “AHo”-system described by HONEOGER, A. and Plückthun, A. Yet another numbering scheme for immunoglobulin variable domains: An automatic modelling and analysis tool. Journal of Molecular Biology 2001, vol. 309, p. 657-670. The publication further provides conversion tables between the AHo and the Kabat system (KABAT, E. A., et al. Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p. 91-3242).
An “isolated” antibody or nucleic acid is one being identified and separated and/or recovered from at least one component of its natural environment.
The term “identity” as used herein refers to the sequence match between two proteins or nucleic acids. The protein or nucleic acid sequences to be compared are aligned to give maximum identity, for example using bioinformatics tools such as EMBOSS Needle (pair wise alignment). When the same position in the sequences to be compared is occupied by the same nucleobase or amino acid residue, then the respective molecules are identical at that very position. Accordingly, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of sequence positions are identical, then the identity is 60%. The percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (NEEDILEMAN, S. B. and Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453) which has been incorporated into EMBOSS Needle, using a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. Two molecules having the same primary amino acid or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition. In case of nucleic acids, for example, two molecules having the same sequence but different linkage components such as thiophosphate instead of phosphate are identical by this definition.
“Similar” protein sequences are those which, when aligned, share similar amino acid residues and most often, but not mandatorily, identical amino acid residues at the same positions of the sequences to be compared. Similar amino acid residues are grouped by chemical characteristics of the side chains into families. Said families are described below for “conservative amino acid substitutions”. The “percent similarity” between sequences is the number of positions that contain identical or similar residues at the same sequence positions of the sequences to be compared divided by the total number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions have identical amino acid residues and 2 out of 10 positions contain similar residues, then the sequences have 80% similarity. The similarity between two sequences can e.g. be determined using EMBOSS Needle.
A “variant” refers to an amino acid or nucleic acid sequence which differs from the parental sequence by virtue of addition (including insertions), deletion and/or substitution of one or more amino acid residues or nucleobases while retaining at least one desired activity of the parent sequence disclosed herein. In the case of antibodies such desired activity may include specific antigen binding. Similarly, a variant nucleic acid sequence may be modified when compared to the parent sequence by virtue of addition, deletion and/or substitution of one or more nucleobases, but the encoded antibody retains the desired activity as described above. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.
As used herein, the term “conservative modifications” refers to modifications that are physically, biologically, chemically or functionally similar to the corresponding reference, e.g., has a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Such conservative modifications include, but are not limited to, one or more nucleobases and amino acid substitutions, additions and deletions.
For example, conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, amino acid residues being non-essential with regard to binding to an antigen can be replaced with another amino acid residue from the same side chain family, e.g. serine may be substituted for threonine. Amino acid residues are usually divided into families based on common, similar side-chain properties, such as:
1. nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, methionine), 2. uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, proline, cysteine, tryptophan), 3. basic side chains (e.g., lysine, arginine, histidine, proline), 4. acidic side chains (e.g., aspartic acid, glutamic acid), 5. beta-branched side chains (e.g., threonine, valine, isoleucine) and 6. aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

A conservative substitution may also involve the use of a non-natural amino acid.


Non-conservative substitutions, i.e. exchanging members of one family against members of another family, may lead to substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the binding member, which may lead to a. significant drop in the binding activity, in particular if amino acids are affected that are essential for binding to the target molecule. A non-conservative substitution may also involve the use of a non-natural amino acid.
Conservative and non-conservative modifications can be introduced into parental binding members by a variety of standard techniques known in the art, such as combinatorial chemistry, site-directed DNA mutagenesis, PCR-mediated and/or cassette mutagenesis, peptide/protein chemical synthesis, chemical reaction specifically modifying reactive groups in the parental binding member. The variants can be tested by routine methods for their chemical, biological, biophysical and/or biochemical properties.
Nucleic acid hybridization reactions can be performed under conditions of different stringency. “Stringent conditions” are widely known and published in the art. Typically, during the hybridization reaction a SSC-based buffer can be used in which SSC is 0.15 M NaCl and 15 mM citrate buffer having a pH of 7.0. Increasing buffer concentrations and the presence of a denaturing agent increase the stringency of the hybridization step. For example, high stringency hybridization conditions can involve the use of: (i) 50% (vol/vol) formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C. with washes at 42° C. in 0.2×SSC and 0.1% SDS; (ii) 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (iii) 10% dextran sulfate, 2×SSC, and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. Additionally or alternatively, one, two or more washing steps using wash solutions of low ionic strength and high temperature can be included in the hybridization protocol using, for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.
Various aspects of the invention are described in further detail in the following subsections. It is understood that the various embodiments, preferences and ranges may be combined at will. Further, depending of the specific embodiment, selected definitions, embodiments or ranges may not apply.
In a first aspect, the invention provides a monovalent antibody fragment binding IL-1 beta which inhibits the biological effect of human IL-1 beta with an IC 50 of lower than 50 pM. Said IC 50 is preferably lower than about 40 pM, more preferably lower than about 30, 20, 10, 5, 4, 3, 2 or 1 pM.
Preferably, said monovalent antibody fragment has a molecular weight of about 50 kDa or lower, such as about 45 kDa, 40 kDa, 35 kDa or lower, preferably about 25 kDa, such as 23, 24, 25, 26, or 27 kDa.
In one aspect, the invention provides an antibody, comprising:
(a) at least one of the VII CDR sequences CDR-H1, CDR-112 or CDR-H3 as set forth in SEQ ID Nos.: 1, 2 and 3, respectively, or variants thereof, and/or
(b) at least one of the VL CDR sequences CDR-L1, CDR-L2 or CDR-L3 as set forth in SEQ ID Nos.: 4, 5, and 6, respectively, or variants thereof.
Such antibody may comprise:
(a) at least one of the VH CDR sequences CDR-H1, CDR-H2 or CDR-H3 as set forth in SEQ ID Nos.: 155, 156 and 157, respectively, or variants thereof; and/or
(b) at least one of the VL CDR sequences CDR-L1, CDR-L2 or CDR-L3,
(i) as set forth in SEQ ID Nos.: 158, 159 and 160, respectively, or variants thereof, or
(ii) as set forth in SEQ ID Nos.: 161, 162 and 163, respectively, or variants thereof.
Preferably, the antibody comprises at least the CDR-H3 of SEQ ID No.: 3 and the CDR-L3 of SEQ ID No.: 6 or SEQ ID No.: 157, or a variant thereof, respectively. Even more preferably, said antibody comprises all six CDRs of:
(i) SEQ ID Nos.: 1 to 6 or variants thereof;
(ii) SEQ ID Nos.: 155 to 160 or variants thereof; or
(iii) SEQ ID Nos.: 155 to 157 and SEQ ID Nos.: 161 to 163 or variants thereof.
Such antibody has a very high inhibitory potency against human IL-1 beta with an IC 50 of lower than 50 pM, more preferably lower than about 40 pM, 30, 20, 10, and even more preferably lower than 5 pM and most preferably about 1 pM and lower.
Preferably, the antibody has an inhibitory potency against human IL-1 beta with an IC 50 of at least 2 pM, more preferably of at least 1 pM.
The IC 50 can, e.g., be determined using a cell based potency assay. In one embodiment, the IC 50 value above is determined by inhibiting the IL-1 beta induced release of IL-6 from human fibroblasts. Such assay is based on the observation that fibroblasts stimulated with IL-1 beta release IL-6. In the presence of IL-1 beta inhibiting antibodies, the concentration of released IL-6 is reduced. In a preferred embodiment, Normal Human Dermal Fibroblasts (NHDF-Neo, e.g., obtainable from Lonza Walkersville USA, cat. no. CC-2509) cells are used. Upon incubation with a mixture of hIL-1 beta and the antibody of interest, supernatants are harvested and examined by an IL-6 ELISA such as the R&D Systems Human IL-6 DuoSet ELISA kit (R&D Systems, cat. no. DY206). In one embodiment, the assay is the IL-1 beta neutralization assay as described in example 3. Preferably, the IC 50 value is the mean value obtained of at least three independent repetitions of such assay.
The antibody described herein may be a full-length immunoglobulin or an antibody fragment, such as a Fab, Fab′, F(ab′) 2 , scFv, Fv fragment, nanobody, VHH or minimal recognition unit.
In a preferred embodiment the antibody and in particular the monovalent antibody fragment above is a scFv. The VH and VL domains can be connected in either orientation, VL-linker-VH or VH-linker-VL, by a flexible linker. In a preferred embodiment, the orientation is VL-linker-VH, i.e. the light chain variable region being at the N-terminal end and the heavy chain variable region being at the C-terminal end of the polypeptide.
The antibody is preferably humanized. Such humanized antibody may, e.g., comprise in the variable light chain the FR-L1 of SEQ ID No.: 18, the FR-L2 of SEQ ID No.: 19, the FR-L3 of SEQ ID No.: 20 and/or the FR-L4 of SEQ ID No.: 21 or variants thereof. Additionally or alternatively, the humanized antibody can comprise the heavy chain variable framework region FR-H1 of SEQ ID No.: 22, 26 or 30; the heavy chain variable framework region FR-H2 of SEQ ID No.: 23, 27 or 31; the heavy chain variable framework region FR-H3 of SEQ ID No.; 24, 28 or 32; and/or the heavy chain variable framework region FR-H4 of SEQ ID No.: 25, 29 or 33.
Thus, in a preferred embodiment, the antibody comprises the VII sequence of SEQ ID No.: 7 or SEQ ID No.: 146, or a variant thereof respectively. Such variant has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity to SEQ ID No.: 7 or SEQ ID No.: 146. Examples of such variant VH sequences include, without being limited to, SEQ ID No.: 121, SEQ ID No.: 122, SEQ ID No.: 124, SEQ ID No.: 126, SEQ ID No.: 128, SEQ ID No.: 130, SEQ ID No.: 132, SEQ ID No.: 134, SEQ ID No.: 142, SEQ ID No.: 144, SEQ ID No.: 146, SEQ ID No.: 148, SEQ ID No.: 150 or SEQ ID No.: 152.
Additionally or alternatively, the antibody disclosed herein comprises the VL sequence selected from the group consisting of SEQ ID No.: 8, SEQ ID No.: 136 and SEQ ID No.: 145, or a variant thereof, respectively. Such variant has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity to SEQ ID No.: 8, SEQ ID No.; 136 or SEQ ID No.: 145. Examples of such variant VL sequences include, without being limited to, SEQ ID No.: 123, SEQ ID No.: 125, SEQ ID No.: 127, SEQ ID No.: 129, SEQ ID No.: 131, SEQ ID No.: 133, SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ ID No.: 139 or SEQ ID No.: 153.
In one embodiment, the antibody comprises a VH sequence having at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence similarity to SEQ ID No.: 7 or SEQ ID No.; 146. Additionally or alternatively, the antibody comprises a VL sequence having at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence similarity to SEQ ID No.: 8, SEQ ID No.: 136 or SEQ ID No.: 145.
In a much preferred embodiment, the antibody comprises the VH as set forth in to SEQ ID No.: 7 and the VL as set forth in SEQ ID No.: 8. The framework sequences of both SEQ ID No.: 7 and SEQ ID No.: 8 are derived from a human immunoglobulin described in WO 03/097697 A (ESBATech AG). Its VH and VL framework sequences have been modified for humanization and stabilization of rabbit antibodies, see, e.g., WO 2009/155726 A (ESBATech, AN ALCON BIOMEDICAL RESEARCH UNIT LLC); BORRAS, L., et al. Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies. Journal of Biological Chemistry 2010, vol. 285, no. 12, p. 9054-9066. In one embodiment, the VL framework of the antibody disclosed herein comprises SEQ ID Nos.: 18-21 or variants thereof. Additionally or alternatively, the VH framework of the antibody comprises SEQ ID Nos.: 22-25, SEQ ID Nos.: 26-29 or SEQ ID Nos.: 30-33 or variants thereof, respectively.
In another preferred embodiment, the antibody comprises the VH as set forth in to SEQ ID No.: 146 and the VL as set forth in SEQ ID No.: 8 or in SEQ ID No.: 145.
In another preferred embodiment, the antibody comprises the VH as set forth in to SEQ ID No.: 146 and the VL as set forth in SEQ ID No.: 136.
The antibody, in particular in case of a scFv, may comprise a linker sequence. Such linker sequence has typically ten to about 25 amino acids. Usually, such linker peptide is rich in glycines, which confer flexibility, as well as serines and/or threonines for improved solubility. In a preferred embodiment, a (GGGGS) 4 linker (SEQ ID No.: 9) or a variant thereof is used. Variations of said motif having three to five repeats may also be used. Further suitable linkers are described, e.g., in ALFTHAN, K. Properties of a single-chain antibody containing different linker peptides, Protein Engineering 1995, vol. 8, no. 7, p. 725-731.
In certain embodiments variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve antigen binding, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), to increase stability or solubility, to decrease immunogenicity and/or to alter other biological, biochemical or biophysical properties of the antibody. In some embodiments the variant does not show any improvement over the parent antibody.
Variants of the antibodies provided herein may be prepared by protein and/or chemical engineering, introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by protein/peptide synthesis. Any combination(s) of deletions, substitutions, additions and insertions can be made to the framework or to the CDRs, provided that the generated antibody possesses the desired characteristics for which it can be screened using appropriate methods. Of particular interest are substitutions, preferably conservative substitutions as described above. Preferred conservative substitutions include:
1. Substituting alanine (A) by valine (V);
2. Substituting arginine (R) by lysine (K);
3. Substituting asparagine (N) by glutamine (Q);
4. Substituting aspartic acid (D) by glutamic acid (E);
5. Substituting cysteine (C) by serine (S);
6. Substituting glutamic acid (E) by aspartic acid (D);
7. Substituting glycine (G) by alanine (A);
8. Substituting histidine (H) by arginine (R) or lysine (K);
9. Substituting isoleucine (I) by leucine (L);
10. Substituting methionine (M) by leucine (L);
11. Substituting phenylalanine (F) by tyrosine (Y);
12. Substituting proline (P) by alanine (A);
13. Substituting serine (S) by threonine (T);
14. Substituting tryptophan (W) by tyrosine (Y);
15. Substituting phenylalanine (F) by tryptophan (W);
and/or

16. Substituting valine (V) by leucine (L)
and vice versa.

The antibody described herein may comprise one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such conservative substitutions.
Non-conservative substitutions may lead to more substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the polypeptide. In one embodiment the antibody comprises one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such non-conservative substitutions.
Modifications may be present in the CDRs or in the framework sequences. For example, the CDRs provided herein may comprise one, two, three, four, five or even more modifications. For example, the CDR-L1, CDR-L2 and CDR-L3 sequences taken as a whole are at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% identical to the CDRs provided herein, in particular to (i) SEQ ID Nos.: 4, 5 and 6, or to (ii) SEQ ID Nos.: 161, 162 and 163. Additionally or alternatively, the CDR-H1, CDR-H2 and CDR-H3 sequences taken as a whole are at least 80%, preferably at least 81%, 82%, 83%, 84%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more 0.5 preferably 99% identical to the CDRs provided herein, in particular to (i) SEQ ID Nos.: 1, 2 and 3, or to (ii) SEQ ID Nos.; 155, 156 and 157.
In one embodiment the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 taken as a whole are at least 85%, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% similar to the CDRs provided herein. Additionally or alternatively, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 taken as a whole are at least 85%, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% similar to the CDRs provided herein.
Therefore, a variant may, e.g., comprise one, two, three, four or five substitutions in SEQ ID No.: 4. Much preferred are substitutions at positions marked with X in SEQ ID No.: 14. The variant may, e.g., comprise:
(i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (I), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 32 of the variable light chain; (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 33 of the variable light chain; and/or (iii) glutamic acid (E), phenylalanine (F), glycine (G), methionine (M), asparagine (N), glutamine (Q), serine (S), tryptophan (W), tyrosine (Y) at AHo position 40 of the variable light chain.

Additionally or alternatively, a variant comprises one, two, three, or four substitutions in SEQ ID No.: 5. Much preferred are substitutions at positions marked with X in SEQ ID No.: 15. Such variant may, e.g., comprise:
(i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), tryptophan (W), tyrosine (Y) at AHo position 58 of the variable light chain; and/or (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 69 of the variable light chain.

Additionally or alternatively, a variant comprises one, two, three, four, five or six substitutions in SEQ ID No.: 6. Much preferred are substitutions at positions marked with X in SEQ ID No.: 16. For example, such variant may comprise:
(i) alanine (A), cysteine (C), isoleucine (I), asparagine (N), serine (S), threonine (T), valine (V) at AHo position 109 of the variable light-chain; (ii) alanine (A), glycine (G), proline (P), serine (S) at AHo position 111 of the variable light chain; (iii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 112 of the variable light chain; (iv) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), scrine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 135 of the variable light chain; and/or (v) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 136 of the variable light chain.

Additionally or alternatively, a variant comprises one, two, three, or four substitutions in SEQ ID No.: 1 or in SEQ ID No.: 155. Much preferred are substitutions at positions marked with X in SEQ ID No.: 11. Such variant may, e.g., comprise:
(i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 33 of the variable heavy chain; and/or (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 39 of the variable heavy chain.

Additionally or alternatively, a variant comprises one, two, three, four, five or six substitutions in SEQ ID No.: 2 or in SEQ ID No.: 156. Much preferred are substitutions at positions marked with X in SEQ ID No.: 12. For example, the variant may comprise:
(i) alanine (A), cysteine (C), glycine (G), methionine (M) or tyrosine (Y) at AHo position 59 of the variable heavy chain; (ii) aspartic acid (D), asparagine (N) or proline (P) at AHo position 60 of the variable heavy chain; and/or (iii) alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), phenylalanine (F), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), serine (S), threonine (T), tryptophan (W) or tyrosine (Y) at AHo position 69 of the variable heavy chain.

Additionally or alternatively, a variant comprises one, two, three, four, five, six, seven, eight, nine, ten or eleven substitutions in SEQ ID No.: 3 or in SEQ ID No.: 157. Much preferred are substitutions at positions marked with X in SEQ ID No.: 13. Such variant may, e.g., comprise:
(i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 110 of the variable heavy chain; (ii) alanine (A), cysteine (C), aspartic acid (D), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 111 of the variable heavy chain; (iii) alanine (A), cysteine (C), phenylalanine (F), histidine (H), isoleucine (I), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine (S), threonine (T), valine (V), tyrosine (Y) at AHo position 112 of the variable heavy chain; (iv) phenylalanine (F) or isoleucine (I) at AHo position 113 of the variable heavy chain; (v) alanine (A), cysteine (C), glutamic acid (E), glycine (G), serine (S), threonine (T), valine (V) at AHo position 114 of the variable heavy chain; (vi) alanine (A), glycine (G), methionine (M) or asparagine (N) at AHo position 115 of the variable heavy chain; (vii) alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), serine (S), threonine (T) at AHo position. 135 of the variable heavy chain; (viii) alanine (A), cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 136 of the variable heavy chain; (ix) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 137 of the variable heavy chain; and/or (x) alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 138 of the variable heavy chain.

A particularly preferred type of variant is one where one or more entire CDRs are replaced. Typically, the CDR-H3 and CDR-L3 contribute most significantly to antigen binding. For example, the entire CDR-L1, CDR-L2, CDR-H1 and/or CDR-H2 may be replaced by a different CDR of natural or artificial origin. In some embodiments, one or more CDRs are replaced by an alanine-cassette.
In one embodiment, the variant described herein has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity to a sequence selected from the group consisting of SEQ ID No.: 10, SEQ ID No.: 73 and SEQ ID No.: 82.
In one embodiment, the variant described herein has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence similarity to SEQ ID No.: 10, SEQ ID No.: 73 and SEQ ID No.; 82.
Additionally or alternatively, the VH of the antibody comprises solubility enhancing point mutations. WO2009/155725 (ESBATech, a Novartis company) describes a motif, which has proven to increase the overall solubility of the antibody. The residues are placed at positions located in the interface of the variable domain and the constant domain of an antibody and stabilize antibody fragments, in particular scFv, lacking the constant domain. In particular, one, preferably all three of the following residues are present:
(i) serine (S) at heavy chain amino acid position 12 (according to AHo numbering);
(ii) serine (S) or threonine (T) at heavy chain amino acid position 103 (according to AHo numbering); and/or
(iii) serine (S) or threonine (T) at heavy chain amino acid position 144 (according to AHo numbering).
In a preferred embodiment the antibody has a serine at VII position 12; a serine at VH position 103; and a threonine at VH position 144 (all AHo numbering).
Thus, in one embodiment the antibody disclosed herein comprises the VH framework sequences of SEQ ID Nos.: 30-33 or variants thereof.
Preferably, a variant antibody as used herein:
(i) retains specific binding to IL-1 beta, in particular to hIL-1 beta;
(ii) has a potency (IC 50 ) with regard to inhibiting the biological effect of human IL-1 beta of lower than 500 pM, preferably lower than 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, more preferably of lower than 25 pM;
(iii) is cross-reactive with cynomolgus IL-1 beta, rhesus monkey IL-1 beta and/or rat IL-1 beta; and/or
(iv) competes with the antibody disclosed herein for binding to IL-1 beta, preferably human IL-1 beta, cynaomolgus IL-1 beta, rhesus monkey IL-1 beta and/or rat IL-1 beta, most preferably hIL-1 beta.
In one embodiment, the variant comprises a VL sequence selected from the group consisting of SEQ ID No.: 96, SEQ ID No.: 97, SEQ ID No.: 98, SEQ ID No.: 99, SEQ ID No.: 100, SEQ ID No.: 101, SEQ ID No.: 102, SEQ ID No.: 103, SEQ ID No.: 104, SEQ ID No.: 105, SEQ ID No.: 123, SEQ ID No.: 125, SEQ ID No.: 127, SEQ ID No.: 129, SEQ ID No.: 131, SEQ ID No.: 133, SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ ID No.: 139, SEQ ID No.: 141, SEQ ID No.: 143, SEQ ID No.; 145, SEQ ID No.: 147, SEQ ID No.: 149, SEQ ID No.: 151 and SEQ ID No.: 153.
Additionally or alternatively, the variant comprises a VII sequence selected from the group consisting of SEQ ID No.: 106, SEQ ID No.: 107, SEQ ID No.: 108, SEQ ID No.: 109, SEQ ID No.: 110. SEQ ID No.: 111, SEQ ID No.: 112, SEQ ID No.: 113, SEQ ID No.: 114, SEQ ID No.: 115, SEQ ID No.: 116, SEQ ID No.: 117, SEQ ID No.: 118, SEQ ID No.: 119, SEQ ID No.: 120, SEQ ID No.: 121, SEQ ID No.: 122, SEQ ID No.: 124, SEQ ID No.: 126, SEQ ID No.: 128, SEQ ID No.: 130, SEQ ID No.: 132, SEQ in No.: 134, SEQ ID No.: 138, SEQ ID No.: 140, SEQ ID No.: 142, SEQ ID No.: 144, SEQ ID No.: 146, SEQ ID No.: 148, SEQ ID No.: 150, SEQ ID No.: 152.
Variants may also be prepared by chain shuffling of light and heavy chains. A single light chain can be combined with a library of heavy chains to yield a library of variants. In one embodiment, said single light chain is selected from the group of VL sequences recited above and/or said library of heavy chains comprises one or more of the VH sequences recited above. Likewise, a single heavy chain can be combined with a library of light chains. Preferably, said single heavy chain is selected from the group of VH sequences recited above and/or said library of light chains comprises one or more of the VL sequences recited above.
In one embodiment, the variant comprises the VL of SEQ ID No.: 135 and/or the VI of SEQ ID No.: 7, SEQ ID No.: 142, SEQ ID No.: 146, SEQ ID No.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ ID No.: 67, SEQ ID No.: 85, SEQ ID No.: 86, SEQ ID No.: 87 or SEQ ID No.: 88.
In one embodiment, the variant comprises the VL of SEQ ID No.: 136 and/or the VI of SEQ ID No.: 7, SEQ ID No.: 142, SEQ ID No.: 146, SEQ ID No.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ ID No.: 68, SEQ ID No.: 81, SEQ ID No.: 82, SEQ ID No.: 83 or SEQ ID No.: 84.
In one embodiment, the variant comprises the VL of SEQ ID No.: 137 and/or the VH of SEQ ID No.: 7, SEQ ID No.: 138, SEQ ID No.: 142, SEQ ID No.: 146, SEQ ID No.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ ID No.: 69, SEQ ID No.: 92, SEQ ID No.: 93, SEQ ID No.: 94 or SEQ ID No.: 95.
In one embodiment, the variant comprises the VL of SEQ ID No.: 139 and/or the VH of SEQ ID No.: 140, SEQ ID No.: 142, SEQ ID No.: 146, SEQ ID No.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ ID No.: 70, SEQ ID No.: 77, SEQ ID No.: 78, SEQ ID No.: 79 or SEQ ID No.: 80.
In one embodiment, the variant comprises the VL of SEQ ID No.: 141 and/or the VH of SEQ ID No.: 142. Preferably, the variant comprises SEQ ID No.: 71.
In one embodiment, the variant comprises the VL of SEQ ID No.: 143 and/or the VH of SEQ ID No.: 144. Preferably, the variant comprises SEQ ID No.: 72.
In one embodiment, the variant comprises the VL of SEQ ID No.: 145 and/or the VH of SEQ ID No.: 146. Preferably, the variant comprises SEQ ID No.: 73.
In one embodiment, the variant comprises the VL of SEQ ID No.: 147 and/or the VI of SEQ ID No.; 148. Preferably, the variant comprises SEQ ID No.: 74.
In one embodiment, the variant comprises the VL of SEQ ID No.: 149 and/or the VH of SEQ ID No. 150. Preferably, the variant comprises SEQ ID No.: 75.
In one embodiment, the variant comprises the VL of SEQ ID No.: 151 and/or the VH of SEQ ID No. 152. Preferably, the variant comprises SEQ ID No.: 76.
In one embodiment, the variant comprises the VL of SEQ ID No.: 8 and/or the VH of SEQ ID No.: 121 or of SEQ ID No.: 122. Preferably, the variant comprises SEQ ID No.; 59 or SEQ ID No.: 60.
In one embodiment, the variant comprises the VL of SEQ ID No.: 153 and/or the VH of SEQ ID No.: 142, SEQ ID No.: 146 or SEQ ID No.: 152. Preferably, the variant comprises SEQ ID No. 89, SEQ ID No.: 90 or 91.
In one embodiment, the variant comprises the VL of SEQ ID No.: 8 and/or the VH of SEQ ID No.: 121, SEQ ID No.; 122, SEQ ID No.: 142, SEQ ID No.: 144, SEQ ID No.: 146, SEQ ID No.: 148, SEQ ID No.: 150 or SEQ ID No.: 152.
In one embodiment, the variant comprises the VH of SEQ ID No.: 7 and/or the VL of SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ ID No.: 139 or SEQ ID No.: 153.
In one embodiment, the variant comprises a sequence selected from the group consisting of SEQ ID No.: 34 to 95 and SEQ ID No.: 154.
A binding member can comprise any of the VL and/or the VH sequences mentioned above. Binding members having a single domain format, such as a nanobody or a VHH, comprise only one of either the VL or VH sequences mentioned above, preferably the VH sequence. Multivalent binding members, in particular F(ab′) 2 fragments, bis-scFv or diabodies, preferably bispecific binding members, may comprise one or more of the VL sequences mentioned above and/or one or more of the VH sequences mentioned above.
The antibodies of the instant invention are particularly stable. As used herein the term “stability” refers to the biophysical property of the antibody to remain monomeric in solution after prolonged incubation and/or incubation at elevated temperature. Unstable antibodies tend to dimerize or oligomerize and even precipitate, thereby decreasing shelf-life and becoming less suitable for pharmaceutical applications.
The antibodies provided herein and in particular the monovalent antibody fragment above remain monomeric at least to 75%, preferably at least to 80%, 85%, and most preferably to 93% after being incubated for 1 month at 37° C. at a concentration of 1 mg/ml in PBS at pH 7.2. Additionally or alternatively, the antibody remains monomeric at least to 90%, preferably at least to 92%, 94%, 96%, 98% more preferably to 100% after 1 month at room temperature at a concentration of 1 mg/ml in PBS at pH 7.2.
The degree of monomers can, e.g., be determined by SEC-HPLC (Size Exclusion Chromatography-High-Performance Liquid Chromatography). A suitable mobile phase for such testing is, e.g., PBS at pH 7.2. The monomer content can be quantified by peak integration of the UV280 signal measured during the protein chromatography. A suitable system is, e.g., a Dionex Summit HPLC controlled by CHROMELEON® 6.5 software that also allows for subsequent chromatogram analysis and peak quantification.
The antibodies disclosed herein and in particular the monovalent antibody fragment above are also stable at higher concentrations, for example, they remain monomeric at least to 50%, preferably at least to 55%, 60%, 65%, 70% and most preferably to 75% after being incubated for 2 weeks at room temperature and/or 4° C. at a concentration of about 50 mg/ml in PBS at pH 7.2.
Moreover, the antibodies provided herein and in particular the monovalent antibody fragment above are particularly soluble and can therefore be highly concentrated without precipitation due to aggregate formation. Preferably, the antibodies can be concentrated in PBS at pH 7.2 to a concentration of more than 20 mg/ml without precipitation, more preferably to a concentration of 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml and most preferably to 70 mg/ml in. PBS at pH 7.2.
In a much preferred embodiment, the antibody has a melting temperature of about 60° C. as determined by differential scanning fluorimetry (DSF), preferably 65° C., 70° C., 71° C., 72° C., 73° C. and most preferably 74° C. This method is based on the properties of certain dyes being fluorescent only in a hydrophobic environment. For example, protein unfolding can be detected as an increase in fluorescence upon binding of the dye SYPRO® Orange to a heat-denatured protein (NIESEN F. H. et al. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2007, vol. 2, p. 2212-2221). The stability of a protein can thus be analyzed by thermal denaturation.
The antibody has preferably a theoretical isoelectric point (pI) in the range of 5 to 10, preferably 7 to 9, most preferably about 8.3. The theoretical pI can, for example, be calculated by using the ProtParam tool on the ExPASy Server (see also GASTEIGER E. et al. Protein Identification and Analysis Tools on the ExPASy Server. (In) The Proteomics Protocols Handbook. Edited by WALKER J. M. Totowa: Humana Press Inc., 2005. ISBN 9781588295934. p. 571-607).
The antibody can be cross-reactive with IL-1 beta from non-human species, such as, without being limited to, cynomolgus IL-1 beta, rhesus monkey IL-1 beta, rat IL-1 beta, murine IL-1 beta, canine IL-1 beta, feline IL-1 beta, marmoset IL-1 beta, swine IL-1 beta and/or guinea pig IL-1 beta. Preferably, the antibody is cross-reactive with cynomolgus IL-1 beta (e.g., recombinantly produced and available from Sino Biological Inc., cat. no. 90010-CNAE), rhesus monkey IL-1 beta (e.g., recombinantly produced and available from R&D Systems, cat. no. 1318-RL/CF) and/or rat IL-1 beta (e.g., recombinantly produced and available from Peprotech, cat. no. 400-01B).
Preferably, there is no residual activity of IL-1 beta when being neutralized with the antibody disclosed herein in an in vivo and/or an in vitro setting, i.e. the antibody completely inhibits the action of IL-1 beta. “No residual activity” as used herein refers to lower than 2% of the potency assay signal corresponding to the IL-6 release from human fibroblasts induced by 10 pg/ml of IL-1 beta, preferably the assay as described in example 3, in presence of 60 ng/ml of the antibody described herein when compared to antibodies of non-relevant specificity or vehicle control at the same concentration.
The invention also provides a binding member competing with the antibodies disclosed herein for binding to human IL-1 beta.
As used herein, the term “competing” refers to the competition between binding members for binding to the same target. Competition can be determined by competitive binding assays in which the binding member of interest prevents or inhibits or reduces specific binding of the antibodies disclosed herein to a common antigen (here, hIL-1 beta or a fragment thereof). Such competitive binding assays are known in the art and include, without being limited to, solid phase direct or indirect radioimmunoassay (RIA) and solid phase direct or indirect enzyme immunoassay (EIA). Typically, such assay involves the use of purified antigen bound to a solid surface, a. binding member to be tested and the reference antibody as described herein. Competitive inhibition is measured by determining the amount of either: (i) the reference antibody bound to the solid surface in the presence of the binding member to be tested, or (ii) the binding member to be tested bound to the solid surface in the presence of the reference antibody. A competing binding member may bind: (i) to the same epitope as the antibody, (ii) to an overlapping epitope, or (iii) to a different epitope on the same target molecule but sterically hindering binding of the antibody to its target.
Usually, when a competing binding member is present in excess, it will reduce specific binding of the antibody as described herein to IL-1 beta by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. Preferably, binding of the antibody is reduced by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
In one embodiment, the competing binding member binds to hIL-1 beta with an affinity K D of at least about 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, 10 nM.
In one embodiment, the binding member is monovalent, such as a scFv or a Fab fragment. In another embodiment, the binding member is multivalent. Such multivalent molecule can be bivalent (such as a full-length immunoglobulin or a F(ab′) 2 fragment) or comprises at least three target binding sites.
The multivalent binding member can be a bispecific antibody such as a diabody, a single-chain diabody or a tandem scFv (see, e.g., KONTERMANN, R. E. Methods in Molecular Biology. Edited by LO, B. Totowa, N. J.: Humana Press, 2004. ISBN 1588290921. p. 227-242). Said bispecific antibodies may well use shorter linkers then those described above for scFv, i.e., having only one to three repeats of the basic motif of SEQ ID NO: 14 (see, e.g., HOLLIGER, P., et al. Diabodies: small bivalent and bispecific antibody fragments. PNAS 1993, vol. 90, no. 14, p. 6444-6448). In another embodiment the multivalent binding member is a triabody, a minibody or tetrabody.
The invention also provides T-bodies comprising the antibodies disclosed herein. T-bodies are immunoglobulin T-cell receptors (cIgTCRs) which combine the antigen recognition of antibodies with the signal and effector properties of the T-cell receptor complex. In such constructs the antibody is preferably an antibody fragment such as a Fv, a Fab, a scFv or a scFv-Fc, most preferably a scFv. For further discussion of the general design of T-bodies and their applications, see, e.g., SCHIRRMANN, T. and Pecher, G. Handbook of Therapeutic Antibodies. Edited by DÜBEL, S. Weinheim: Wiley-VCH, 2009. ISBN 3527314539, p. 533-561.
The invention further provides a naïve (i.e., being not engineered for increased affinity or potency) binding member against IL-1 beta having a Monovalent Potency (measured, e.g., as affinity (K D ) or biological potency in cell-based assays (IC 50 ) in units of mol/l) at a certain Molecular Weight (in g/mol) after normalization to the Number of Binding Sites per binding member, as determined by the equation
K =Monovalent Potency (mol/l)*Molecular Weight (g/mol)/Number of Binding Sites
As described above, monovalent antibody fragments having potency values in the picomolar range are particular and not routinely obtained. Potency often correlates with the size of the binding member: high potency in the picomolar range can be obtained by full-length immunoglobulins, whereas very small antibody fragments such as nanobodies or minimal recognition units, or small non-antibody scaffolds such as affilins often show lower potency values, i.e., in the nanomolar range. Seemingly, there is a minimum for said function K provided by scFv as described herein: the smaller the binding member, and the higher its monovalent potency or affinity, and the more binding sites per molecule, the smaller K. For example, for scFv as described herein, the lower limit of K equals about 50 ng/1 whereas the upper limit of K equals about 12,500 ng/l; for the respective full-length immunoglobulins, the lower limit equals about 150 ng/l and the upper K limit equals about 37,500 ng/I; for other binding members having molecular weights smaller than the scFv as described herein, the K value is K>500,000 ng/l. In a preferred embodiment, the K value is about 50 ng/l, 100 ng/l, 200 ng/l, 500 ng/l, 750 ng/l, 1,000 ng/l, 1,250 ng/l, 1,500 ng/l, 1,750 ng/l, 2,000 ng/l, 2,250 ng/l, or 2,500 ng/l.
Nucleic Acids, Vectors, Host Cells and Method of Production
The antibodies described herein are encoded by a single nucleic acid or by two or more nucleic acids, for example each encoding at least one variable region. Knowing the sequence of the antibody or of its parts, cDNAs encoding the polypeptide sequence can be generated by methods well known in the art, e.g. by gene synthesis. These cDNAs can be cloned by standard cloning and mutagenesis techniques into a suitable vector such as an expression vector or a cloning vector. Optionally, the variable light chain is encoded by a separate nucleic acid than the variable heavy chain of the antibody. Further, additional sequences such as tags (e.g., a His-tag), constant domains for the production of a Fab or a full-length immunoglobulin, linkers, the coding sequence of a second binding specificity or another functional polypeptide such as an enzyme to generate a fusion construct or a bispecific molecule may be included into the genetic construct.
Based on the cloning strategy chosen, genetic constructs may generate an antibody having one or more additional residues at the N-terminal or C-terminal end. For example, an N-terminal methionine derived from the start codon or an additional alanine may be present in an expressed polypeptide, unless it has been clipped off post-translationally. It is therefore to be understood that the antibodies disclosed herein comprise the disclosed sequences rather than consist of them.
In one embodiment, the invention provides a nucleic acid sequence comprising at least 300 nucleobases, more preferably at least 350, 400, 450, or 500 nucleobases and having at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No.: 17. In a much preferred embodiment the nucleic acid sequence is SEQ ID No.: 17.
Additionally or alternatively, the invention provides a nucleic acid sequence comprising at least 300 nucleobases, more preferably at least 350, 400, 450, or 500 nucleobases, which hybridizes with the nucleic acid of SEQ ID No.; 17 under high stringency conditions.
Basic protocols of standard cloning, mutagenesis and molecular biology techniques are described in, e.g., Molecular Cloning, A Laboratory Manual (GREEN, M. and Sambrook, J. Molecular Cloning: a Laboratory Manual. 4th edition. Cold Spring Harbor Laboratory, 2012. ISBN 1936113422).
Appropriate host cells for the expression of the genetic constructs can be prokaryotic or eukaryotic. Suitable prokaryotic host cells are gram-negative or gram-positive and include species of the Escherichia, Erwinina, Enterobacter, Klebsiella, Pseudomonas or Bacillus families. Much preferred is Escherichia coli , in particular E. coli strains BL21 (DE3) (LIFE TECHNOLOGIES™, cat. no. C6000.03) and ORIGAMI™ 2(DE3) (Novagen, cat. no 71345).
If post-translational modifications such as glycosylation or phosphorylation are desired, eukaryotic host cells are preferable. For example, eukaryotic microbes such as commonly used Saccharomyces cerevisiae or Pichia pastoris strains may serve as host cells. Host cells can also include plant or animal cells, in particular insect or mammalian cells. Suitable mammalian cells include, without being limited to, Chinese Hamster Ovary Cells (CHO), Human Embryonic Kidney Cells (HEK), Human Umbilical Vein Endothelial Cells (HUVEC) or NS0 myeloma cells.
The antibody can be produced by expression in a suitable host cell. For example, the expression vectors described above are introduced into a host cell by standard techniques such as electroporation or chemical transformation. The transformed cells are then cultivated under conditions adequate for recombinant protein expression, typically in appropriate nutritional media, optionally modified for inducing promoters, selecting transformants, or amplifying encoding sequences of interest. The antibody is recovered from the culture and optionally purified using standard techniques in the art. The yield of recombinant protein may be improved by optimizing media and culture conditions such as temperature or oxygen supply. In prokaryotes the antibody can be produced in the periplasm, intracellularly as inclusion bodies or be secreted into the medium. Upon harvest, the protein can be purified using methods well known in that art such as gel filtration, ion exchange chromatography, reversed phase chromatography, hydrophobic interaction, mixed mode chromatography and/or affinity chromatography.
In one embodiment the antibody is produced in a cell-free system. This typically involves in vitro transcription followed by in vitro translation of nucleic acid product templates encoding the proteins described herein, e.g., plasmid DNA or PCR product templates. For example, crude lysates from growing cells are used, providing the necessary enzymes as well as the cellular protein synthesis machinery. The necessary building blocks such as amino acids or nucleobases as well as energy delivering molecules and others can be exogenously supplied. Cell-free expression systems can, for example, be based on lysed rabbit reticulocytes (e.g., Rabbit Reticulocyte Lysate System, Promega, cat. no. L4540), HeLa cells (e.g., 1-Step Human In Vitro Translation Kit, Thermo Scientific, cat. no. 88881), insect cells (e.g., EasyXpress Insect Kit II, Qiagen, cat. no. 32561), wheat germs (e.g., Wheat Germ Extract, Promega, cat. no, L4380), or E. coli cells (e.g., PUREXPRESS® In Vitro Protein Synthesis Kit, NEB, cat. no. E6800S). Also, optimized cell-free antibody expression systems for improved disulfide bond generation can be used for production. Commercially available kits include insect cell lysates (e.g., EasyXpress Disulfide Insect Kit, Qiagen, cat. no. 32582) or E. coli cell lysates (e.g., EasyXpress Disulfide E. coli Kit, Qiagen, cat. no. 32572). Cell-free protein synthesis has, e.g., the advantage of being fast, achieving high product yields, allowing for easy modification of reaction conditions, forming a low degree of or even no byproducts. Cell-free protein synthesis may involve biological and/or chemical steps which cannot be conducted in purely biological or chemical production systems. For example, non-natural or chemically-modified amino acids can be incorporated into the protein at desired positions. ScFv-toxin fusion proteins have been successfully produced in cell-free systems (NICHOLLS, P. J., et al. Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate. Journal of Biological Chemistry 1993, vol. 268, pp. 5302-5308). Thus, in one embodiment a method of producing the antibody described herein, the binding member above or the T-body above is provided comprising the steps of: (a) providing a cell-free system, (b) providing a nucleic acid product template encoding the antibody described herein, the binding member above or the T-body above, (c) allowing for transcription and translation of said nucleic acid product template; (d) recovering, and optionally (e) purifying said antibody, said binding member or said T-body, respectively.
Additionally or alternatively, a method of producing the antibody described herein comprises at least one step of chemical synthesis. For example, the method may be entirely chemical. In another embodiment, the cell-based or the cell-free production systems described above comprise such at least one step of chemical synthesis.
In a preferred embodiment the antibodies described herein are produced in a cell-based system using an expression vector for intracellular expression in E. coli . Upon expression the polypeptide is generated as inclusion bodies within the cells which are separated from further cell particles followed by solubilisation in a denaturing agent such as guanidine hydrochloride (GndHCl) and refolded by renaturation procedures well known to the skilled person.
It is to be understood that the nucleic acids, vectors, host cells and method of production described above also apply to the binding members (insofar as they are a protein) and/or to T-bodies described herein.
Chemical and/or Biological Modifications
In one aspect the antibody of the instant invention is chemically and/or biologically modified. Such modification may comprise, but is not limited to, glycosylation, PEGylation, HESylation, Albumin fusion technology, PASylation, labelling with dyes and/or radioisotopes, conjugation with enzymes and/or toxins, phosphorylation, hydroxylation and/or sulfation. Likewise, any binding member, the nucleic acid sequence, the vector and/or the host cell described above can be modified accordingly.
Chemical and/or biological modifications may be conducted to optimize pharmacodynamics or water solubility of the protein or to lower its side effects. For example, PEGylation, PASylation and/or HESylation may be applied to slow down renal clearance and thereby increase plasma half-life time of the antibody. Additionally or alternatively, a modification may add a different functionality to the protein, e.g. a toxin to more efficiently combat cancer cells, or a detection molecule for diagnostic purposes.
Glycosylation refers to a process that attaches carbohydrates to proteins. In biological systems, this process is performed enzymatically within the cell as a form of co-translational and/or post-translational modification. A protein, here the antibody, can also be chemically glycosylated. Typically, but not limited to, glycosylation is (i) N-linked to a nitrogen of asparagine or arginine side-chains; (ii) O-linked to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains; (iii) involves the attachment of xylose, fucose, mannose, and N-acetylglucosamine to a phospho-serine; or (iv) in form of C-mannosylation wherein a mannose sugar is added to a tryptophan residue found in a specific recognition sequence. Glycosylation patterns can, e.g., be controlled by choosing appropriate cell lines, culturing media, protein engineering manufacturing modes and process strategies (HOSSLER, P. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009, vol. 19, no. 9, p. 936-949).
Protein engineering to control or alter the glycosylation pattern may involve the deletion and/or the addition of one or more glycosylation sites. The creation of glycosylation sites can conveniently be accomplished by introducing the corresponding enzymatic recognition sequence into the amino acid sequence of the antibody or by adding or substituting one or more of the above enumerated amino acid residues.
It may be desirable to PEGylate the antibody. PEGylation may alter the pharmacodynamic and pharmacokinetic properties of a protein. Polyethylene-glycol (PEG) of an appropriate molecular weight is covalently attached to the protein backbone (see, e.g., PASUT, G. and Veronese, F. State of the art in PEGylation: the great versatility achieved after forty years of research. Journal of Controlled Release 2012, vol. 161, no. 2, p. 461-472). PEGylation may additionally reduce the immunogenicity by shielding the PEGylated protein from the immune system and/or alter its pharmacokinetics by, e.g. increasing the in vivo stability of the antibody, protecting it from proteolytic degradation, extending its half-life time and by altering its biodistribution.
Similar effects may be achieved by PEG Mimetics, e.g., HESylating or PASylating the antibody. HESylation utilises hydroxyethyl starch (“HES”) derivatives, whereas during PASylation the antibody becomes linked to conformationally disordered polypeptide sequences composed of the amino acids proline, alanine and scrine. Said PEG Mimetics and related compounds are, e.g., described in BINDER, U. and Skerra, A. Half-Life Extension of Therapeutic Proteins via Genetic Fusion to Recombinant PEG Mimetics, in Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives. Edited by KONTERMANN, R., Weinheim, Germany: Wiley-VCH, 2012. ISBN: 9783527328499. p. 63-81.
The antibody may include an epitope and in particular a salvage receptor binding epitope. Such salvage receptor binding epitope typically refers to an epitope of the Fe region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) and has the effect of increasing the in vivo half-life of the molecule.
Additionally or alternatively, the antibody is labelled with or conjugated to a second moiety which ascribes ancillary functions following target binding. Said second moiety may, e.g., have an additional immunological effector function, be effective in drug targeting or useful for detection. The second moiety can, e.g., be chemically linked or fused genetically to the antibody using known methods in the art.
Molecules which may serve as second moiety include, without being limited to, radionuclides, also called radioisotopes (e.g., 35 S 32 P, 14 C, 18 F, 125 I); apoenzymes; enzymes (such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase or angiogenin); co-factors; peptides (e.g., HIS-tags); proteins (incl. lectins); carbohydrates (incl. mannose-6-phosphate tag); fluorophores (including fluorescein isothiocyanate (FITC); phycoerythrin; green/blue/red and other fluorescent proteins; allophycocyanin (APC)); chromophores; vitamins (including biotin); chelators; antimetabolites (e.g., methotrexate), liposomes; toxins including cytotoxic drugs such as taxol, gramicidin D or colchicine; or a radiotoxin.
A labelled antibody is particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, an antibody labelled with a suitable radioisotope, enzyme, fluorophore or chromophore can be detected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single cell analysis (e.g., FACS analysis), respectively. Similarly, the nucleic acids and/or vectors disclosed herein can be used for detection or diagnostic purposes, e.g. using labelled fragments thereof as probes in hybridization assays. Labelling protocols may, e.g., be found in JOHNSON, I. and Spence, M. T. Z. Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies. Life Technologies, 2010. ISBN: 0982927916.
Compositions
The antibody of the instant invention, any binding member, the nucleic acid sequences or the vector disclosed herein can be provided in a composition which further comprises a suitable carrier, excipient or diluent. Much preferred is a composition comprising an antibody described herein.
Such composition can, e.g., be a diagnostic, a cosmetic or a pharmaceutical composition. For therapeutic or cosmetic purposes, said composition is a pharmaceutical composition comprising a pharmaceutical carrier, excipient or diluent, i.e. not being toxic at the dosages and a concentration employed.
Suitable “carrier”, “excipients” or “diluents” include, without being limited to: (i) buffers such as phosphate, citrate, or other, organic acids; (ii) antioxidants such as ascorbic acid and tocopherol; (iii) preservatives such as 3-pentanol, hexamethonium chloride, benzalkonium chloride, benzyl alcohol, alkyl paraben, catechol, or cyclohexanol; (iv) amino acids, such as e.g. histidine, arginine; (v) peptides, preferably up to 10 residues such as polylysine; (vi) proteins, such as bovine or human serum albumin: (vii) hydrophilic polymers such as polyvinylpyrrolidone; (viii) monosaccharides, disaccharides, polysaccharides and/or other carbohydrates including glucose, mannose, sucrose, mannitol, trehalose, sorbitol, aminodextran or polyamidoamines; (ix) chelating agents, e.g. EDTA; (x) salt-forming ions such as sodium; (xi) metal complexes (e.g. Zn-protein complexes); and/or (xii) ionic and non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Many of said exemplary compounds have different functions and may, e.g., act as carrier and as diluent. It is also to be understood that the composition may comprise more than one of each carrier, diluent or excipient.
The antibody, the binding member, the nucleic acid sequences or the vector may be provided on solid support materials such as beads and microparticles. Typically, the molecules are linked to such carrier via a covalent bond (optionally involving a linker), noncovalently or admixture. Said beads and microparticles can comprise, for example, starch, cellulose, polyacrylate, polylacetate polyglycolate, poly(lactide-co-glycolide), latex, or dextran.
Therapeutic Applications
The molecules described herein, in particular the antibody, binding member, nucleic acid or vector, are useful as a medicament. Typically, such medicament comprises a therapeutically effective amount of the molecules provided herein. Accordingly, said molecules can be used for the production of a medicament useful in the treatment of IL-1 beta-related disorders.
In one aspect, a method of treating an IL-1 beta-related disorder is provided comprising the steps of administering a pharmaceutically effective amount of the molecules described herein, in particular the antibody, to a subject in need thereof. In one embodiment, the pharmaceutical composition above (i.e., medicament) comprising such pharmaceutically effective amount of the antibody is administered to said subject.
The term “treat” or “treatment” as used herein refers to the administration of a pharmaceutically effective amount of the antibody, binding member, nucleic acid, vector or host cell of the instant invention, to a subject in need thereof to prevent, cure, delay the onset and/or progression, reduce the severity of, stabilize, modulate, cure or ameliorate one or more symptoms of an IL-1 beta-related disorder. Typically, the antibody, binding member, nucleic acid, vector or host cell is provided in a pharmaceutical composition including those previously described herein.
A “therapeutically effective amount” refers to an amount which at the dosage regimen applied yields the desired therapeutic effect, i.e., to reach treatment goals as defined above. The dosage will depend on various factors including patient and clinical factors (e.g., age, weight, gender, clinical history of the patient, severity of the disorder and/or response to the treatment), the nature of the disorder being treated, the particular composition to be administered, the route of administration, and other factors.
The subject in need of such treatment can be a human or a non-human animal, e.g., a mouse, rat, rabbit, monkey, dog, horse, cow, chicken, guinea pig or pig. Typically, the subject is diagnosed with an IL-1 beta-related disorder or may acquire such a disorder.
Examples of IL-1 beta-related disorders, in which antagonist of IL-1 beta have shown therapeutic effects include, without being limited to, proliferative diabetic retinopathy, gouty arthritis, Schnitzler syndrome, systemic juvenile idiopathic arthritis, rheumatoid arthritis, acute gouty arthritis, chronic gouty arthritis, urticaria, vasculitis, type 1 diabetes, type 2 diabetes, ankylosing spondylitis, recurrent multifocal osteomyelitis, relapsing polychondritis, cyropyrin-associated periodic syndrome (CAPS), Behçet's disease, familial mediterranean fever, chronic obstructive pulmonary disease, polymyalgia rheumatic, NALP3-mutations, pyoderma gangrenosum, chronic idiopathic urticarial, osteoarthritis, wet age-related macular degeneration, dry eye syndrome, pustular psoriasis, synovitis-acne-pustulosis-hyperostosis-osteitis syndrome, macrophage activation syndrome, periodic fever, adenitis, pharyngitis, aphthous ulcer syndrome, adult-onset Still's disease, mevalonate kinase deficiency, atherosclerosis, TNF-receptor associated periodic syndrome (TRAPS), acne vulgaris and/or acne inversa.
The term “CAPS” or cryopyrin-associated periodic syndrome is to be understood to include each of familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disease, also known as chronic infantile neurological, cutaneous and articular (CINCA) syndrome.
The pharmaceutical composition may be applied by different administration routes. Administration can be conducted, for example, but not limited to, parenterally, e.g., intramuscularly, subcutaneously, intravenously as a bolus or by continuous infusion, intraarticularly, intrasynovially, intracerebrally, intracerebrospinally, intrathecally, epidurally, or intraperitoneally; orally; rectally; locally, urogenitally; topically, e.g., to the skin or the eye; Intravitreally; intravenously; intraocularly; oticly; intranasally; by inhalation; dermally such as intradermally, subcutaneously or transdermally; sublingually; buccally, for example. Preferred are the topical, rectal, local, intranasal, intravenous and/or intradermal routes of administration.
The antibody of the instant invention, the binding member, the nucleic acid sequences, the vector or host cell can be combined with one or more further therapeutically effective compound. Said compound may either be capable of disrupting signalling via the IL-1 receptor, or alternatively inhibit one or more different targets such as, e.g., other mediators of inflammatory responses. Such compound(s) can be administered simultaneously or sequentially.
For therapeutic applications, the antibody may also be radiolabelled or linked to a toxin or linked to another effector function as described above.
Diagnostic Applications and/or Detection Purposes
The antibody of the instant invention may be used for detection or diagnostic purposes in vivo and/or in vitro. For example, a wide range of immunoassays involving antibodies for detecting the expression in specific cells or tissues are known to the skilled person. Likewise, any binding member, the nucleic acid sequence, the vector and/or the host cell described previously can be used accordingly as detailed in this section.
For such applications the antibody, binding member, the nucleic acid sequence, the vector or the host cell disclosed herein may be either labelled or unlabelled. E.g., an unlabelled antibody may be used and detected by a secondary antibody recognizing an epitope on the antibody described herein.
In another embodiment the antibody, binding member, nucleic acid sequence, vector and/or host cell is conjugated with one or more substances which can be recognized by a detector substance(s), e.g., the antibody being conjugated with biotin which can be detected by streptavidin. Likewise, the nucleic acids and/or vectors disclosed herein can be used for detection or diagnostic purposes, e.g., by using labelled fragments thereof as probes in hybridization assays.
In certain embodiments, any of the molecules provided herein, in particular the antibody, is useful for detecting the presence of IL-1 beta, preferably including full-length IL-1 beta, fragments thereof and/or precursors thereof, in a sample, preferably biological sample. The term “detecting” encompasses quantitative and/or qualitative detection. In certain embodiments a biological sample comprises a cell or tissue from human patients. Non limiting examples of biological samples include blood, urine, cerebrospinal fluid, biopsy, lymph and/or non-blood tissues.
In certain embodiments, the method comprises contacting the biological sample with an anti-IL-1 beta antibody as described herein under conditions permissive for binding of the antibody to IL-1 beta, if present, and detecting whether a complex is formed between the antibody and IL-1 beta. Such method may be an in vitro or in vivo method. In one embodiment an anti-IL-1 beta antibody is used to select subjects eligible for therapy with the antibody described herein, e.g., where IL-1 beta is a biomarker for selection of patients. Similarly, instead of the antibody, such method may involve the use of the binding member above or a T-body described herein.
In another aspect, the antibody is used in cosmetic applications, e.g., for improving the aesthetic appearance of skin.
In a further aspect, a kit is provided comprising the antibody, a packaged combination of reagents with instructions for performing the detection or diagnostic assay. The reagents are typically provided in predetermined amounts of dry powders, usually lyophilized, including excipients which after dissolution will provide a reagent solution having the appropriate concentration. Other additives such as stabilizers and/or buffers may also be included. If the antibody is labelled with an enzyme, the kit will typically include the according substrates and cofactors. Likewise, any binding member, the nucleic acid sequence, the vector and/or the host cell described previously can be used accordingly as detailed in this section.







Sequence listing







The sequences disclosed herein are:

SEQ ID No: 1- VH CDR1

FSLSSAAMA



SEQ ID No: 2- VH CDR2

IIYDSASTYYASWAKG



SEQ ID No: 3- VH CDR3

ERAIFSGDFVL



SEQ ID No: 4- VL CDR1

QASQSIDNWLS



SEQ ID No: 5- VL CDR2

RASTLAS



SEQ ID No: 6- VL CDR3

QNTGGGVSIA



SEQ ID No: 7- VH

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQIVfNSLRAEDTAVYYCARE



RAIFSGDFVLWGQGTLVTVSS



SEQ ID No: 8- VL

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No: 9- linker

GGGGSGGGGSGGGGSGGGGS



SEQ ID No: 10- DLX2323

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No: 11- CDR variant of VH CDR1

FSLSXXAMA



SEQ ID No: 12- CDR variant of VH CDR2

IIXXSASTXYASWAKG



SEQ ID No: 13- CDR variant of VH CDR3

EXXXXXXXXXX



SEQ ID No: 14- CDR variant of VL CDR1

QASQSIXXXLS



SEQ ID No: 15- CDR variant of VL CDR2

XASXLAS



SEQ ID No: 16- CDR variant of VL CDR3

QNXGXXXXIA



SEQ ID No: 17- DNA sequence of DLX2323

GAAATTGTTATGACCCAGAGCCCGAGCACCCTGAGCGCAAGCGTTGGTGA



TCGTGTGATTATTACCTGTCAGGCAAGCCAGAGCATTGATAATTGGCTGA



GCTGGTATCAGCAGAAACCGGGTAAAGCACCGAAACTGCTGATTTATCGT



GCAAGCACCCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAG



TGGTGCAGAATTTACCCTGACCATTAGCAGCCTGCAGCCGGATGATTTTG



CAACCTATTATTGTCAGAATACCGGTGGTGGTGTTAGCATTGCATTTGGT



CAGGGCACCAAACTGACCGTTCTGGGTGGTGGCGGTGGATCCGGTGGGGG



TGGTAGCGGAGGTGGTGGTTCAGGCGGTGGTGGCAGCGAAGTTCAGCTGG



TTGAAAGTGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGC



TGTACCGCAAGCGGTTTTAGCCTGAGCAGCGCAGCAATGGCATGGGTTCG



TCAGGCACCTGGTAAAGGTCTGGAATGGGTTGGTATTATCTATGATAGCG



CAAGCACCTATTATGCAAGCTGGGCAAAAGGTCGTTTTACCATTAGCCGT



GATACCAGTAAAAATACCGTTTACCTGCAGATGAATAGTCTGCGTGCAGA



GGATACCGCAGTGTATTATTGTGCACGTGAACGTGCAATTTTCAGCGGTG



ATTTTGTTCTGTGGGGTCAGGGAACCCTGGTTACCGTTAGCAGC



SEQ ID No.: 18- FR-L1 of FW1.4

EIVMTQSPSTLSASVGDRVIITC



SEQ ID No.: 19- FR-L2 of FW1.4

WYQQKPGKAPKLLIY



SEQ ID No.: 20- FR-L3 of FW1.4

GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC



SEQ ID No.: 21- FR-L4 of FW1.4

FGQGTKLTVLG



SEQ ID No.: 22- FR-H1 of rFW1.4

EVQLVESGGGLVQPGGSLRLSCTASG



SEQ ID No.: 23- FR-H2 of rFW1.4

WVRQAPGKGLEWVG



SEQ ID No.: 24- FR-H3 of rFW1.4

RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR



SEQ ID No.: 25- FR-H4 of rFW1.4

WGQGTLVTVSS



SEQ ID No.: 26- FR-H1 of rFW1.4(V2)

EVQLVESGGGLVQPGGSLRLSCTVSG



SEQ ID No.: 27- FR-H2 of rFW1.4(V2)

WVRQAPGKGLEWVG



SEQ ID No.: 28- FR-H3 of rFW1.4(V2)

RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR



SEQ ID No.: 29- FR-H4 of rFW1.4(V2)

WGQGTLVTVSS



SEQ ID No.: 30- FR-H1 of rFW1.4-SST

EVQLVESGGGSVQPGGSLRLSCTASG



SEQ ID No.: 31- FR-H2 of rFW1.4-SST

WVRQAPGKGLEWVG



SEQ ID No.: 32- FR-H3 of rFW1.4-SST

RFTISRDTSKNTVYLQMNSLRAEDTASYYCAR



SEQ ID No.: 33- FR-H4 of rFW1.4-SST

WGQGTTVTVSS



SEQ ID No.: 34- CDR-L1_D32X

EIVMTQSPSTLSASVGDRVIITCQASQSIXNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 35- CDR-L1_N33X

EIVMTQSPSTLSASVGDRVIITCQASQSIDXWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 36- CDR-L1_W40X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNXLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of glutamic acid (E), phenylalanine

(F), glycine (G), methionine (M), asparagine (N),

glutamine (Q), serine (S), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 37- CDR-L2_R58X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYX



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), tryptophan (W) and tyrosine

(Y).



SEQ ID No.: 38- CDR-L2_T69X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASXLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 39- CDR-L3_T109X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNXGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

isoleucine (I), asparagine (N), serine (S),

threonine (T) and valine (V).



SEQ ID No.: 40- CDR-L3_G111X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGXGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), glycine (G), proline

(P) and serine (S).



SEQ ID No: 41- CDR-L3_G112X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGXVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 42- CDR-L3_V135X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGXSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No: 43- CDR-L3_S136X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVXIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I),

leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 44- CDR-H1_S33X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSXAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 45- CDR-H1_A39X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSXAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

glutamine (Q), arginine (R), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No: 46- CDR-H2_Y59X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIXDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), glycine

(G), methionine (M) and tyrosine (Y).



SEQ ID No: 47- CDR-H2_D60X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYXSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of aspartic acid (D), asparagine (N)

and proline (P).



SEQ ID No.: 48- CDR-H2_Y69X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTXYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), aspartic acid (D),

glutamic acid (E), glycine (G), phenylalanine (F),

histidine (H), isoleucine (I), lysine (K), leucine

(L), methionine (M), proline (P), asparagine (N),

serine (S), threonine (T), tryptophan (W) and

tyrosine (Y).



SEQ ID No: 49- CDR-H3_R110X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCAREXAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

glutamine (Q), arginine (R), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 50- CDR-H3_A111X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERXIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), phenylalanine (F), glycine (G),

histidine (H), isoleucine (I), lysine (K),

methionine (M), asparagine (N), proline (P),

glutamine (Q), arginine (R), serine (S), threonine

(T), valine (V), tryptophan (W) and tyrosine (Y).



SEQ ID No.: 51- CDR-H3_I112X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAXFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

phenylalanine (F), histidine (H), isoleucine (I),

leucine (L), methionine (M), asparagine (N),

glutamine (Q), serine (S), threonine (T), valine

(V) and tyrosine (Y).



SEQ ID No.: 52- CDR-H3_F113X

EIVMTQSPSTLSASVGDRVIITCQASOSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIXSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of phenylalanine (F) and isoleucine

(I).



SEQ ID No.: 53- CDR-H3_S114X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERA1FXGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

glutamic acid (E), glycine (G), serine (S),

threonine (T) and valine (V).



SEQ ID No.: 54- CDR-H3_G115X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSXDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), glycine (G),

methionine (M) and asparagine (N).



SEQ ID No.: 55- CDR-H3_D135X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGXFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), aspartic acid (D),

glutamic acid (E), histidine (H), asparagine (N),

serine (S) and threonine (T).



SEQ ID No: 56- CDR-H3_F136X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDXVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

phenylalanine (F), glycine (G), histidine (H),

isoleucine (I), leucine (L), methionine (M),

asparagine (N), glutamine (Q), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 57- CDR-H3_V137X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFXLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 58- CDR-H3_L138X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVXWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 59- DLX2464

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDMAGWGQGTLVTVSS



SEQ ID No.: 60- DLX2465

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMDLWGQGTLVTVSS



SEQ ID No.: 61- DLX2466

EIVMTQSPSTLSASVGDRVIITCQASQSIGKYLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSDAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMAGWGQGTLVTVSS



SEQ ID No.: 62- DLX2467

EIVMTQSPSTLSASVGDRVIITCQASQSIHNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGSSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSRAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERMIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 63- DLX2468

EIVMTQSPSTLSASVGDRVIITCQASQSIGNYLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGTSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMVLWGQGTLVTVSS



SEQ ID No.: 64- DLX2475

EIVMTQSPSTLSASVGDRVIITCQASQSIDKWLSWYQQKPGKAPKLLIYQ



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVHIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSYAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFKLWGQGTLVTVSS



SEQ ID No.: 65- DLX2476

EIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERDIFSGDFVGWGQGTLVTVSS



SEQ ID No.: 66- DLX2480

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDFVLWGQGTLVTVSS



SEQ ID No: 67- DLX2543

EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 68- DLX2529

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 69- DLX2547

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAF



GQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



RDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 70- DLX2528

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 71- DLX2585

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISK



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 72- DLX2545

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTASGFSLSSAAMAWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISR



DTSKNTLYLQMNSLRAEDTAVYFCARERNIFSGDMVLWGQGTTVTVSS



SEQ ID No.: 73- DLX2531

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 74- DLX2586

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISK



DTSKNTVYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTLVTVSS



SEQ ID No.: 75- DLX2530

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISR



DNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS



SEQ ID No.: 76- DLX2548

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISK



DTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No.: 77- DLX2676

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISR



DNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS



SEQ ID No.: 78- DLX2677

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 79- DLX2678

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISK



DTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No.: 80- DLX2679

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISK



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 81- DLX2680

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISR



DNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS



SEQ ID No.: 82- DLX2681

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 83- DLX2682

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISK



DTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No.: 84- DLX2683

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISK



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 85- DLX2684

EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISR



DNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS



SEQ ID No.: 86- DLX2685

EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLS



CTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISR



DNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 87- DLX2686

EIVMTQSPSTLSASVGDRVTITCQASOSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISK



DTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No.: 88- DLX2687

EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS



CTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISK



DTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 89- DLX2689

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



KASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAF



GQGTKVEEKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRL



SCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



RDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 90- DLX2690

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



KASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAF



GQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTIS



KDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No.: 91- DLX2691

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



KASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAF



GQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



KDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 92- DLX2692

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGIN1AF



GQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRL



SCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTIS



RDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS



SEQ ID No.: 93- DLX2693

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAF



GQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRL



SCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



RDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS



SEQ ID No.: 94- DLX2694

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAF



GQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTIS



KDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTVSS



SEQ ID No: 95- DLX2695

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAF



GQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



KDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS



SEQ ID No.: 96- VL CDR-L1_D32X

EIVMTQSPSTLSASVGDRVIITCQASQSIXNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No: 97- VL CDR-L1_N33X

EIVMTQSPSTLSASVGDRVIITCQASQSIDXWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No: 98- VL CDR-L1_W40X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNXLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of glutamic acid (E), phenylalanine

(F), glycine (G), methionine (M), asparagine (N),

glutamine (Q), serine (S), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 99- VL CDR-L2_R58X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYX



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), tryptophan (W) and tyrosine

(Y).



SEQ ID No.: 100- VL CDR-L2_T69X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASXLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 101- VL CDR-L3_T109X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNXGGGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

isoleucine (I), asparagine (N), serine (S),

threonine (T) and valine (V).



SEQ ID No.: 102- VL CDR-L3_G111X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGXGVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), glycine (G), proline

(P) and serine (S).



SEQ ID No.: 103- VL CDR-L3_G112X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGXVSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 104- VL CDR-L3_V135X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGXSIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 105- VL CDR-L3_S136X

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVXIAFG



QGTKLTVLG

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I),

leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 106- VH CDR-H1_S33X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSXAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 107- VH CDR-H1_A39X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSXAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

glutamine (Q), arginine (R), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 108- VH CDR-H2_Y59X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IXDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), glycine

(G), methionine (M) and tyrosine (Y).



SEQ ID No: 109- VH CDR-H2_D60X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYXSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of aspartic acid (D), asparagine (N)

and proline (P).



SEQ ID No.: 110- VH CDR-H2_Y69X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTXYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), aspartic acid (D),

glutamic acid (E), glycine (G), phenylalanine (F),

histidine (H), isoleucine (I), lysine (K), leucine

(L), methionine (M), proline (P), asparagine (N),

serine (S), threonine (T), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 111- VH CDR-H3_R110X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAREXA



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

glutamine (Q), arginine (R), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 112- VH CDR-H3_A111X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERX



IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), phenylalanine (F), glycine (G),

histidine (H), isoleucine (I), lysine (K),

methionine (M), asparagine (N), proline (P),

glutamine (Q), arginine (R), serine (S), threonine

(T), valine (V), tryptophan (W) and tyrosine (Y).



SEQ ID No.: 113- VH CDR-H3_I112X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



XFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

phenylalanine (F), histidine (H), isoleucine (I),

leucine (L), methionine (M), asparagine (N),

glutamine (Q), serine (S), threonine (T), valine

(V) and tyrosine (Y).



SEQ ID No.: 114- VH CDR-H3_F113X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IXSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of phenylalanine (F) and isoleucine

(I).



SEQ ID No.: 115- VH CDR-H3_S114X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFXGDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

glutamic acid (E), glycine (G), serine (S),

threonine (T) and valine (V).



SEQ ID No.: 116- VH CDR-H3_G115X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSXDFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), glycine (G),

methionine (M) and asparagine (N).



SEQ ID No.: 117- VH CDR-H3_D135X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGXFVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), aspartic acid (D),

glutamic acid (E), histidine (H), asparagine (N),

serine (S) and threonine (T).



SEQ ID No.: 118- VH CDR-H3_F136X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDXVLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C),

phenylalanine (F), glycine (G), histidine (H),

isoleucine (I), leucine (L), methionine (M),

asparagine (N), glutamine (Q), serine (S),

threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 119- VH CDR-H3_V137X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFXLWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 120- VH CDR-H3_L138X

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVXWGQGTLVTVSS

Preferably, X is selected from the group

consisting of alanine (A), cysteine (C), aspartic

acid (D), glutamic acid (E), phenylalanine (F),

glycine (G), histidine (H), isoleucine (I), lysine

(K), leucine (L), methionine (M), asparagine (N),

proline (P), glutamine (Q), arginine (R), serine

(S), threonine (T), valine (V), tryptophan (W) and

tyrosine (Y).



SEQ ID No.: 121- VH DLX2464

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQ



IFSGDMAGWGQGTLVTVSS



SEQ ID No.: 122- VH DLX2465

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERN



IFSGDNIDLWGQGTLVTVSS



SEQ ID No.: 123- VL DLX2466

EIVMTQSPSTLSASVGDRVIITCQASQSIGKYLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 124- VH DLX2466

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSDAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERN



IFSGDMAGWGQGTLVTVSS



SEQ ID No.: 125- VL DLX2467

EIVMTQSPSTLSASVGDRVIITCQASQSIHNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGSSIAFG



QGTKLTVLG



SEQ ID No.: 126- VH DLX2467

EVQLVESGGGLVQPGGSLRLSCTASGFSLSRAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERM



IFSGDFVLWGQGTLVTVSS



SEQ ID No.: 127- VL DLX2468

EIVMTQSPSTLSASVGDRVIITCQASQSIGNYLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGTSIAFG



QGTKLTVLG



SEQ ID No.: 128- VH DLX2468

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSSAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERN



IFSGDMVLWGQGTLVTVSS



SEQ ID No.: 129- VL DLX2475

EIVMTQSPSTLSASVGDRVIITCQASQSIDKWLSWYQQKPGKAPKLLIYQ



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVHIAFG



QGTKLTVLG



SEQ ID No.: 130- VH DLX2475

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSYAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFKLWGQGTLVTVSS



SEQ ID No.: 131- VL DLX2476

EIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 132- VH DLX2476

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERD



IFSGDFVGWGQGTLVTVSS



SEQ ID No.: 133- VL DLX2480

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFG



QGTKLTVLG



SEQ ID No.: 134- VH DLX2480

EVQLVESGGGLVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQ



IFSGDFVLWGQGTLVTVSS



SEQ ID No.: 135- VL DLX2543

EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYK



ASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 136- VL DLX2529

EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYR



ASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFG



QGTKLTVLG



SEQ ID No.: 137- VL DLX2547

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



RASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAF



GQGTKLEIKR



SEQ ID No.: 138- VH DLX2547

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS



SEQ ID No.: 139- VL DLX2528

EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQ



ASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFG



QGTKLTVLG



SEQ ID No.: 140- VH DLX2528

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFVLWGQGTLVTVSS



SEQ ID No.: 141- VL DLX2585

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 142- VH DLX2585

EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERA



IFSGDFDYWGQGTLVTVSS



SEQ ID No.: 143- VL DLX2545

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 144- VH DLX2545

EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWIGI



IYDSASTYYASWAKGRFTISRDTSKNTLYLQMNSLRAEDTAVYFCARERN



IFSGDMVLWGQGTTVTVSS



SEQ ID No.: 145- VL DLX2531

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 146- VH DLX2531

EVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGI



IYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERA



IFSGDFALWGQGTLVTVSS



SEQ ID No.: 147- VL DLX2586

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 148- VH DLX2586

EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGI



IYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYFCARERQ



IFSGDMDGWGQGTLVTVSS



SEQ ID No.: 149- VL DLX2530

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 150- VH DLX2530

EVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGI



IYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERN



IFSGDMALWGQGTTVTVSS



SEQ ID No.: 151- VL DLX2548

EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYR



ASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG



QGTKLTVLG



SEQ ID No.: 152- VH DLX2548

EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGI



IYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQ



IFSGDMDGWGQGTTVTVSS



SEQ ID No.: 153- VL DLX2544

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIY



KASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAF



GQGTKVEKR



SEQ ID No.: 154- DLX2544

ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYOQKPGKAPKLLIY



KASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAF



GQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL



SCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTIS



RDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS



SEQ ID No.: 155- CDR-H1 of DLX2531

FSLSNSAMA



SEQ ID No.: 156- CDR-H2 of DLX2531

IIYDSASTYYASWAKG



SEQ ID No.: 157- CDR-H3 of DLX2531

ERAIFSGDFAL



SEQ ID No.: 158- CDR-L1 of DLX2531

QASQSIDNWLS



SEQ ID No.: 159- CDR-L2 of DLX2531

RASTLAS



SEQ ID No.: 160- CDR-L3 of DLX2531

QNTGGGVSIA



SEQ ID No.: 161- CDR-L1 of DLX2681

RASQSIGNWLS



SEQ ID No.: 162- CDR-L2 of DLX2681

RASNLAS



SEQ ID No.: 163- CDR-L3 of DLX2681

QNTGGGINIA






























































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































EXAMPLES
Example 1
Identification of rhIL-1 Beta Neutralizing scFv
Immunization of rabbits: Rabbits were immunized with recombinant human IL-1 beta protein (Peprotech, USA, cat. no. 200-01B), Lymph node and spleen cells were isolated after the final boost and the cells were cryopreserved.
Flow cytometry sorting of rabbit B cells and culturing: IL-1 beta-specific memory B cells were sorted as single cells into 96-well microplates using FACSAria III (BD Biosciences). Single B cell clones were cultured in the presence of feeder cells.
Screening of B cell clones: Cell culture supernatants were analyzed by ELISA for the presence of anti-IL-1 beta-specific IgGs. Briefly, rhIL-1 beta (Peprotech, cat. no. 200-01B) was coated at a concentration of 2 mcg/mil overnight at 4° C. on Maxisorp 96-well microplates in PBS. After blocking with $% non-fat dry milk, cell culture supernatants were added. IL-1 beta-specific IgGs were detected by anti-rabbit IgG-HRP (Southern Biotech, cat. no. 4050-05). The ELISA was developed with BM Blue POD substrate (Roche Applied Science). B cell clones specific for rhIL-1 beta were further analyzed for their neutralization capacity in a human fibroblast assay.
Sequencing of IL-1 beta-neutralizing IgGs: all rabbit B cell clones producing neutralizing anti-IL-1 beta antibodies were subjected to mRNA isolation using the RNeasy Mini Kit (Qiagen Germany, cat. no. 74106). mRNA was used as template for reverse transcription according to the manufacture's protocol (OneStep RT-PCR kit, Qiagen Germany, cat. no. 21.0212). Subsequently, PCR reactions using oligonucleotides to specifically amplify rabbit IgG heavy and light chain encoding sequences were carried out (Biometra Thermocycler T3). Heavy and light chain PCR fragments were independently sequenced (ABI, Sanger 3730xl; Microsynth AG, Balgach, Switzerland), and obtained DNA sequences were translated into protein sequences using EMBOSS Transeq and aligned using CLUSTALW2.
Construction of anti-IL-1 beta scFv genes, and scFv protein expression: rabbit IgG CDR regions of the light and the heavy chains as defined above were identified and grafted into the human light and heavy chain acceptor frameworks comprising SEQ ID Nos.: 18-21 and 22-25, respectively. Bacterial expression vectors were generated encoding scFv proteins with the N-terminal variable light chain linked by the sequence SEQ ID No: 9 to the C-terminal variable heavy chain. ScFv proteins were expressed in E. coli BL21 (DE3); Novagen, USA, cat. no. 69450-3) as inclusion bodies, which were purified, solubilized and the proteins were refolded. The refolded scFvs were purified by standard size exclusion chromatography and monomeric peak fractions were collected. Purified scFvs were analyzed for IL-1 beta binding by ELISA. ScFv that were found to specifically bind rhIL-1 beta were tested for IL-1 beta neutralization in a human fibroblast assay. By this procedure, the scFv DLX2323 and other anti-IL-1 beta scFvs were identified as potent inhibitors of IL-1 beta.
Example 2
Recognition of Human IL-1 Beta
Firstly, the specific recognition of rhIL-1 beta by DLX2323 was confirmed by ELISA ( FIG. 1 ). Briefly, rhIL-1 beta (Peprotech, cat. no. 200-01B) was coated at a concentration of 2 mcg/mil overnight at 4° C. on Maxisorp 96-well microplates in PBS. After blocking with 5% non-fat dry milk, increasing concentrations of scFvs (10 to 300 ng/ml) were added, and scFvs were detected by Protein L-HRP (Sigma-Aldrich, cat. no. P3226). The ELISA was developed with BM Blue POD substrate (Roche Applied Science). As a negative control, scFv of irrelevant specificity was used. This result shows that DLX2323 specifically binds to rhIL-1 beta.
To confirm that DLX2323 and the control scFv were recognized by Protein L-HRP and, thus, a lack of signal for rhIL-1 beta binding in the ELISA above was not due to a detection problem, another experiment was conducted. The scFvs were directly coated on the plate and detected by Protein L-HRP as described above. All scFvs were coated at a concentration of 2 mcg/ml in PBS. This ELISA experiment showed that DLX2323 and the control scFv are recognized by the detection agent Protein L-HRP.
In another ELISA ( FIG. 2 ), the recognition of human natural IL-1 beta by DLX2323 was confirmed. As commonly known, expression of human proteins in cells other than human cells might cause changes, e.g., in post-translational modifications and/or conformation. This might lead to a differential recognition of recombinant and natural proteins by antibodies. Natural human IL-1 beta was secreted by THP-1 cells (DSMZ Germany, cat. no. ACC 16) after stimulation with 10 ng/ml of PMA (Sigma-Aldrich, cat. no. P1585), 1 mg/ml of LPS (Sigma-Aldrich, cat. no. L4391) and 2 mM of ATP (Sigma-Aldrich, cat. no. A6559-25UMO). Cell supernatants were harvested and secreted human natural IL-1 beta was quantified using the Human IL-1 beta/IL-1F2 ELISA DuoSet (R&D Systems, cat no. DY201). DLX2323 was coated on 96-well microplates (Maxisorp, Nunc) at a coating density of 5 mcg/ml in DPBS (pH 7.4). Human IL-1 beta either as recombinantly expressed version (Peprotech, cat, no. 200-01B) or as natively secreted version were applied at final concentrations ranging from 0.5 to 4 ng/ml. Bound IL-1 beta was detected by a biotinylated goat anti-hIL-1 beta antibody (R&D Systems, cat. no. DY201) and Streptavidin-HRP (BD Pharmingen, cat. no. 554060). The ELISA was developed using BM Blue POD substrate (Roche Applied Science). For quantification purposes the absorbance was measured at 450 nm using a VersaMax microplate reader (Molecular Devices, USA). The result (see FIG. 2 ) shows that both, recombinant and natural human IL-1 beta are recognized by DLX2323 at comparable levels.
Example 3
Neutralization of rhIL-1 Beta Biological Activity
Antibodies and scFvs were tested for their IL-1 beta neutralization capacity in a human dermal fibroblast assay (NHDF-Neo, cat. no. CC-2509, Lonza Walkersville USA). Activation of such fibroblasts with IL-1 beta leads to specific IL-6 release which is quantified by ELISA. Inhibition of IL-1 beta by specific antibodies decreases the amount of IL-6 released from such fibroblasts. The inhibitory potency of the anti-IL-1 beta antibody is quantified by measuring the half-maximal reduction (IC 50 ) of IL-1 beta-induced IL-6 release. Human dermal fibroblasts were seeded in 96-well microplates at 5′000 cells/well 16-20 hours prior to addition of IL-1 beta. The fibroblasts were cultured in fibroblast basal medium (FBM; Lonza, cat. no. CC-3131) with supplements (hFGF-B, Insulin, FBS, GA-1000) as described by the cell supplier (Lonza Walkersville USA: CLONETICS™ Dermal Fibroblast Cell Systems). FBM then was removed and cells were washed once with Dulbecco's Modified Eagle Medium (DMEM; Gibco, Life Technologies, cat. no. 11880) to remove growth factors. Cells were then incubated for 7 hours in DMEM media. Antibodies or scFvs and rhIL-1 beta were pre-incubated in DMEM for 1 hour at 37° C. The mixture was added, to the cells at a final concentration of 10 pg/ml of IL-1 beta. As negative control, 10 pg/ml of IL-1 beta was added to cells without any anti-IL-1 beta antibody. As positive control, a mouse monoclonal antibody against IL-1 beta was applied (R&D systems, USA, cat. no. MAB201). The cells were incubated with the IL-1 beta/anti-IL-1 beta antibody mixture for 18-24 hours, and cell culture supernatants were analyzed for IL-6 release using the Human IL-6 DuoSet ELISA Kit according to the manufacturer's instructions (R&D Systems, USA, cat. no. DY206).
The IC 50 of DLX2323 was determined to be 3 pM±1.05 in eight independent assays.
Example 4
Comparison of Neutralization Potency with Commercially Available IL-1 Beta Inhibitors
DLX2323 was identified as an IL-1 beta neutralizing scFv. The biological potency of DLX2323 and other inhibitors was assessed in the human dermal fibroblast assay as described in Example 3. Recombinant human IL-1 beta was pre-incubated with increasing concentrations of the scFv DLX2323, the anti-human IL-1 beta monoclonal IgG antibody MAB201, the IL-1 beta receptor antagonist (rhIL-1ra) (R&D systems, cat. no. 280-RA-010/CF) or the FDA approved, marketed canakinumab IgG (Novartis, ILARIS®) prior to addition to the wells. Two independent experiments were performed: FIG. 3A depicts the comparison of DLX2323 with MAB201, whereas FIG. 3B shows the comparison of DLX2323 with rhIL-1ra and canakinumab. The following IC 50 values were determined; MAB201: 2-3 pM; DLX2323: 2-4 pM; rhIL-1ra: 40 pM and Canakinumab: 90 pM. In conclusion, the monovalent monomeric scFv DLX2323 is almost as potent as the bivalent monoclonal mouse antibody MAB201. It shows distinctly higher potency in neutralizing human IL-1 beta than the marketed inhibitor Canakinumab and the rhIL-1ra. Furthermore, DLX2323 could fully block IL-1 beta-induced IL-6 release.
Example 5
Solubility
DLX2323 was stored in PBS buffer pH 7.2 (Phosphate Buffered Saline 1×, Gibco, LIFE TECHNOLOGIES™, cat. no. 20012). To determine its maximum solubility, DLX2323 was concentrated using Vivaspin 20 centrifuge concentrators (Sartorius Stedim Biotech, cat. no, VS2001) at room temperature. The concentration process was stopped at 71 mg/ml due to the high viscosity of the sample. The obtained DLX2323 protein solution was viscous, clear, and no precipitates were observed by visual inspection.
Example 6
Stability
Regarding the stability of scFvs, two different processes can be observed that contribute to their instability. Firstly, the scFv could be prone to dimerization, often followed by oligomerization and eventually aggregation. Secondly, scFv degradation, leading to smaller fragments, can occur over time. To judge whether DLX2323 is stable, HPLC (Dionex, Summit system) size exclusion chromatography (Tosoh, TSKgel G2000SWxl, cat. no. 08540) was deployed to determine the percentage of monomeric, non-degraded scFv protein at certain time points at different protein concentrations and temperatures (e.g., 4° C., RT and 37° C.). The percentage of monomer was measured at the starting point of the study (T0) and after one month for 1 mg/ml of DLX2323, and, in another experiment, after two weeks for a 50 mg/ml solution of DLX2323. The protein was formulated in PBS, pH 7.2. The results of the stability study are listed in tables 1 and 2.








TABLE 1





monomer content



after 1 month
Observation










DLX2323, 1 mg/ml, T0
97%


DLX2323, 1 mg/ml, stored at RT
98%

DLX2323, 1 mg/ml, stored at 37° C.
93%
Very low levels



of degradation

































TABLE 2





monomer



content


after 2 weeks
Observations










DLX2323, 50 mg/ml, T0
97%


DLX2323, 50 mg/ml, stored at 4° C.
78%
dimerization

DLX2323, 50 mg/ml, stored at RT
76%
oligomerization

DLX2323, 50 mg/ml, stored at 37° C.
43%
oligomerization


























DLX2323's thermal stability was assessed by differential scanning fluorimetry (DSF). For this measurement a real-time PCR device (Corbett, Rotor-Gene) heated DLX2323 in a temperature gradient from 30° C. to 95° C. (raising in 1° C. steps, waiting 5 seconds per step). The protein sample contained 0.5 mg/ml of DLX2323 and 20× SYPRO® Orange (Sigma-Aldrich, cat. no. 5692, 5000×) in PBS. As soon as the protein started melting, Sypro Orange turned fluorescent. This fluorescence was online measured (excitation wavelength of 470 nm; emission wavelength of 555 nm) during the gradient run. Using Rotor-Gene 6000 Series Software 1.7 the midpoint melting temperature (Tm) of DLX2323 was calculated to be 74° C.
Example 7
Cross-Reactivity
Cross-reactivity of DLX2323 to IL-1 beta homologs of other species than human beings was assessed in ELISA. Binding to the recombinantly expressed IL-1 beta proteins of the following species was investigated: cynomolgus (Sino Biological Inc., USA, cat. no. 90010-CNAE), rhesus macaque (R&D Systems, USA, cat. no. 1318-RL/CF), swine (Kingfisher Biotech, USA, cat. no, RP0297S-025), canine (Kingfisher Biotech, USA, cat. no. RP0085D-025), guinea pig (Kingfisher Biotech, cat. no. RP0343GP-025), rat (Peprotech, cat no. 400-01B) and mouse (BioLegend, cat. no. 575102). Binding of DLX2323 was compared to ELISA-positive control antibodies (R&D Systems, USA, goat anti-human IL-1 beta polyclonal IgG, cat. no. AB-201-NA; BioLegend, Inc., USA, biotin anti-mouse/rat IL-1 beta antibody, cat. no. 503505). Briefly, proteins were coated at a concentration of 2 mcg/ml over night at 4° C. on Maxisorp 96-well microplates in PBS. After blocking with 5% non-fat dry milk, increasing concentrations (0.1 mcg/ml, 0.3 mcg/ml and 1.0 mcg/ml) of DLX2323 were added to the wells. Successful coating of every protein was separately confirmed exploiting IL-1 beta-specific control antibodies. Whereas DLX2323 was detected by Protein L-HRP (Sigma-Aldrich, USA, cat. no. P3226), the control antibodies were detected by either Streptavidin-HRP (BD Pharmingen, USA, cat, no. 554060) or other eligible secondary antibodies labelled with HRP. The ELISA was developed with BM Blue POD substrate (Roche Applied Science) and the absorbance was measured at 450 nm. DLX2323 recognized four species orthologs of IL-1 beta, namely human, cynomolgus, rhesus macaque and rat IL-1 beta. No cross-reactivity could be observed for porcine, guinea pig, canine and mouse IL-1 beta.
Besides the cross-reactivity of DLX2323 to IL-1 beta homologs of other species than human beings, the recognition pattern of DLX2323 regarding various human IL-1 family members and other cytokines was measured: rhIL-1ra (R&D systems, USA, cat. no. 280-RA-010/CF), rhIL-1 alpha (PeproTech, cat. no. 200-01A), rhIL-18 (BioVision, cat. no. 4179-25), rhIL-33 (Peprotech, cat. no. 200-33), IL-36ra (R&D Systems, cat. no, 1275-IL/CF), rhTNF alpha (Peprotech, Hamburg, Germany cat. no. 300-01A) and rhIL-6 (Peprotech, cat. no. 200-06). In the applied ELISA assay the following antibodies served as positive controls: biotin anti-human IL-1ra (BioLegend, cat, no. 509501), biotin anti-human IL-1 alpha (BioLegend, cat. no. 515703), anti-human IL-18 polyclonal antibody (BioVision, cat. no. 5179-100), biotin anti-human IL-33 antibody (Peprotech, cat. no. 500-P261Bt), anti-human TNF alpha scFv DLX105 (Delenex propriety antibody described in WO 2006/131013 A), biotin anti-human IL-6 (R&D systems, DY206, cat. no. 840114), anti-human IL-36ra (R&D systems, cat. no. AF1275). The ELISA was carried out essentially as described above. No cross-reactivities of DLX2323 for any of these tested human IL-1 family members and cytokines could be detected.
Example 8
In Vivo Efficacy
In this example, the in vivo inhibition of human IL-1 beta activity by DLX2323 is demonstrated. Human IL-1 beta can bind and activate the mouse IL-1 receptor thereby inducing an inflammatory response in the mouse in vivo. The inflammation leads to elevated levels of cytokines in the serum including mouse IL-6 (mIL-6). Recombinant human IL-1 beta (Peprotech, cat, no. 200-01B) was administered subcutaneously at a dose of 1.5 mcg/kg body weight to 8 weeks old male BALB/c mice (Charles River, Germany). After 2 hours mL-6 levels were significantly elevated in serum. To test the neutralization capacity of DLX2323 in vivo, it was intraperitoneally (i.p.) injected two hours prior to the IL-1 beta dosing. One group of mice was injected with a 5 mg/kg dose of DLX2323, the second group was injected with a 15 mg/kg dose of DLX2323. Negative control groups were treated with either PBS i.p. or scFvs of irrelevant specificity. A fifth group of mice was intravenously injected with 10 mg/kg of canakinumab (Novartis, ILARIS®) as a positive control. Two hours after the rhIL-1 beta application blood samples were taken and serum levels of mIL-6 were measured using the Mouse IL-6 DuoSet ELISA kit according to the manufacturer's instructions (R&D Systems, cat. no. DY406). For the groups of mice treated with DLX2323 and canakinumab only very low amounts of mIL-6 or even no mIL-6 could be detected (0.0-2 pg/ml of mIL-6; FIG. 4 ). Mice receiving PBS or control scFv showed significantly elevated IL-6 levels of 50 to 170 pg/ml. DLX2323 was very efficiently neutralizing human IL-1 beta in an in vivo setting, even at a 5 mg/kg dose.
Example 9
CDR Libraries
To better characterize the association of DLX2323 with human IL-1 beta, amino acid mutations were designed and site-specifically inserted into the CDR regions of DLX2323. CDR positions were chosen for mutagenesis based on their surface exposure, anticipated interaction with human IL-1 beta as deduced from homology models or sequence comparisons with antecedent rabbit IgGs. In the light chain, the motif DNW in CDR-L1 (SEQ ID No: 14), the amino acid residues R and T in CDR-L2 (SEQ ID No: 15), the threonine in CDR-43 as well as the motif GGVS were selected (SEQ ID No: 16) for the design of variants of DLX2323. In the heavy chain, the motif SA was chosen in CDR-H1 (SEQ ID No: 11), in the CDR-H2 the motif YD and the following tyrosine (SEQ ID No: 12). In CDR-H3, all positions were selected for substitution (SEQ ID No: 13) except for the N-terminal glutamic acid (E). All said positions are indicated with an X in SEQ ID Nos. 11-16, respectively. Site directed mutagenesis, sequencing of clones and library assembly were carried out by GeneArt (LIFE TECHNOLOGIES™, Regensburg, Germany).
The resulting scFv mutants are expressed in 96-well format in 1 ml cultures in E. coli BL21 (DE3) (Novagen, USA, cat. no. 69450-3). During expression in this system most of the scFv proteins form insoluble inclusion bodies within the cell but a considerable amount of protein can be retrieved from the soluble fractions after cell lyses, which was found to be sufficient for the analysis by the rhIL-1 beta ELISA (see example 2 for details). Cells were lysed in lysis buffer (1 mM EDTA, 0.1 mg/ml lysozyme, PBS pH 7.2) in a 96-well format by freezing and subsequent thawing. Derived crude extracts were cleared by centrifugation. Supernatants were added to wells of a microtiterplate coated with 50 ng/ml of rhIL-1 beta (Peprotech) per well. After washing, bound scFvs are detected by ProteinL-HRP and binding to rhIL-1 beta quantified by BM Blue POD Substrate (Roche Applied Science).
Table 3 lists single-site mutations that clearly permit binding of rhIL-1 beta as defined by an ELISA signal at least 2-fold over the negative control, but not smaller than 0.1 optical units.






TABLE 3



Residue position
amino acid substitutions



CDR-L1_D32
A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,


Y

CDR-L1_N33
A, C, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, Y

CDR-L1_W40
E, F, G, M, N, Q, S, Y

CDR-L2_R58
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, W, Y

CDR-L2_T69
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W,


Y

CDR-L3_T109
A, C, I, N, S, V

CDR-L3_G111
A, P, S

CDR-L3_G112
A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W,


Y

CDR-L3_V135
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W,


Y

CDR-L3_S136
A, C, D, E, F, G, H, I, L, M, N, P, Q, R, T, V, W, Y

CDR-H1_S33
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W,


Y

CDR-H1_A39
C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y

CDR-H2_Y59
A, C, G, M

CDR-H2_D60
N, P

CDR-H2_Y69
A, D, E, G, F, H, I, K, L, M, N, P, S, T, W

CDR-H3_R110
A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, Y

CDR-H3_A111
C, D, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y

CDR-H3_I112
A, C, F, H, L, M, N, Q, S, T, V, Y

CDR-H3_F113
I

CDR-H3_S114
A, C, E, G, T, V

CDR-H3_G115
A, M, N,

CDR-H3_D135
A, E, H, N, S, T

CDR-H3_F136
A, C, G, H, I, L, M, N, Q, S, T, V, W, Y

CDR-H3_V137
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W,


Y

CDR-H3_L138
A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W,


Y
















































These binding data have predictive value also concerning the neutralization of rhIL-t beta biological function as verified by the expression of five exemplary single-site mutants in large scale format, purified to homogeneity, and subjected to cell-based analysis alongside with likewise purified DLX2323 protein.
DLX2323_CDR-H1_A39N (SEQ ID No. 45 with X=N), DLX2323_CDR-H3_A111N (SEQ ID No.: 50 with X=N), DLX2323 CDR-H3_F136N (SEQ ID No. 56 with X=N), DLX2323_CDR-H3 V137D (SEQ ID No.: 57 with X=D), and DLX2323_CDR-H3_L38D (SEQ ID No.: 58 with X=D) and the corresponding parental scFv, DLX2323, were expressed in E. coli BL21(DE3) (Novagen) cells at a 2 liter scale. Following cell lysis by sonication, inclusion bodies were enriched by centrifugation, washed, protein solubilized in guanidine buffer (6 M guanidine/HCl, 100 mM Tris, 1 mM EDTA, pH 8.5), refolded in urea buffer (4 M urea, 50 mM glycine, 2 mM cystine, 2 mM cysteine, pH10), concentrated in 50 mM glycine, 50 mM NaCl, pH10, and purified to homogeneity by size exclusion chromatography. Monomeric peak fractions of this procedure were concentrated to a final protein content of approximately 0.5-5 mg/ml, passed over a sterile filter unit, and as such subjected to cell-based analyses as described in Example 3. Table 4 shows the neutralization potency IC 50 of each of the mutants as determined in two independent runs.








TABLE 4





Clone ID
IC 50












DLX2323
2-4
pM


DLX2323_CDR-H1_A39N
10
pM


DLX2323_CDR-H3_A111N
2-4
pM


DLX2323_CDR-H3_F136N
2-4
pM


DLX2323_CDR-H3_V137D
5
pM


DLX2323_CDR-H3_L138D
10
pM



























In overall good agreement with the preceding ELISA results, an IC 50 value in the low pM range was observed for all five single-site mutants selected. Each member of the selected mutant subset had a neutralizing capacity towards rhIL-1 beta comparable to that of the parental scFv DLX2323. Accordingly, the binding of DLX2323 and its variants to rhIL-1 beta shows that a significant degree of sequence variability is allowed while concomitantly preserving key functional features.
Example 10
Generation of Combinatorial Variants
Based on the above, combinatorial mutants of DLX2323 were designed combining 3 to 9 of the previously investigated CDR singles-site changes. Individual residue changes were selected on basis of; (i) strong ELISA binding results comparable to or better than that of the parent antibody DLX2323; (ii) combinations of far-spread single site changes that would simultaneously affect multiple CDR regions; and (iii) a cumulative modulation of CDR-H3, alone. The designed mutants were expressed in large scale format and purified as described above for the single mutants, before the neutralization capacity was assessed in the human fibroblast assay of Example 3. Individual amino acid changes per sequence and IC 50 data of corresponding scFv proteins are detailed in Table No.: 5. All these isolated scFv proteins had neutralizing activity towards rhIL1-beta in vitro. The five best candidates displayed IC 50 values in the low picomolar range, and are thus highly comparable to the parental scFv DLX2323.







TABLE 5



scFv
Amino acid changes (AHo annotation)
IC50










DLX2464
VL: parental
4
pM


VH: A111Q, F136M, V137A, L138G

DLX2465
VL: parental
4
pM


VH: A111N, F136M, V137D

DLX2466
VL: D32G, N33K, W40Y, T109A
300
pM


VH: A39D, A111N, F136M, V137A, L138G

DLX2467
VL: D32H, T69N, V135S
25
pM


VH: S33R, A111M

DLX2468
VL: D32G, W40Y, T109A, V135T
15
pM


VH: A39S, A111N, F136M

DLX2475
VL: N33K, R58Q, S136H
200
pM


VH: A39Y, V137K

DLX2476
VL: D32S, N33S
100
pM


VH: A111D, L138G

DLX2480
VL: V135I, S136N
5
pM


VH: S33D, A111Q




































Variants carrying more than 6 amino acid substitutions in the CDR and flanking framework regions of the variable light and/or the variable heavy chain were designed. In total, combinatorial candidates (four VL and six VH mutant sequences) were generated, expressed, and functionally analysed for rhIL1-beta binding (ELISA) and neutralization (human fibroblast assay) in vitro as described in Example 3. Table 6 summarizes the respective point mutations as well as the neutralization potency as measured in two independent experiments of each clone.







TABLE 6



ScFv
Amino acid changes (AHo annotation)
IC50










DLX2543
VL: I20T, D32S, N33S, R58K, A87T, T109A
3-4
pM


VH: parental

DLX2529
VL: Q24R, D32G, T69N, D99E, V135I, S136N
1-2
pM


VH: parental

DLX2547
VL: E1D, I20T, D32S, N33S, W40Y, V135I,
30
pM


S136N, T146E, V147I, L148K, G149R


VH: parental

DLX2528
VL: I20T, D32G, S42A, R58Q, T69N, A87T,
8
pM


E88D, T109A, G112A, V135T, S136T


VH: parental

DLX2585
VL: parental
1-2
pM


VH: A25V, A39Y, A42S, R82K, V137D, L138Y

DLX2545
VL: parental
7
pM


VH: V55I, V89L, Y105F, A111N, F136M,


L144T

DLX2531
VL: parental
0.6-1
pM


VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2586
VL: parental
2
pM


VH: A25V, A39Y, A42S, V55I, R82K, Y105F,


A111Q, F136M, V137D, L138G

DLX2530
VL: parental
1-2
pM


VH: L12N, S33D, Y69F, T84N, V89L, V103T,


A111N, F136M, V137A, L144T

DLX2548
VL: parental
1-2
pM


VH: A25V, A39Y, A42S, V55I, R82K, V89L,


Y105F, A111Q, F136M, V137D, L138G, L144T














































All 10 scFv proteins were found to effectively neutralize rhIL1-beta in the human fibroblast assay in vitro with IC 50 values in the high femtomolar to low picomolar range. Clones DLX2531, DLX2548, DLX2585, DLX2530, DLX2529, and DLX2586 reproducibly yielded lower IC 50 values than the parental scFv DLX2323.
Finally, the above VL and VII sequences were chain shuffled. Binding to rhIL1-beta was confirmed for cleared lysates of E. coli BL21 origami cells transformed with 19 chain shuffled VL and VH mutants. Table 7 summarizes the substitutions.







TABLE 7



ScFv
VL-VH fusion
Amino acid substitutions



DLX2676
DLX2528_2530
VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A,



G112A, V135T, S136T



VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,



V137A, L144T

DLX2677
DLX2528_2531
VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A,



G112A, V135T, S136T



VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2678
DLX2528_2548
VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A,



G112A, V135T, S136T



VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F, A111Q,



F136M, V137D, L138G, L144T

DLX2679
DLX2528_2585
VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A,



G112A, V135T, S136T



VH: A25V, A39Y, A42S, R82K, V137D, L138Y

DLX2680
DLX2529_2530
VL: Q24R, D32G, T69N, D99E, V135I, S136N



VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,



V137A, L144T

DLX2681
DLX2529_2531
VL: Q24R, D32G, T69N, D99E, V135I, S136N



VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2682
DLX2529_2548
VL: Q24R, D32G, T69N, D99E, V135I, S136N



VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F, A111Q,



F136M, V137D, L138G, L144T

DLX2683
DLX2529_2585
VL: Q24R, D32G, T69N, D99E, V135I, S136N



VH: A25V, A39Y, A42S, R82K, V137D, L138Y

DLX2684
DLX2543_2530
VL: I20T, D32S, N33S, R58K, A87T, T109A



VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,



V137A, L144T

DLX2685
DLX2543_2531
VL: I20T, D32S, N33S, R58K, A87T, T109A



VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2686
DLX2543_2548
VL: I20T, D32S, N33S, R58K, A87T, T109A



VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F, A111Q,



F136M, V137D, L138G, L144T

DLX2687
DLX2543_2585
VL: I20T, D32S, N33S, R58K, A87T, T109A



VH: A25V, A39Y, A42S, R82K, V137D, L138Y

DLX2689
DLX2544_2531
VL: BID, I20T, D32S, N33S, W40Y, R58K, A87T, E88D, D99E,



T109A, V135I, S136N, L145V, T146E, V147I, L148K, G149R



VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2690
DLX2544_2548
VL: E1D, I20T, D32S, N33S, W40Y, R58K, A87T, E88D, D99E,



T109A, V135I, S136N, L145V, T146E, V147I, L148K, G149R



VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F, A111Q,



F136M, V137D, L138G, L144T

DLX2691
DLX2544_2585
VL: E1D, I20T, D32S, N33S, W40Y, R58K, A87T, E88D, D99E,



T109A, V135I, S136N, L145V, T146E, V147I, L148K, G149R



VH: A25V, A39Y, A42S, R82K, V137D, L138Y

DLX2692
DLX2547_2530
VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E,



V147I, L148K, G149R



VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,



V137A, L144T

DLX2693
DLX2547_2531
VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E,



V147I, L148K, G149R



VH: L12N, S33N, A39S, T84N, V103T, V137A

DLX2694
DLX2547_2548
VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E,



V147I, L148K, G149R



VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F, A111Q,



F136M, V137D, L138G, L144T

DLX2695
DLX2547_2585
VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E,



V147I, L148K, G149R



VH: A25V, A39Y, A42S, R82K, V137D, L138Y










































































Binding was considered specific if an ELISA signal of ≧0.1 optical units was obtained which was at least 2-fold higher than the negative control. Results are shown in FIG. 6 . For example, DLX2690 and DLX2691 slightly passed the cut-off filter, but are tested positive. The remaining collective VL and VH chain-shuffled mutants bind rhIL1-beta with absolute ELISA signals comparable to or even higher than DLX2323.
The ICs values of DLX2681 and DLX2693 were determined in the human fibroblast assay as described above. DLX2681 was found to neutralize rhIL1-beta with an IC 50 value of 0.6 pM and the IC 50 value of DLX2693 was determined to be 12.5 pM.
While there are shown and described presently preferred embodiments of the invention, it is to be understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. Since numerous modifications and alternative embodiments of the present invention will be readily apparent to those skilled in the art, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the best mode for carrying out the present invention. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the following claims.