Antibodies Directed To GPNMB And Uses Thereof

Abstract

The present invention provides fully human monoclonal antibodies that specifically bind to GPNMB, and uses thereof. Nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDRs) are provided. The present invention also provides immunoconjugates comprising anti-GPNMB antibodies and methods of using such immunoconjugates. The present invention further provides bi-specific antibodies comprising an anti-GPNMB antibody component and an anti-CD3 component, and methods of using such bispecific antibodies.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 14/454,179, filed Aug. 7, 2014, which is a divisional of U.S. patent application Ser. No. 13/355,366, filed Jan. 20, 2012 (now U.S. Pat. No. 8,846,873), which is a continuation of U.S. patent application Ser. No. 13/151,690, filed Jun. 2, 2011, which is a continuation of U.S. patent application Ser. No. 12/911,269, filed Oct. 25, 2010, which is a continuation of U.S. patent application Ser. No. 12/721,099, filed Mar. 10, 2010, which is a continuation of U.S. patent application Ser. No. 12/506,029, filed Jul. 20, 2009, which is a continuation of U.S. patent application Ser. No. 12/290,779, filed Nov. 3, 2008, which is a continuation of U.S. patent application Ser. No. 11/792,032, which is a national stage application, filed under 35 U.S.C. .sctn.371, of International Application No. PCT/US2005/043482, filed on Nov. 30, 2005, which claims priority to U.S. Provisional Application No. 60/632,023, filed Nov. 30, 2004, and U.S. Provisional Application No. 60/733,779, filed Nov. 7, 2005; the contents of each of which are hereby incorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The contents of the text file named "669USSeqList.txt", which was created on Jan. 20, 2012 and is 167 KB in size, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to antibodies with specificity to GPNMB, and uses of such antibodies. In particular, the present invention provides fully human monoclonal antibodies that specifically bind to GPNMB, and uses thereof. Nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDRs) are provided. The present invention also provides immunoconjugates comprising anti-GPNMB antibodies and methods of using such immunoconjugates. The present invention further provides bi-specific antibodies comprising an anti-GPNMB antibody component and an anti-CD3 component, and methods of using such bispecific antibodies.

BACKGROUND OF THE INVENTION

GPNMB

[0004] A putative transmembrane glycoprotein called "nmb" (Acc. No. X76534 EMBL), referred to herein as GPNMB, was identified and described by Weterman et al., (Int J Cancer 60:73-81, 1995) as differentially expressed in low-metastatic human melanoma cancer cell lines and xenografts, compared to a more aggressive melanoma cell line. GPNMB shares 33% identity with the precursor of pMe 117 melanocyte-specific protein (Kwon et al., 1991, PNAS 88:9228-9232). GPNMB is 71% homologous to a dendritic cell-associated transmembrane protein, DC-HIL (Shikano et al., 2001 Biol. Chem. 276:8125-8134). GPNMB is also known as the hematopoietic growth factor inducible neurokinin-1 protein HGFIN (Bandari et al, Reg. Peptides 111:169-178) and the bone-related gene osteoactivin (Owen et al. Crit Rev Eukaryot Gene Expr 2003, 13(2-4):205-220)

[0005] It was also reported that nmb could reduce the metastatic potential of a highly metastatic nmb-negative melanoma cell line (Weterman, 1995). GPNMB was considered a candidate glioblastoma tumor marker after public database mining and expression profiling (Loging et al., 2000, Genome Research 10:1393-1402). This gene was found overexpressed in lung tumors (US Patent Publication No. US20030064947), as well as breast, rectal and colon cancers (US Patent Publication No. US2003100720). NCBI SAGE data also shows overexpression of this gene in stomach and pancreatic carcinoma. The mouse ortholog has been shown to be highly upregulated in a neural stem cell line NSC, derived from the TSC2 knockout model for Tuberous Sclerosis Complex Syndrome (International Publication No. WO 2003/080856).

Antibodies

[0006] Antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains (about 25 kDa) and two heavy (H) chains (about 50-70 kDa). The amino-terminal portion of each chain includes a variable domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the L and H chain has one and three or four constant domains, respectively that are primarily responsible for effector function. There are two types of human L chains, classified as kappa and lambda. H chains are classified as mu, delta, gamma, alpha, or epsilon based upon the constant domain amino acid sequence, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Isotypes may be further divided into subclasses e.g. IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4.

[0007] Immunoglobulins can be produced naturally in vivo by B lymphocytes. Each clone of B cells produces antibody with an antigen receptor having a unique prospective antigen binding structure. The repertoire of antigen receptors, approximately 10.sup.7 possibilities, exists in vivo prior to antigen stimulation. This diversity is produced by somatic recombination, i.e., the joining of different antibody gene segments Immunoglobulin H chain, kappa L chain and lambda L chain are encoded by three separate genetic loci and each locus has multiple copies of at least 3 types of gene segments encoding variable (V), constant (C) and joining (J) regions, the heavy chain gene also includes a diversity (D) region. The selection of specific V, C and J regions (and D for the heavy chain) from amongst the various gene segments available (45 heavy chain V; 35 kappa V; 23 heavy chain D; 6 heavy chain J; 5 kappa J) generates approximately 10.sup.11 possible specificities of germline sequences exhibited in B cells. The joining of V, C and J regions can result in the loss or addition of residues at the junctions. The L and H chain V region of human antibodies consists of relatively conserved framework regions (FR) that form a scaffold for three hypervariable regions also known as complementary determining regions (CDR). From the amino terminus of either the heavy or light chain, the V domain is made up of FR and CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3. Joining of the V domain with a D (heavy chain only) and J domain adds CDR3-FR4. The CDRs are generally responsible for antigen binding.

[0008] The specificity of monoclonal antibodies have made them attractive agents for targeting cancer in vivo with the hopes of eradicating disease while sparing normal tissue. The approach, which initially utilized mouse monoclonal antibodies has encountered limitations to potential effectiveness such as immunogenicity; inefficient effector functions and short half-life in vivo. Technologies were developed for chimeric antibodies which sought to utilize the antigen binding variable domains of mouse monoclonal antibodies combined with the constant regions of human antibodies (Boulianne, et al. 1984 Nature 312:643-646; Morrison et al, 1984 PNAS USA 81:6851-6855); humanized antibodies which grafted antigen binding complementary determining regions (CDRs) from mouse antibodies to human immunoglobulin (Jones, et al, 1986 Nature 321: 522-525; Riechmann, et al, 1988 Nature 332:323-327; Verhoeyen, et al, 1988 Science 239:1534-1536; Vaughan, et al, 1998 Nature Biotechnol. 16:535-539); and phage display libraries of single chain scFvs or Fab fragments of antibodies (de Haard, et al, 1999 J Biol. Chem. 274: 18218-18230; Knappik, et al, 2000 J. Mol. Biol. 296:57-86; Sheets, et al, 1998 PNAS USA 95:6157-6162; Vaughan, et al, 1994 Nature Biotechnol 14:309-314, 1996; Griffiths et al EMBO J. 13:3245-3260). Additionally, transgenic animals having human immunoglobulin genes and nonfunctional endogenous genes have been developed for immunization and production of fully human monoclonal antibodies (Fishwild, et al, 1996 Nature Biotechnol 14:845-851; Mendez, et al, 1997 Nature Genet. 15:146-156; Nicholson, et al, 1999 J. Immunol 163, 6898-6906).

[0009] Single Chain Antibodies:

[0010] Single chain Fv antibodies (scFvs) were first described in the late 1980's (Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). A polypeptide linker, typically ranging in length from 5 to 27 amino acid residues, is used to join the C-terminus of the variable light chain domain (V.sub.L) to the N-terminus of the variable heavy chain domain (V.sub.H). Alternatively, the linker joins the C-terminus of the V.sub.H to the N-terminus of the V.sub.L. Both formats (V.sub.L-V.sub.H and V.sub.H-V.sub.L) have been used successfully in the literature. The most common linker used in the literature is the (Gly.sub.4Ser).sub.3 15 amino acid linker, however there are several other linkers that have been utilized, including a 25 amino acid linker called 205C (Pantoliano et al., Biochemistry 30:10117-10125 (1991)). Single chain antibodies are currently in the clinic; one of the most advanced is h5G1.1 or Pexelizumab. This scFv is specific for human C5 complement and is being used in clinical trials for cardiac patients undergoing cardiopulmonary bypass surgery (Shernan et al., Ann. Thorac Surg. 77:942-949 (2004)).

[0011] Bispecific Antibodies (Bi-Abs):

[0012] An area of mAb research where considerable progress has been made is in the development of bispecific antibodies (biAbs). There are distinct advantages to developing therapeutic antibody molecules with dual specificity. For example, biAbs can serve as mediators to target immune effector cells such as CTLs to unwanted cells (Baeuerle et al., Curr. Opin. Mol. Ther. 5:413-419 (2003)). In another example, chemically linked bispecific antibodies directed against Fc gamma receptors CD16, CD64, and CD89, were significantly more effective in vitro than conventional IgG antibodies (Peipp and Valerius, Biochem. Soc. Trans. 30:507-511 (2002)). One of the challenges in developing biAbs as viable therapeutics has been producing large enough quantities of a stable moiety for clinical applications. Another challenge has been in determining the right combination of validated targets and the underlying biology that would lead to a therapeutic product. For recent reviews on the difficulties experienced with biAbs, see (Kontermann, Acta Pharmacol Sin 26:1-9 (2005); Peipp and Valerius, Soc. Trans. 30:507-511 (2002)).

[0013] Bispecific Single Chain Antibodies (Bi-scFv):

[0014] A notable type of biAb that can be made is a bi-specific single chain antibody or bi-scFv. For a review on the generation of bi-scFv's see (Kipriyanov and Le Gall, Curr Opin Drug Discov Devel 7:233-242 (2004)). Bi-scFvs are typically comprised of 4 variable domains, 2 heavy (V.sub.H) and 2 light (V.sub.L), which are derived from 2 different antibodies. The 4 domains are linked together with 3 short linkers, ranging in length from 5-27 amino acids. The biological activity of this type of antibody depends on several features concerning the construction of the molecule. For example, both the linker sequences between the antibody V domains and the order of the 4 antibody V domains themselves (for the 2 antibodies) can vary, as well as the expression system that is used; all of which can greatly affect the solubility and biological activity of the various resulting products (Kipriyanov et al., J. Mol. Biol. 330:99-111 (2003); Le Gall et al., Protein Eng. Des. Sel. 17:357-366 (2004); Pavlinkova et al., Clin Cancer Res. 5:2613-1619 (1999)).

[0015] Cytotoxic T Lymphocytes:

[0016] Under normal circumstances, T cells are activated when the CD3/T cell receptor (CD3/TCR) complex binds to a relevant MHC molecule associated with a specific Ag peptide. Engagement of CD3/TCR with MHC results in intracellular signals necessary to trigger an immune response against a pathogen or tumor. Similar signals that cause T cell activation can also be achieved by antibodies that can bind certain structures of the CD3/TCR complex. In the literature, it has been shown that biAbs recognizing both the TCR/CD3 complex and tumor associated antigen (TAA) can trigger the activation program in CTLs in the presence of target cells (Chapoval et al., J Immunol 155:1296-1303 (1995)).

[0017] Recombinant technologies are being utilized to enable further improvements upon antibody molecules with the goal of enhancing in vivo efficacy. Such technologies provide, for example, for optimizing molecular size, affinity, pharmacokinetics, toxicity, specificity, valency, effector functions, direct and indirect arming, combination therapy, and various prodrug approaches.

[0018] It would be desirable to have an antibody suitable for in vivo targeting of GPNMB expressing pathologies and to enable therapeutic efficacy.

SUMMARY OF THE INVENTION

[0019] The current invention provides human monoclonal antibodies that specifically bind GPNMB as well as variants, derivatives and antigen binding fragments of such antibodies.

[0020] The invention provides preferred somatic recombinations of human antibody gene segments to provide specificity for GPNMB and genetically engineered anti-GPNMB antibody variants and derivatives that originate from these gene segments. In addition, the current invention provides multiple affinity matured human antibodies with binding specificity for GPNMB.

[0021] In one embodiment, the present invention provides an antibody, or binding fragment thereof, that binds to GPNMB, wherein said antibody, or binding fragment thereof, neutralizes a GPNMB-induced activity, and wherein said antibody, or binding fragment thereof, cross-reacts with a fully human anti-GPNMB antibody selected from the group consisting of Mab1.2.1, Mab1.10.1, and Mab2.22.1 or an antibody in the same antigen-binding bin as fully human anti-GPNMB antibody Mab1.2.1, Mab1.10.1, or Mab2.22.1.

[0022] In another embodiment, the present invention provides an antibody, or binding fragment thereof, that binds to GPNMB, wherein said antibody, or binding fragment thereof, neutralizes a GPNMB-induced activity, and wherein said antibody, or binding fragment thereof, cross-reacts with a fully human anti-GPNMB antibody selected from the group consisting of Mab2.3.1 and Mab1.15.1 or an antibody in the same antigen-binding bin as fully human anti-GPNMB antibody Mab2.3.1 or Mab1.15.1.

[0023] In yet another embodiment, the present invention provides an antibody, or binding fragment thereof, that binds to GPNMB, wherein said antibody, or binding fragment thereof, neutralizes a GPNMB-induced activity, and wherein said antibody, or binding fragment thereof, cross-reacts with fully human anti-GPNMB antibody Mab2.10.1 or an antibody in the same antigen-binding bin as fully human anti-GPNMB antibody Mab2.10.1.

[0024] In one embodiment, the present invention provides naked IgG1 anti-GPNMB antibodies that have cytotoxic effect to cells overexpressing GPNMB. In a specific embodiment, the present invention provides methods of treating or preventing diseases associated with overexpression of GPNMB comprising administering to a subject in need thereof a composition comprising a naked IgG1 anti-GPNMB antibody and an immunomodulator (such as, but not limited to, interferons and cytokines).

[0025] In another embodiment, the present invention provides immunoconjugates that comprise an anti-GPNMB antibody or a fragment thereof, and a cytotoxic agent. In a specific embodiment, the cytotoxic agent is auristatin E (dolastatin-10) or a derivative thereof. Methods of using such immunoconjugated are also provided.

[0026] In one embodiment, the present invention provides bispecific antibodies comprising an anti-GPNMB component and an anti-CD3 antibody component, which enable the cytotoxic killing of target tumor cells by T cells. In another embodiment, the present invention provides single chain Fv antibody conjugated to a cytotoxic agent. In a specific embodiment, the cytotoxic agent is auristatin E (dolastatin-10) or a derivative thereof. Methods of using such bispecific antibodies and conjugated single chain Fv antibodies are also provided.

[0027] Amino acid sequences for anti-GPNMB human monoclonal antibodies of the invention and nucleic acid sequences encoding them are provided.

[0028] Compositions comprising human anti-GPNMB antibodies, including therapeutic compositions comprising same, and methods of use are provided. Particularly, therapeutic immunoconjugates comprising anti-GPNMB antibodies and a cytotoxic or cytostatic agent for treating GPNMB expressing cancers and other GPNMB related disorders are provided. Dosage regimens are also provided.

[0029] Additional aspects of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practicing the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1: Tumor growth inhibition and complete regression of SK-MEL-2 xenografts in athymic mice after treatment with 2.50 to 20 mg/kg i.v. every 4 days for 4 treatments. The responses of tumor-bearing animals to reference drugs such as vinblastine (1.7 mg/kg i.v. q4 d.times.4) and paclitaxel (24 mg/kg i.v. q2 d.times.4) are also shown. Control groups are treated with either phosphate-buffered saline (PBS) or physiological saline.

[0031] FIG. 2: Indirect immunotoxin killing of UACC-62 melanoma cells by anti-GPNMB antibodies

[0032] FIG. 3: Inhibition of colony formation of UACC-62 cells incubated with Auristatin E (AE) conjugated anti-GPNMB antibodies.

[0033] FIG. 4: Tumor growth inhibition and complete regression of SK-MEL-2 xenografts in athymic mice after treatment with CR011-vcMMAE 5.0 mg/kg i.v. every 4 days for 4 treatments. The lack of responses of tumor-bearing animals to unconjugated CR011 or to free monomethylauristatin E demonstrate that the intact immunoconjugate is essential for anti-tumor effects.

[0034] FIG. 5: Tumor size reduction and complete regression of SK-MEL-2 xenografts in athymic mice after treatment with 1.25 to 20 mg/kg i.v. every 4 days for 4 treatments. The responses of tumor-bearing animals to reference drugs such as Vinblastine (1.7 mg/kg i.v. q4 d.times.4) and paclitaxel (24 mg/kg i.v. q2 d.times.4) are also shown. Control groups are treated with either phosphate-buffered saline (PBS) or physiological saline.

[0035] FIG. 6: The serum concentration-time profile of the antibody of CR011-vcMMAE after intravenous administration of 1 and 10 mg/kg in athymic mice. Detection was achieved with a sandwich ELISA assay, which employed the CR011 antigen (CG56972, GPNMB) and a horseradish peroxidase-conjugated anti-human globulin. Results shown are the serum concentrations expressed as .mu.g/mL (left x-axis) and micromolarmolar concentration (right X-axis).

[0036] FIG. 7: Aggregate responses, expressed as percent cures, were recorded for test animals treated with 5 different, graduated dosing intervals (i.e., 0, 1, 4, 8, and 16 days between treatments). The slope of the line is not significantly different from 0 (p<0.2904).

[0037] FIG. 8: The proportions of complete regressors as a function of dosing interval and stratified by cumulative dose. For each group, n=6 mice/group. Athymic mice bearing established SK-MEL-2 tumor implants (day 14, 80 mg) were treated i.v. with CR011-vcMMAE and the incidence of complete regressions is recorded.

[0038] FIGS. 9A-C: Effects of ectopic expression of GPNMB or sensitivity to CR011-vcMMAE. HEK293 cells are transfected with empty vector (vector) or GPNMB-containing plasmid (GPNMB) as described in Materials and Methods. (FIG. 9A) Cell lysates are prepared from the transfected HEK293 cells and the expression of GPNMB (upper panel) or actin (lower panel) is determined by immunoblotting. Lane 1: Empty vector transfectants. Lane 2: GPNMB transfectants. (FIG. 9B) Flow cytometry analysis of GPNMB expression on empty vector or GPNMB transfected cells. (FIG. 9C) CR011-vcMMAE in vitro growth inhibition of transfected cells. Cells are treated with various concentrations of CR011-vcMMAE (diamonds: vector or circles: GPNMB) or IgG2-vcMMAE (triangles: vector or squares: GPNMB) for 96 hours. After a clonogenic assay, the surviving fraction is normalized to the untreated control and expressed as a percentage of the control using GraphPad Prism graphing software. Each treatment is performed in triplicate. A representative graph from two independent experiments is shown.

[0039] FIGS. 10A-C: Effect of GPNMB siRNA on endogenous GPNMB expression and sensitivity to CR011-vcMMAE. SK-Mel-2 cells are transfected with 50 nM of control siRNA or siRNA targeting GPNMB. (FIG. 10A) Cell lysates are prepared from the transfected SK-Mel-2 cells 2 and 4 days post-transfection and the expression of GPNMB (upper panel) or actin (lower panel) is determined by immunoblotting. Lane 1: Mock (oligofectamine) transfection. Lane 2: Control siRNA transfection. Lane 3: GPNMB siRNA transfection. (FIG. 10B) Flow cytometry analysis of GPNMB expression 2 and 4 days after transfection. SK-Mel-2 cells are transfected with mock, control siRNA or GPNMB siRNA as indicted in the Materials and Methods. (FIG. 10C) CR011-vcMMAE in vitro growth inhibition of mock (diamonds), control siRNA (circles) or GPNMB siRNA (triangles) transfected SK-Mel-2 cells is determined by a clonogenic assay as described in Materials and Methods. The surviving fraction is normalized to the untreated control and expressed as a percentage of control using GraphPad Prism graphing software. Each treatment is performed in triplicate. A representative experiment from two independent studies is shown.

[0040] FIGS. 11A-D: FACS analysis of SK-MEL-2 with isotype control (FIG. 11A), hybridoma IgG2 (B2; FIG. 11B), recombinant IgG2 (B19; FIG. 11C) and recombinant IgG1 (B16; FIG. 11D) to CG56972/GPNMB relative to IgG2 (B2, B19) or IgG1 (Control, B16) controls.

[0041] FIGS. 12A-B: (FIG. 12A) PBMC and mAb (IgG1) mediated ADCC of SK-MEL-2 cells. ADCC effector functions are measured as described above at 2, 5 and 10 .mu.g/200 .mu.l using target:effector ratios of 10, 30, 60 and 100 as indicated. (FIG. 12B) PBMC and mAb (IgG2) do not cause ADCC to SK-MEL-2 cells. ADCC effector functions are measured as described above at 0, 2, 5 and 10 .mu.g/200 .mu.l using target: effector ratios of 10, 30, 60 and 100 as indicated.

[0042] FIGS. 13A-C: Expression of CG56972 in human cancer cell lines and tissues. RTQ PCR analysis of (FIG. 13A) human brain cancer cell lines or (FIG. 13B) human brain cancer glioma and medulloblastoma biopsies. (FIG. 13C) Microarray analysis of CG56972 expression in human brain cancer and oligodendroglioma tissues. Tissues or cell lines are harvested, mRNA prepared and RTQ PCR or CuraChip analysis performed as described in Materials and Methods.

[0043] FIGS. 14A-F: FACS analysis of cell surface binding of CR011 mAb to CG56972. SK-MEL-2 (FIG. 14A), XF-498 (FIG. 14B), U-118-MG (FIG. 14C), SNB-78 (FIG. 14D), SF-539 (FIG. 14E) and SF-268 (FIG. 14F) cells are labeled with a saturating concentration (10 .mu.g/mL) of CR011 mAb or control IgG2. Bound mAb is detected by flow cytometry with PE-conjugated goat-anti-human secondary antibody as described in Materials and Methods. GM: Geometric mean. The SF-268 cell line is CG56972 transcript negative and used as a negative control.

[0044] FIG. 15: Immunoblot analysis of CG5672 expression in human brain cancer cell lines. Cell lysates are resolved on Tris-glycine gels and transferred to membranes. Immunoblot analysis is carried out with a polyclonal antibody to CG56972 followed by enhanced chemiluminescence detection as described in Materials and Methods. Arrowheads indicate the relative mobility of the p100 and 120 CG56972 species. The SF-268 cell line is CG56972 transcript negative and used as a negative control.

[0045] FIGS. 16A-F: CR011-vcMMAE in vitro growth inhibition of astocytoma/glioblastoma cell growth. XF-498 (FIG. 16A), SNB-78 (FIG. 16B), U-118-MG (FIG. 16C), SF-539 (FIG. 16D), LOXIMVI (FIG. 16E) and SF-268 (FIG. 16F) cells are incubated with the indicated concentration of CR011-vcMMAE. Cells are also incubated with control PK16.3 mAb (data shown in Table I) as described in the Materials and Methods. Cell growth was determined by clonogenic assay. The surviving colonies are counted and plotted using GraphPad Prism graphing software. The experiment is performed in triplicate wells and repeated twice. vA representative experiment is shown. IC50s for cell killing is presented in ng/mL concentrations. The LOXIMVI and SF-268 cell lines are CG56972 transcript negative and used as negative controls.

[0046] FIG. 17: Development of CR011 Engineered Antibodies. Four antibody variable (V) domains (shown in C for the bi-scFv) are derived from the light and heavy chain variable domains (V.sub.L and V.sub.H) making up the antigen binding sites of CR011 and anti-CD3 whole IgGs. The middle linker joining the 2 individual scFv components together (shown in dashed line) may play a key role in determining the resulting activity of each of the scFv components, including the effective cytolytic activity provided by the cytotoxic T cells engaged by the anti-CD3 scFv component of the bi-scFv.

[0047] FIGS. 18A-B: (FIG. 18A) ELISA results for CR011 scFv (squares) and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv (diamonds). Both engineered CR011 antibodies bound to the GPNMB target. (FIG. 18B) Western blotting of 2 of the CR011 engineered antibody products (arrows). Clone 16 corresponded to the CHOK1 line expressing CR011 scFv (monomer), while clone 17 corresponded to the CHOK1 line expressing CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv (dimer). Clones 16 and 17 are used to produce the engineered antibody products.

[0048] FIGS. 19A-D: Flow cytometry analysis of binding of CR011 scFv and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv products to native GPNMB protein expressed on the cell surface of target cells. Human T cells (FIGS. 19A, 19C (Top and Bottom panels on the left, respectively)) are used as a source of CD3, while SK-Mel-5 (FIGS. 19B, 19D) (Top and Bottom panels on the right, respectively)) cells are used as a source of GPNMB.

[0049] FIG. 20: Cytotoxicity analysis showed that purified CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv, but not CR011 scFv, causes killing of GPNMB positive SK-Mel-5 tumor cells by T lymphocytes.

[0050] FIG. 21: The chemical structure of Maleimidocoaproyl-Valine-Citrullin-Monomethyl-Auristatin E (vcMMAE).

[0051] FIG. 22: Disulfides on CR011 antibody are gently reduced in the presence of TCEP to generate .about.4 thiols per Ab. vcMMAE is then added to antibody solution. Nucleophilic attack of thiolates on maleimide-groups results in a stable thioester linkage. The resulting conjugate is purified from the mixture.

[0052] FIGS. 23A-I: Reaction of vcMMAE with NAcCys at pH 7.0 and pH 9.0 in the presence or absence of TCEP. FIG. 23A: VCMMAE converts fully into NAcCys-adduct following an incubation in phosphate pH 7 buffer. FIGS. 23B-E: Appearance of a side product in a course of incubation of vcMMAE in borate buffer. FIGS. 23F-I: Appearance of side products in borate pH 9 and in the presence of TCEP.

[0053] FIG. 24: LCMS identification of the side product with retention time of 9.2 min not capable of reaction with cysteine and therefore, not capable of conjugation to CR011.

[0054] FIG. 25: Kinetics of the formation of NAcCys-vcMMAE and of the side product (succinimidyl-vcMMAE) following incubation in borate pH 9.0 buffer in the presence or absence of TCEP.

DETAILED DESCRIPTION OF THE INVENTION

[0055] As used herein, the term "antibody" refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, engineered, and grafted antibodies. Unless otherwise modified by the term "intact," as in "intact antibodies," for the purposes of this disclosure, the term "antibody" also includes antibody fragments such as Fab, F(ab').sub.2, Fv, scFv, bi-scFv, bi-Ab, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind GPNMB specifically. Typically, such fragments would comprise an antigen-binding domain.

[0056] As used herein, the terms "antigen-binding domain," "antigen-binding fragment," and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant."

[0057] An antigen-binding domain typically comprises an antibody light chain variable region (V.sub.L) and an antibody heavy chain variable region (V.sub.H), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a V.sub.H domain, but still retains some antigen-binding function of the intact antibody.

[0058] As used herein, the term "repertoire" refers to a genetically diverse collection of nucleotides derived wholly or partially from sequences that encode expressed immunoglobulins. The sequences are generated by in vivo rearrangement of, e.g., V, D, and J segments for H chains and, e.g., V and J segment for L chains. Alternatively, the sequences may be generated from a cell line by in vitro stimulation, in response to which the rearrangement occurs. Alternatively, part or all of the sequences may be obtained by combining, e.g., unrearranged V segments with D and J segments, by nucleotide synthesis, randomised mutagenesis, and other methods, e.g., as disclosed in U.S. Pat. No. 5,565,332.

[0059] As used herein, the terms "specific interaction" and "specific binding" refer to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant K.sub.A is higher than 10.sup.6 M.sup.-1, or more preferably higher than 10.sup.8 M.sup.-1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

[0060] As used herein, the term "substantially as set out" refers that the relevant CDR, V.sub.H, or V.sub.L domain of the invention will be either identical to or have only insubstantial differences in the specified regions (e.g., a CDR), the sequence of which is set out. Insubstantial differences include minor amino acid changes, such as substitutions of 1 or 2 out of any 5 amino acids in the sequence of a specified region.

[0061] As used herein, the term "CR011" refers to a fully human monoclonal antibody that specifically binds to GPNMB. In some embodiments, CR011 refers to those antibodies that are identified in Tables 2A-2D of the present application. In some embodiments, CR011 refers to Mab 1.15.1 as described in the instant invention.

[0062] The terms "GPNMB" and "CG56972" are used interchangeably herein. As used herein, the terms "GPNMB" or "CG56972" refer to a transmembrane glycoprotein that has an amino acid sequence as set forth in SEQ ID NO: 289, an analog, derivative or a fragment thereof, or a fusion protein comprising GPNMB, an analog, derivative or a fragment thereof. In certain embodiments, the term "GPNMB" refers to the mature, processed form of GPNMB. In other embodiments, the term "GPNMB" refers to the extracellular domain of GPNMB.

[0063] As used herein, the term "GPNMB activity" refers to one or more activities associated with GPNMB. To "modulate" GPNMB activity is to alter the baseline results observed with, and that can be attributed to GPNMB. To "neutralize" GPNMB is to cancel one or more effects, e.g. activity observed with, and that can be attributed to GPNMB.

[0064] As used herein, the term "isolated" refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to preparations where the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or at least 70-80% (w/w) pure, more preferably, at least 80-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

[0065] As used herein, the term "inhibit" or "inhibition of" refers to reducing by a measurable amount, or to prevent entirely.

[0066] As used herein, the term "Cytotoxic effect" in reference to the effect of an agent on a cell, means killing of the cell. "Cytostatic effect" refers to an inhibition of cell proliferation. A "cytotoxic agent" refers an agent that has a cytotoxic or cytostatic effect on a cell, thereby depleting or inhibiting the growth of, respectively, cells within a cell population.

[0067] As used herein, the terms "prevent," "preventing," and "prevention" refer to the inhibition of the development or onset of a disorder associated with aberrant expression and/or activity of GPNMB (e.g., cancer) or the prevention of the recurrence, onset, or development of one or more symptoms of a disorder associated with aberrant expression and/or activity of GPNMB (e.g., cancer) in a subject resulting from the administration of a therapy or the administration of a combination of therapies.

[0068] As used herein, the term "effective amount" refers to a dosage or amount that is sufficient to reduce the activity of GPNMB to result in amelioration of symptoms in a patient or to achieve a desired biological outcome.

[0069] As used herein, the term "prophylactically effective amount" refers to the amount of a therapy which is sufficient to result in the prevention of the development, recurrence, or onset of a disorder associated with aberrant expression and/or activity of GPNMB (e.g., cancer) or one or more symptoms thereof, or to enhance or improve the prophylactic effect(s) of another therapy.

[0070] As used herein, a "protocol" includes dosing schedules and dosing regimens. The protocols herein are methods of use and include prophylactic and therapeutic protocols.

[0071] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, such as a cynomolgous monkey, chimpanzee, and a human), and more preferably a human.

[0072] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to an agent that can be used in the prevention, treatment, management, or amelioration of a disorder associated with aberrant expression and/or activity of GPNMB (e.g., cancer) or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to an antibody that immunospecifically binds to GPNMB. In certain other embodiments, the term "therapeutic agent" refers an agent other than an antibody that immunospecifically binds to GPNMB.

[0073] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s), and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disorder associated with aberrant expression and/or activity of GPNMB (e.g., cancer) or one or more symptoms thereof. In certain embodiments, the terms "therapies" and "therapy" refer to anti-cancer therapy, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of cancer or one or more symptoms thereof known to one of skill in the art such as medical personnel.

[0074] As used herein, the terms "treat," "treatment," and "treating" refer to the eradication, removal, modification, or control of primary, regional, or metastatic cancer tissue, or the reduction or amelioration of the progression, severity, and/or duration of a disorder associated with aberrant expression and/or activity of GPNMB or amelioration of one or more symptoms thereof resulting from the administration of one or more therapies. In certain embodiments, such terms in the context of cancer refer to a reduction in the growth of cancerous cells, a decrease in number of cancerous cells and/or a reduction in the growth, formation and/or volume of a tumor. In other embodiments, such terms refer to the minimizing or delay of the spread of cancer resulting from the administration of one or more therapies to a subject with such a disease. Treatment can include, for example, a decrease in the severity of a symptom, the number of symptoms, or frequency of relapse.

[0075] Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. (See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0076] The current invention provides germline human antibody heavy chain V, D, J combinations and light chain V, J combinations including nucleotide and amino acid sequence of the V.sub.H and V.sub.L domain FR and CDR regions with specificity for GPNMB.

[0077] Upon exposure to antigen, those B cells with antigen binding specificity based on germline sequences are activated, proliferate, and differentiate to produce immunoglobulins of different isotypes as well as undergo somatic mutation and/or affinity maturation to produce immunoglobulins of higher affinity for the antigen. The current invention provides the nucleotide and amino acid sequence of such affinity matured V domain FR and CDR regions having specificity to GPNMB.

[0078] Fab type antibody fragments containing the antigen binding portion of the antibody molecule may consist of the L chain covalently linked by a disulfide bond to a portion of the H chain which has the V domain and first constant domain. Single chain Fv antibody fragment (scFv) has the H variable domain linked to the L variable domain by a polypeptide linker. The invention provides antibody fragments such as Fab and scFv molecules having sequences derived from germline or affinity matured V domains of antibodies binding specifically to GPNMB.

[0079] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments (see, e.g., Songsivilai & Lachmann, 1990 Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992 J. Immunol. 148:1547-1553). Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).

[0080] It will be appreciated that such bifunctional or bispecific antibodies are contemplated and encompassed by the invention. A bispecific single chain antibody with specificity to GPNMB and to the CD3 antigen on cytotoxic T lymphocytes can be used to direct these T cells to tumor cells expressing GPNMB and cause apoptosis and eradication of the tumor. Bispecific scFv constructs for this purpose are described herein. The scFv components specific for GPNMB can be derived from anti-GPNMB antibodies described herein. In some embodiments, the anti-GPNMB antibody components disclosed herein can be used to generate a biologically active scFv directed against GPNMB. The anti-CD3 scFv component of the therapeutic bispecific scFv was derived from a sequence deposited in Genbank (accession number CAE85148). Alternative antibodies known to target CD3 or other T cell antigens may similarly be effective in treating malignancies when coupled with anti-GPNMB, whether on a single-chain backbone or a full IgG.

[0081] GPNMB binding human antibodies may include H or L constant domains including L kappa or lambda constant regions, or any isotype H constant domain. In one embodiment of the invention, a human antibody with binding specificity to GPNMB contains germline sequences such as the heavy chain V regions: VH1-2 (SEQ ID NO: 308), VH2-5 (SEQ ID NO: 360), VH3-11 (SEQ ID NO: 361), VH3-21 (SEQ ID NO: 362), VH3-30 (SEQ ID NO:363), VH3-33 (SEQ ID NO: 364), VH4-31 (SEQ ID NO: 365), VH4-59 (SEQ ID NO:366) or VH5-51 (SEQ ID NO:367); the heavy chain D region: D1-20 (amino acid sequences translated by SEQ ID NO: 375), D1-26 (amino acid sequences translated by SEQ ID NO:376), D3-10 (amino acid sequences translated by SEQ ID NO:377), D3-16 (amino acid sequences translated by SEQ ID NO:378), D3-22 (amino acid sequences translated by SEQ ID NO: 379), D3-9 (amino acid sequences translated by SEQ ID NO:380), D4-17 (amino acid sequences translated by SEQ ID NO: 381), D5-24 (amino acid sequences translated by SEQ ID NO: 382), D6-13 (amino acid sequences translated by SEQ ID NO:383), or D6-19 (amino acid sequences translated by SEQ ID NO: 384); the heavy chain J region: JH3b (SEQ ID NO: 385), JH4b (SEQ ID NO:386), JH5b (SEQ ID NO: 387) or JH6b (SEQ ID NO: 388); the light chain V kappa regions A2 (SEQ ID NO:373), A3 (SEQ ID NO: 371), A20 (SEQ ID NO: 370), A27 (SEQ ID NO: 369), A30 (SEQ ID NO:374), L2 (SEQ ID NO:372) or O1 (SEQ ID NO: 368); and the J region JK1 (SEQ ID NO:389), JK2 (SEQ ID NO: 390), JK3 (SEQ ID NO: 391), JK4 (SEQ ID NO: 392) or JK5 (SEQ ID NO: 393). (generally, see Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. 1987 and 1991; also see Chothia & Lesk 1987 J. Mol. Biol. 196:901-917; Chothia et al. 1989 Nature 342:878-883). In a particular embodiment of the invention human antibodies with binding specificity to GPNMB are combined germline regions as shown in Table 1.

TABLE-US-00001 TABLE 1 Human anti-GPNMB antibody germline region combinations. Ab VH D JH VL JL 1.10.2 VH4-59 D6-19 JH4b A3 JK5 1.15.1 VH4-31 D1-20 JH4b L2 JK1 1.2.2 VH2-5 D3-16 JH4b O1 JK5 1.7.1 VH4-31 D1-20 JH4b L2 JK1 2.10.2 VH3-30 D3-10 JH6b A3 JK5 2.15.1 VH3-33 D4-17 JH4b A20 JK4 2.16.1 VH3-11 D6-13 JH3b L2 JK3 2.17.1 VH1-2 D6-19 JH5b A2 JK4 2.21.2 VH3-21 D1-26 JH4b A20 JK5 2.22.1 VH4-31 D3-22 JH6b A30 JK1 2.24.1 VH5-51 D5-24 JH4b A27 JK1 2.3.1 VH1-2 D3-10 JH4b A2 JK4 2.7.1 VH3-33 D3-10 JH4b A20 JK4 2.8.1 VH2-5 D3-9 JH4b O1 JK4

[0082] In an embodiment of the invention, the isolated antibody has a heavy chain variable region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265, 270, 274, 277, 281 and 285. Such amino acid sequences can be encoded by nucleotide sequences selected from the group consisting of SEQ ID NOs: 1, 19, 37, 55, 73, 91, 109, 127, 145, 163, 181, 199, 217 and 235. In another embodiment, the invention provides an isolated antibody that specifically binds to GPNMB and has a light chain variable region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. Such amino acid sequences can be encoded by nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 28, 46, 64, 82, 100, 118, 136, 154, 172, 190, 208, 226 and 244. In yet another embodiment, the invention provides an isolated antibody that specifically binds to GPNMB and has a heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265, 270, 274, 277, 281 and 285 and has a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. In yet another embodiment of the invention, anti-GPNMB antibodies comprise at least one CDR of any of the H or L CDR polypeptide sequences SEQ ID NOs: 4, 6, 8, 13, 15, 17, 22, 24, 26, 31, 33, 35, 40, 42, 44, 49, 51, 53, 58, 60, 62, 67, 69, 71, 76, 78, 80, 85, 87, 89, 94, 96, 98, 103, 105, 107, 112, 114, 116, 121, 123, 125, 130, 132, 134, 139, 141, 143, 148, 150, 152, 157, 159, 161, 166, 168, 170, 175, 177, 179, 184, 186, 188, 193, 195, 197, 202, 204, 206, 211, 213, 215, 220, 222, 224, 229, 231, 233, 238, 240, 242, 247, 249, 251, 254, 257, 261, 266, 271, 278, 282, 286, 255, 258, 262, 267, 272, 275, 279, 283, 287, 259, 263, 264, 268, 269, 273, 276, 280, 284 and 288.

[0083] In particular embodiments, human anti-GPNMB antibodies are Mab1.10.2, Mab1.15.1, Mab1.2.2, Mab1.7.1, Mab2.10.2, Mab2.15.1, Mab2.16.1, Mab2.17.1, Mab2.21.2, Mab2.22.1, Mab2.24.1, Mab2.3.1, Mab2.7.1, and Mab2.8.1. These antibodies have amino acid sequences and nucleic acid sequences encoding them identified in this application as shown in Tables 2A-2D.

TABLE-US-00002 TABLE 2A Antibody Nucleotide (DNA) and Amino Acid (AA) Sequences Gene Segment 1.10.2 1.15.1 1.2.2 1.7.1 H variable DNA SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO: 37 SEQ ID NO: 55 H variable AA SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 38 SEQ ID NO: 56 H FR1 SEQ ID NO: 3 SEQ ID NO: 21 SEQ ID NO: 39 SEQ ID NO: 57 H CDR1 SEQ ID NO: 4 SEQ ID NO: 22 SEQ ID NO: 40 SEQ ID NO: 58 H FR2 SEQ ID NO: 5 SEQ ID NO: 23 SEQ ID NO: 41 SEQ ID NO: 59 H CDR2 SEQ ID NO: 6 SEQ ID NO: 24 SEQ ID NO: 42 SEQ ID NO: 60 H FR3 SEQ ID NO: 7 SEQ ID NO: 25 SEQ ID NO: 43 SEQ ID NO: 61 H CDR3 SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 44 SEQ ID NO: 62 H FR4 SEQ ID NO: 9 SEQ ID NO: 27 SEQ ID NO: 45 SEQ ID NO: 63 L variable DNA SEQ ID NO: 10 SEQ ID NO: 28 SEQ ID NO: 46 SEQ ID NO: 64 L variable AA SEQ ID NO: 11 SEQ ID NO: 29 SEQ ID NO: 47 SEQ ID NO: 65 L FR1 SEQ ID NO: 12 SEQ ID NO: 30 SEQ ID NO: 48 SEQ ID NO: 66 L CDR1 SEQ ID NO: 13 SEQ ID NO: 31 SEQ ID NO: 49 SEQ ID NO: 67 L FR2 SEQ ID NO: 14 SEQ ID NO: 32 SEQ ID NO: 50 SEQ ID NO: 68 L CDR2 SEQ ID NO: 15 SEQ ID NO: 33 SEQ ID NO: 51 SEQ ID NO: 69 L FR3 SEQ ID NO: 16 SEQ ID NO: 34 SEQ ID NO: 52 SEQ ID NO: 70 L CDR3 SEQ ID NO: 17 SEQ ID NO: 35 SEQ ID NO: 53 SEQ ID NO: 71 L FR4 SEQ ID NO: 18 SEQ ID NO: 36 SEQ ID NO: 54 SEQ ID NO: 72

TABLE-US-00003 TABLE 2B Antibody Nucleotide (DNA) and Amino Acid (AA) Sequences Gene Segment 2.10.2 2.15.1 2.16.1 2.17.1 H variable DNA SEQ ID NO: 73 SEQ ID NO: 91 SEQ ID NO: 109 SEQ ID NO: 127 H variable AA SEQ ID NO: 74 SEQ ID NO: 92 SEQ ID NO: 110 SEQ ID NO: 128 H FR1 SEQ ID NO: 75 SEQ ID NO: 93 SEQ ID NO: 111 SEQ ID NO: 129 H CDR1 SEQ ID NO: 76 SEQ ID NO: 94 SEQ ID NO: 112 SEQ ID NO: 130 H FR2 SEQ ID NO: 77 SEQ ID NO: 95 SEQ ID NO: 113 SEQ ID NO: 131 H CDR2 SEQ ID NO: 78 SEQ ID NO: 96 SEQ ID NO: 114 SEQ ID NO: 132 H FR3 SEQ ID NO: 79 SEQ ID NO: 97 SEQ ID NO: 115 SEQ ID NO: 133 H CDR3 SEQ ID NO: 80 SEQ ID NO: 98 SEQ ID NO: 116 SEQ ID NO: 134 H FR4 SEQ ID NO: 81 SEQ ID NO: 99 SEQ ID NO: 117 SEQ ID NO: 135 L variable DNA SEQ ID NO: 82 SEQ ID NO: 100 SEQ ID NO: 118 SEQ ID NO: 136 L variable AA SEQ ID NO: 83 SEQ ID NO: 101 SEQ ID NO: 119 SEQ ID NO: 137 L FR1 SEQ ID NO: 84 SEQ ID NO: 102 SEQ ID NO: 120 SEQ ID NO: 138 L CDR1 SEQ ID NO: 85 SEQ ID NO: 103 SEQ ID NO: 121 SEQ ID NO: 139 L FR2 SEQ ID NO: 86 SEQ ID NO: 104 SEQ ID NO: 122 SEQ ID NO: 140 L CDR2 SEQ ID NO: 87 SEQ ID NO: 105 SEQ ID NO: 123 SEQ ID NO: 141 L FR3 SEQ ID NO: 88 SEQ ID NO: 106 SEQ ID NO: 124 SEQ ID NO: 142 L CDR3 SEQ ID NO: 89 SEQ ID NO: 107 SEQ ID NO: 125 SEQ ID NO: 143 L FR4 SEQ ID NO: 90 SEQ ID NO: 108 SEQ ID NO: 126 SEQ ID NO: 144

TABLE-US-00004 TABLE 2C Antibody Nucleotide (DNA) and Amino Acid (AA) Sequences Gene Segment 2.21.2 2.22.1 2.24.1 2.3.1 H variable DNA SEQ ID NO: 145 SEQ ID NO: 163 SEQ ID NO: 181 SEQ ID NO: 199 H variable AA SEQ ID NO: 146 SEQ ID NO: 164 SEQ ID NO: 182 SEQ ID NO: 200 H FR1 SEQ ID NO: 147 SEQ ID NO: 165 SEQ ID NO: 183 SEQ ID NO: 201 H CDR1 SEQ ID NO: 148 SEQ ID NO: 166 SEQ ID NO: 184 SEQ ID NO: 202 H FR2 SEQ ID NO: 149 SEQ ID NO: 167 SEQ ID NO: 185 SEQ ID NO: 203 H CDR2 SEQ ID NO: 150 SEQ ID NO: 168 SEQ ID NO: 186 SEQ ID NO: 204 H FR3 SEQ ID NO: 151 SEQ ID NO: 169 SEQ ID NO: 187 SEQ ID NO: 205 H CDR3 SEQ ID NO: 152 SEQ ID NO: 170 SEQ ID NO: 188 SEQ ID NO: 206 H FR4 SEQ ID NO: 153 SEQ ID NO: 171 SEQ ID NO: 189 SEQ ID NO: 207 L variable DNA SEQ ID NO: 154 SEQ ID NO: 172 SEQ ID NO: 190 SEQ ID NO: 208 L variable AA SEQ ID NO: 155 SEQ ID NO: 173 SEQ ID NO: 191 SEQ ID NO: 209 L FR1 SEQ ID NO: 156 SEQ ID NO: 174 SEQ ID NO: 192 SEQ ID NO: 210 L CDR1 SEQ ID NO: 157 SEQ ID NO: 175 SEQ ID NO: 193 SEQ ID NO: 211 L FR2 SEQ ID NO: 158 SEQ ID NO: 176 SEQ ID NO: 194 SEQ ID NO: 212 L CDR2 SEQ ID NO: 159 SEQ ID NO: 177 SEQ ID NO: 195 SEQ ID NO: 213 L FR3 SEQ ID NO: 160 SEQ ID NO: 178 SEQ ID NO: 196 SEQ ID NO: 214 L CDR3 SEQ ID NO: 161 SEQ ID NO: 179 SEQ ID NO: 197 SEQ ID NO: 215 L FR4 SEQ ID NO: 162 SEQ ID NO: 180 SEQ ID NO: 198 SEQ ID NO: 216

TABLE-US-00005 TABLE 2D Antibody Nucleotide (DNA) and Amino Acid (AA) Sequences Gene Segment 2.7.1 2.8.1 H variable DNA SEQ ID NO: 217 SEQ ID NO: 235 H variable AA SEQ ID NO: 218 SEQ ID NO: 236 H FR1 SEQ ID NO: 219 SEQ ID NO: 237 H CDR1 SEQ ID NO: 220 SEQ ID NO: 238 H FR2 SEQ ID NO: 221 SEQ ID NO: 239 H CDR2 SEQ ID NO: 222 SEQ ID NO: 240 H FR3 SEQ ID NO: 223 SEQ ID NO: 241 H CDR3 SEQ ID NO: 224 SEQ ID NO: 242 H FR4 SEQ ID NO: 225 SEQ ID NO: 243 L variable DNA SEQ ID NO: 226 SEQ ID NO: 244 L variable AA SEQ ID NO: 227 SEQ ID NO: 245 L FR1 SEQ ID NO: 228 SEQ ID NO: 246 L CDR1 SEQ ID NO: 229 SEQ ID NO: 247 L FR2 SEQ ID NO: 230 SEQ ID NO: 248 L CDR2 SEQ ID NO: 231 SEQ ID NO: 249 L FR3 SEQ ID NO: 232 SEQ ID NO: 250 L CDR3 SEQ ID NO: 233 SEQ ID NO: 251 L FR4 SEQ ID NO: 234 SEQ ID NO: 252

VH4-31 Derived Anti-GPNMB Antibodies:

[0084] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH4-31 or are derived therefrom and have an amino acid sequence of the formula:

TABLE-US-00006 (SEQ ID NO: 253) X.sub.1SGPGLVKPSQX.sub.2LSLTCTVS GGSIS SX.sub.3X.sub.4YX.sub.5WX.sub.6 WIRX.sub.7HPGK GLEWIG YIYYSGX.sub.8TYX.sub.9NPSLKS RVX.sub.10ISVDTSKNQFSLX.sub.11LSSV TAADTAVYYCAR. Where: X1 is E or Q; X2 is T or N; X3 is A, F or G; X4 is N or G; X5 is Y or F; X6 is T or S; X7 is Q or H; X8 is S or N; X9 is C, S or Y; X10 is I or T; X11 is K or T;

[0085] In specific embodiments SEQ ID NO:253 is combined with D3-22 or D1-20. Furthermore the combination of SEQ ID NO:253 with D3-22 or D1-20 is combined with JH6b or JH4b and in specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab1.15.1, Mab1.7.1 and Mab2.22.1, have amino acid sequences SEQ ID NOs:20, 56 and 164 and can be encoded by nucleotide sequences SEQ ID NO:19, 55 and 163.

[0086] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH4-31 CDR or affinity matured sequences thereof, of the formula:

TABLE-US-00007 (SEQ ID NO: 254) CDR1: GGSIS SX.sub.3X.sub.4YX.sub.5WX.sub.6. Where: X3 is A, F or G; X4 is N or G; X5 is Y or F; X6 is T or S;

[0087] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence selected from the following: SEQ ID NO:22, 58, 166.

[0088] In particular embodiments H chain CDR2 sequences are the germline VH4-31 CDR or affinity matured sequences thereof of the formula:

TABLE-US-00008 (SEQ ID NO: 255) CDR2: YIYYSGX.sub.8TYX.sub.9NPSLKS. Where: X8 is S or N; X9 is C, S or Y;

In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR2 sequence selected from the following: SEQ ID NO: 24, 60, and 168.

[0089] In particular embodiments, the H chain CDR3 sequence is a D3-22, JH6b combination having SEQ ID NO:170. Alternatively, in particular embodiments the H chain CDR3 sequence is a D1-20, JH4b combination having SEQ ID NO:26 or 62.

VH1-2 Derived Anti-GPNMB Antibodies:

[0090] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH1-2 or are derived therefrom and include an amino acid sequence of the formula:

TABLE-US-00009 (SEQ ID NO: 256) QLVQSGAEVKKPGASVKVSCKAS GYTFT GX.sub.1YMH WVRQX.sub.2PGQGLEW MGWINPNSGGTX.sub.3YX.sub.4QKFQX.sub.5 RVTMTRDTSISTX.sub.6YMELSRLRSDDTA VYYCAR. Where: X1 is Y or F; X2 is A or T; X3 is N or Y; X4 is A or V; X5 is D or G; X6 is A or V;

[0091] In specific embodiments SEQ ID NO:256 is combined with D3-10 or D6-19. Furthermore the combination of SEQ ID NO:256 with D3-10 or D6-19 is combined with JH4b or JH5b and in specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab2.3.1 and Mab 2.17.1 have amino acid sequences: SEQ ID NO:128 and 200 and can be encoded by nucleotide sequences SEQ ID NO:127 and 199.

[0092] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH1-2 CDR or affinity matured sequences thereof, of the formula:

TABLE-US-00010 (SEQ ID NO: 257) CDR1: GYTFTGX.sub.1YMH Where: X1 is Y or F,

[0093] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence selected from SEQ ID NO: 130 and 202.

[0094] In particular embodiments H chain CDR2 sequences are the germline VH1-2 CDR or affinity matured sequences thereof of the formula:

TABLE-US-00011 (SEQ ID NO: 258) CDR2: WINPNSGGTX.sub.3YX.sub.4QKFQX.sub.5. Where: X3 is N or Y; X4 is A or V; X5 is D or G

[0095] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR2 sequence selected from SEQ ID NO:132 and 204.

[0096] In particular embodiments H chain CDR3 sequences are germline D3-10, JH4b combinations or affinity matured sequences thereof, having the amino acid sequence of the formula:

TABLE-US-00012 (SEQ ID NO: 259) CDR3: X.sub.1X.sub.2X.sub.3GSGSX.sub.4X.sub.5. Where: X1 is Y or D; X2 is Y or F; X3 is Y or F; X4 is Y or L; X5 is Y or L

[0097] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR3 sequence selected from SEQ ID NO:134 and 206.

VH2-5 Derived Anti-GPNMB Antibodies:

[0098] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH2-5 or are derived therefrom and include an amino acid sequence of the formula:

TABLE-US-00013 (SEQ ID NO: 260) ITLKESGPTLVX.sub.1PTQTLTLTCTFS GFSLS X.sub.2X.sub.3GX.sub.4GVG WIRQPPG KALX.sub.5WLX.sub.6 LIYWNDDKX.sub.7YSPSLX.sub.8S RLTITKDTSKNQVVLX.sub.9X.sub.10 TNMDPVDTATYYCAH. Where: X1 is K or T; X2 is T or A; X3 is S or G; X4 is M or V; X5 is D or E; X6 is A or T; X7 is R or H; X8 is K or R; X9 is T or R; X10 is M or I;

[0099] In specific embodiments SEQ ID NO:260 is combined with D3-9 or D3-16 and furthermore is combined with JH4b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example, Mab 2.8.1 and Mab 1.2.2 have amino acid sequences SEQ ID NO: 38 and 236 and can be encoded by nucleotide sequences SEQ ID NO: 37 and 235.

[0100] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH2-5 CDR or affinity matured sequences thereof, of the formula:

TABLE-US-00014 (SEQ ID NO: 261) CDR1: GFSLS X.sub.2X.sub.3GX.sub.4GVG. Where: X2 is T or A; X3 is S or G; X4 is M or V;

[0101] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence selected from SEQ ID NO: 40 and 238.

[0102] In particular embodiments H chain CDR2 sequences are the germline VH2-5 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00015 (SEQ ID NO: 262) CDR2: LIYWNDDKX.sub.7YSPSLX.sub.8S. Where: X7 is R or H; X8 is K or R;

[0103] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR2 sequence selected from SEQ ID NO:42 and 240.

[0104] In particular embodiments H chain CDR3 sequences are germline D3-9, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00016 (SEQ ID NO: 263) CDR3: X.sub.1YDILTGX.sub.2X.sub.3. Where: X1 is Y or H; X2 is Y or F; and X3 is Y or N

[0105] In a specific embodiments an anti-GPNMB antibody of the invention comprises a CDR3 amino acid sequence SEQ ID NO:242.

[0106] In yet another particular embodiment H chain CDR3 sequences are germline D3-16, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00017 (SEQ ID NO: 264) CDR3: YDYX.sub.1WGS. Where: X1 is V or D

[0107] In a specific embodiment an anti-GPNMB antibody of the invention comprises a CDR3 amino acid sequence SEQ ID NO: 44.

VH3-33 Derived Anti-GPNMB Antibodies:

[0108] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH3-33 or are derived therefrom and have an amino acid sequence of the formula:

TABLE-US-00018 (SEQ ID NO: 265) QVQLX.sub.1X.sub.2SGGGVVQPGRSLRLSCAAS GFTFX.sub.3X.sub.4YGX.sub.5H WVRQAPGK GLEWVA VIWX.sub.6DGX.sub.7NKYYADSVKG RFTISRDNSKNTLYLQMNSLRAE DX.sub.8AVYYCAX.sub.9. Where: X1 is V or E; X2 is E or Q; X3 is S or N; X4 is S or N; X5 is M or I; X6 is Y or F; X7 is S or R; X8 is T or A; X9 is R or K

[0109] In specific embodiments SEQ ID NO:265 is combined with D3-10 or D4-17 and furthermore with JH4b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 2.7.1 and Mab2.15.1 have amino acid sequences: SEQ ID NO:92 and 218 and can be encoded by nucleotide sequences SEQ ID NO:91 and 217.

[0110] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH3-33 CDR or affinity matured sequences thereof, of the formula:

TABLE-US-00019 (SEQ ID NO: 266) CDR1: GFTFX.sub.3X.sub.4YGX.sub.5H. Where: X3 is S or N; X4 is S or N; X5 is M or I;

[0111] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 amino acid sequence selected from SEQ ID NO:94 and 220.

[0112] In particular embodiments H chain CDR2 sequences are the germline VH3-33 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00020 (SEQ ID NO: 267) CDR2: VIWX.sub.6DGX.sub.7NKYYADSVKG. Where: X6 is Y or F; X7 is S or R;

[0113] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR2 sequence selected from SEQ ID NO:96 and 222.

[0114] In particular embodiments H chain CDR3 sequences are D3-10, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00021 (SEQ ID NO: 268) CDR3: YYYGSGX.sub.1. Where: X1 is S or L

[0115] A specific embodiment is anti-GPNMB antibody 2.7.1 having a CDR3 amino acid sequence SEQ ID NO:224.

[0116] In an alternative embodiment H chain CDR3 sequences are D4-17, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00022 (SEQ ID NO: 269) CDR3: DYGDX.sub.1. Where: X1 is Y or S

[0117] A specific embodiment is anti-GPNMB antibody 2.15.1 having a CDR3 amino acid sequence SEQ ID NO: 98.

VH3-11 Derived Anti-GPNMB Antibodies:

[0118] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH3-11 or are derived therefrom and have an amino acid sequence of the formula:

TABLE-US-00023 (SEQ ID NO: 270) QVQLVESGGGLVKPGGSLRLSCAAS GFTFS X.sub.1YX.sub.2MX.sub.3 WIRQAPGKG LEWVS YISX.sub.4SGSX.sub.5X.sub.6X.sub.7YADSVKG RFTX.sub.8SRDNAKNSLYLQMNSLR AEDTAVYYCAR. Where: X1 is D or S; X2 is S or Y; X3 is S or T; X4 is S or I; X5 is T or I; X6 is T or I; X7 is Y or H; X8 is I or M;

[0119] In specific embodiments SEQ ID NO:270 is combined with D6-13 and furthermore with JH3b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 2.16.1 have amino acid sequence SEQ ID NO:110 and can be encoded by nucleotide sequence SEQ ID NO:109.

[0120] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH3-11 CDR1 or affinity matured sequences thereof, of the formula:

TABLE-US-00024 (SEQ ID NO: 271) CDR1: GFTFS X.sub.1YX.sub.2MX.sub.3. Where: X1 is D or S; X2 is S or Y; X3 is S or T;

[0121] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 amino acid sequence SEQ ID NO:112.

[0122] In particular embodiments H chain CDR2 sequences are the germline VH3-11 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00025 (SEQ ID NO: 272) CDR2: YISX.sub.4SGSX.sub.5X.sub.6X.sub.7YADSVKG. Where: X4 is S or I; X5 is T or I; X6 is T or I; X7 is Y or H;

[0123] In specific embodiments an anti-GPNMB antibody of the invention comprises a CDR2 sequence SEQ ID NO:114.

[0124] In particular embodiments H chain CDR3 sequences are D6-13, JH3b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00026 (SEQ ID NO: 273) CDR3: X.sub.1X.sub.2AAAG--AFDI. Where: X1 is G or D; X2 is I or G;

[0125] A specific embodiment is anti-GPNMB antibody 2.16.1 having a CDR3 amino acid sequence SEQ ID NO:116.

VH3-21 Derived Anti-GPNMB Antibodies:

[0126] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH3-21 or are derived therefrom and have an amino acid sequence of the formula:

TABLE-US-00027 (SEQ ID NO: 274) X.sub.1VQLX.sub.2X.sub.3SGGGLVKPGGSLRX.sub.4 SCAAS GFTFS SYSMN WVRQAPG KGLEWVS X.sub.5ISS SSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRA EDTAVYYCAR. Where: X1 is E or Q; X2 is V or E; X3 is E or Q; X4 is F or L; X5 is S or F;

[0127] In specific embodiments SEQ ID NO:274 is combined with D1-26 and furthermore with JH4b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 2.21.1 have amino acid sequence SEQ ID NO:146 and can be encoded by nucleotide sequence SEQ ID NO:145.

[0128] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH3-21 CDR1, SEQ ID NO:148 or affinity matured sequences thereof.

[0129] In particular embodiments H chain CDR2 sequences are the germline VH3-21 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00028 (SEQ ID NO: 275) CDR2: X.sub.5ISS SSSYIYYADSVKG. Where: X5 is S or F;

[0130] In specific embodiments an anti-GPNMB antibody of the invention comprises a CDR2 amino acid sequence SEQ ID NO:150.

[0131] In particular embodiments H chain CDR3 sequences are D1-26, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00029 (SEQ ID NO: 276) CDR3: X.sub.1X.sub.2VGAT-FDY. Where: X1 is G or D; X2 is I or W;

[0132] A specific embodiment is anti-GPNMB antibody 2.21.1 having a CDR3 amino acid sequence SEQ ID NO:152.

VH3-30 Derived Anti-GPNMB Antibodies:

[0133] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH3-30 or are derived therefrom and include an amino acid sequence of the formula:

TABLE-US-00030 (SEQ ID NO: 277) QLVESGGGVVQPGRSLRLSCAAS GFX.sub.1FS SYGMH WVRQAPGKGLEW VAVISYDGX.sub.2NKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTA VYYCAK. Where: X1 is T or A; X2 is S or N;

[0134] In specific embodiments SEQ ID NO:277 is combined with D3-10 and furthermore with JH6b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 2.10.2 have amino acid sequence SEQ ID NO:74 and can be encoded by nucleotide sequence SEQ ID NO:73.

[0135] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH3-30 CDR1, or affinity matured sequences thereof having an amino acid sequence of the formula:

TABLE-US-00031 (SEQ ID NO: 278) GFX.sub.1FS SYGMH. Where: X1 is T or A;

[0136] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence SEQ ID NO:76.

[0137] In particular embodiments H chain CDR2 sequences are the germline VH3-30 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00032 (SEQ ID NO: 279) CDR2: VISYDGX.sub.2NKYYADSVKG. Where: X2 is S or N;

[0138] In specific embodiments an anti-GPNMB antibody of the invention comprises a CDR2 amino acid sequence SEQ ID NO:78.

[0139] In particular embodiments H chain CDR3 sequences are D3-10, JH6b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00033 (SEQ ID NO: 280) CDR3: X.sub.1X.sub.2X.sub.3VRGX.sub.4X.sub.5X.sub.6. Where: X1 is I or D; X2 is T or L; X3 is M or V; X4 is V or I; X5 is I or R; X6 is I or G;

[0140] A specific embodiment is anti-GPNMB antibody 2.10.2 having a CDR3 amino acid sequence SEQ ID NO:80.

VH4-59 Derived Anti-GPNMB Antibodies:

[0141] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH4-59 or are derived therefrom and include an amino acid sequence of the formula:

TABLE-US-00034 (SEQ ID NO: 281) QVQLQESGPGLVKPSETLSLTCTVS GX.sub.1SIS X.sub.2YYWS WIRQPPGKGL EWIGYX.sub.3YYSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTA VYYCAR. Where: X1 is G or D; X2 is S or N; X3 is I or F;

[0142] In specific embodiments SEQ ID NO:281 is combined with D6-19 and furthermore with JH4b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 1.10.2 have amino acid sequence SEQ ID NO:2 and can be encoded by nucleotide sequence SEQ ID NO:1.

[0143] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH4-59 CDR1, or affinity matured sequences thereof having an amino acid sequence of the formula:

TABLE-US-00035 (SEQ ID NO: 282) GX.sub.1SIS X.sub.2YYWS. Where: X1 is G or D; X2 is S or N;

In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence SEQ ID NO:4.

[0144] In particular embodiments H chain CDR2 sequences are the germline VH4-59 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00036 (SEQ ID NO: 283) CDR2: YX.sub.3YYSGSTNYNPSLKS. Where: X3 is I or F;

[0145] In specific embodiments an anti-GPNMB antibody of the invention comprises a CDR2 amino acid sequence SEQ ID NO:6.

[0146] In particular embodiments H chain CDR3 sequences are D6-19, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00037 (SEQ ID NO: 284) CDR3: X.sub.1X.sub.2GW---DY. Where: X1 is S or D; X2 is S or R;

[0147] A specific embodiment is anti-GPNMB antibody 1.10.2 having a CDR3 amino acid sequence SEQ ID NO:8.

VH5-51 Derived Anti-GPNMB Antibodies:

[0148] In a particular embodiment, GPNMB-binding human antibodies of the invention comprise germline V heavy chain region VH5-51 or are derived therefrom and include an amino acid sequence of the formula:

TABLE-US-00038 (SEQ ID NO: 285) QLVQSGAEVKKPGESLKISCX.sub.1GS GYX.sub.2FT X.sub.3YWIG WVRQMPGKGLE WMGX.sub.4IYPX.sub.5DSDTRYSPSFQG QVTISADKSISTAYLQWSSLKASDTA X.sub.6YYCAR. Where: X1 is K or Q; X2 is S or I; X3 is S or N; X4 is I or V; X5 is G or D; X6 is M or I;

[0149] In specific embodiments SEQ ID NO:285 is combined with D5-24 and furthermore with JH4b. In specific embodiments, after affinity maturation these GPNMB-binding human antibodies, for example Mab 2.24.1 have amino acid sequence SEQ ID NO:182 and can be encoded by nucleotide sequence SEQ ID NO:181.

[0150] Furthermore, in particular embodiments H chain CDR1 sequences are the germline VH5-51 CDR1, or affinity matured sequences thereof having an amino acid sequence of the formula:

TABLE-US-00039 (SEQ ID NO: 286) GYX.sub.2FT X.sub.3YWIG. Where: X2 is S or I; X3 is S or N;

[0151] In specific embodiments an anti-GPNMB antibody of the invention comprise a CDR1 sequence SEQ ID NO:184.

[0152] In particular embodiments H chain CDR2 sequences are the germline VH5-51 CDR2 or affinity matured sequences thereof of the formula:

TABLE-US-00040 (SEQ ID NO: 287) CDR2: X.sub.4IYPX.sub.5DSDTRYSPSFQG. Where: X4 is I or V; X5 is G or D;

[0153] In specific embodiments an anti-GPNMB antibody of the invention comprises a CDR2 amino acid sequence SEQ ID NO:186.

[0154] In particular embodiments H chain CDR3 sequences are D5-24, JH4b combinations or affinity matured sequences thereof and include an amino acid sequence of the formula:

TABLE-US-00041 (SEQ ID NO: 288) CDR3: X.sub.1WLQX2--FDY. Where: X1 is R or K; X2 is L or H;

[0155] A specific embodiment is anti-GPNMB antibody 2.24.1 having a CDR3 amino acid sequence SEQ ID NO:188.

[0156] The antibodies of the invention bind an epitope of GPNMB (SEQ ID NO:289), preferably within the mature sequence of GPNMB and more preferably within the extracellular domain (ECD) of GPNMB.

[0157] Antibodies of the invention bind GPNMB with an affinity of 10.sup.-6 to 10.sup.-11. Preferably with an affinity of 10.sup.-7 or greater and even more preferably 10.sup.-8 or greater. In a preferred embodiment, antibodies described herein bind to GPNMB with very high affinities (Kd), for example a human antibody that is capable of binding GPNMB with a Kd less than, but not limited to, 10.sup.-7, 10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12, 10.sup.-13 or 10.sup.-14 M, or any range or value therein. Affinity and/or avidity measurements can be measured by KinExA.RTM. and/or BIACORE.RTM., as described herein. In particular embodiments antibodies of the invention bind to GPNMB with Kds ranging from 50 to 150 pM.

[0158] Epitope mapping and secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the disclosed antibodies and their complexes with antigens (see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996). Such methods include, but are not limited to, X-ray crystallography (Biochem. Exp. Biol., 11:7-13, 1974) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0159] Furthermore, the specific part of the protein immunogen recognized by antibody may be determined by assaying the antibody reactivity to parts of the protein, for example an N terminal and C terminal half. The resulting reactive fragment can then be further dissected, assaying consecutively smaller parts of the immunogen with the antibody until the minimal reactive peptide is defined. Alternatively, the binding specificity, that is the epitope, of anti-GPNMB antibodies of the invention may be determined by subjecting GPNMB immunogen to SDS-PAGE either in the absence or presence of a reduction agent and analyzed by immunoblotting. Epitope mapping may also be performed using SELDI. SELDI ProteinChip.RTM. (LumiCyte) arrays used to define sites of protein-protein interaction. GPNMB protein antigen or fragments thereof may be specifically captured by antibodies covalently immobilized onto the PROTEINCHIP array surface. The bound antigens may be detected by a laser-induced desorption process and analyzed directly to determine their mass.

[0160] The epitope recognized by anti-GPNMB antibodies described herein may be determined by exposing the PROTEINCHIP Array to a combinatorial library of random peptide 12-mer displayed on Filamentous phage (New England Biolabs). Antibody-bound phage are eluted and then amplified and taken through additional binding and amplification cycles to enrich the pool in favor of binding sequences. After three or four rounds, individual binding clones are further tested for binding by phage ELISA assays performed on antibody-coated wells and characterized by specific DNA sequencing of positive clones.

Derivatives

[0161] This disclosure also provides a method for obtaining an antibody specific for GPNMB. CDRs in such antibodies are not limited to the specific sequences of H and L variable domains identified in Table 1 and may include variants of these sequences that retain the ability to specifically bind GPNMB. Such variants may be derived from the sequences listed in Table 1 by a skilled artisan using techniques well known in the art. For example, amino acid substitutions, deletions, or additions, can be made in the FRs and/or in the CDRs. While changes in the FRs are usually designed to improve stability and immunogenicity of the antibody, changes in the CDRs are typically designed to increase affinity of the antibody for its target. Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes may be determined empirically by routine techniques that involve altering the CDR and testing the affinity of the antibody for its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various alterations can be made according to the methods described in the art (Antibody Engineering, 2.sup.nd ed., Oxford University Press, ed. Borrebaeck, 1995). These include but are not limited to nucleotide sequences that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a "silent" change. For example, the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 3). Furthermore, any native residue in the polypeptide may also be substituted with alanine (Acta Physiol. Scand. Suppl. 643:55-67, 1998; Adv. Biophys. 35:1-24, 1998).

TABLE-US-00042 TABLE 3 Amino acid substitutions Original aa Residue Possible Substitutions Prefered substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, 1,4-Diamino- Arg butyric Acid, Gln, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, Norleucine Leu

[0162] Derivatives and analogs of antibodies of the invention can be produced by various techniques well known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2.sup.nd ed., Spring Verlag, Berlin, Germany).

[0163] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in the art (for example, Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)).

[0164] In one embodiment, a method for making an H variable domain which is an amino acid sequence variant of an H variable domain of the invention comprises a step of adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of the presently disclosed H variable domain, optionally combining the H variable domain thus provided with one or more L variable domains, and testing the H variable domain or H variable/L variable combination or combinations for specific binding to GPNMB or and, optionally, testing the ability of such antigen-binding domain to modulate GPNMB activity. The L variable domain may have an amino acid sequence that is identical or is substantially as set out according to Table 1.

[0165] An analogous method can be employed in which one or more sequence variants of a L variable domain disclosed herein are combined with one or more H variable domains.

[0166] A further aspect of the disclosure provides a method of preparing antigen-binding fragment that specifically binds with GPNMB. The method comprises: (a) providing a starting repertoire of nucleic acids encoding a H variable domain that either includes a CDR3 to be replaced or lacks a CDR3 encoding region; (b) combining the repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for a H variable CDR3 such that the donor nucleic acid is inserted into the CDR3 region in the repertoire, so as to provide a product repertoire of nucleic acids encoding a H variable domain; (c) expressing the nucleic acids of the product repertoire; (d) selecting a binding fragment specific for GPNMB; and (e) recovering the specific binding fragment or nucleic acid encoding it.

[0167] Again, an analogous method may be employed in which a L variable CDR3 of the invention is combined with a repertoire of nucleic acids encoding a L variable domain, which either include a CDR3 to be replaced or lack a CDR3 encoding region. The donor nucleic acid may be selected from nucleic acids encoding an amino acid sequence substantially as set out in SEQ ID NOs: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, 182, 200, 218, 236, 253, 256, 260, 265, 270, 274, 277, 281, 285, 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, 191, 209, 227 and 245. A sequence encoding a CDR of the invention (e.g., CDR3) may be introduced into a repertoire of variable domains lacking the respective CDR (e.g., CDR3), using recombinant DNA technology, for example, using methodology described by Marks et al. (Bio/Technology (1992) 10: 779-783). In particular, consensus primers directed at or adjacent to the 5' end of the variable domain area can be used in conjunction with consensus primers to the third framework region of human H variable genes to provide a repertoire of H variable domains lacking a CDR3. The repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences may be shuffled with repertoires of H variable or L variable domains lacking a CDR3, and the shuffled complete H variable or L variable domains combined with a cognate L variable or H variable domain to make the GPNMB specific antibodies of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system such as described in WO92/01047 so that suitable antigen-binding fragments can be selected.

[0168] Analogous shuffling or combinatorial techniques may be used (e.g. Stemmer, Nature (1994) 370: 389-391). In further embodiments, one may generate novel H variable or L variable regions carrying one or more sequences derived from the sequences disclosed herein using random mutagenesis of one or more selected H variable and/or L variable genes, such as error-prone PCR (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580). Another method that may be used is to direct mutagenesis to CDRs of H variable or L variable genes (Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813; J. Mol. Biol. (1996) 263: 551-567) Similarly, one or more, or all three CDRs may be grafted into a repertoire of H variable or L variable domains, which are then screened for an antigen-binding fragment specific for GPNMB.

[0169] A portion of an immunoglobulin variable domain will comprise at least one of the CDRs substantially as set out herein and, optionally, intervening framework regions as set out herein. The portion may include at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains to further protein sequences including immunoglobulin heavy chain constant regions, other variable domains (for example, in the production of diabodies), or proteinaceous labels as discussed in further detail below.

[0170] Although the embodiments illustrated in the Examples comprise a "matching" pair of H variable and L variable domains, a skilled artisan will recognize that alternative embodiments may comprise antigen-binding fragments containing only a single CDR from either L variable or H variable domain. Either one of the single chain specific binding domains can be used to screen for complementary domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to GPNMB. The screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO92/01047, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding domain is selected in accordance with phage display techniques as described.

[0171] Anti-GPNMB antibodies described herein can be linked to another functional molecule, e.g., another peptide or protein (albumin, another antibody, etc.), toxin, radioisotope, cytotoxic or cytostatic agents. For example, the antibodies can be linked by chemical cross-linking or by recombinant methods. The antibodies may also be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods to attach them are also shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285, and 4,609,546.

[0172] The disclosed antibodies may also be altered to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties can be deleted and/or one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody (WO 87/05330; CRC Crit. Rev. Biochem., 22: 259-306, 1981). Removal of any carbohydrate moieties from the antibodies may be accomplished chemically or enzymatically (Arch. Biochem. Biophys., 259: 52, 1987; Anal. Biochem., 118: 131, 1981; Meth. Enzymol., 138: 350, 1987). The antibodies may also be tagged with a detectable, or functional, label. Detectable labels include radiolabels such as .sup.131I or .sup.99Tc, which may also be attached to antibodies using conventional chemistry. Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

[0173] The valency of the antibodies may be custom designed to affect affinity and avidity, retention time at binding sites (see e.g. Am H. Pathol, 2002 160:1597-1608; J. Med. Chem. 2002 45:2250-2259; Br. J. Cancer 2002 86:1401-1410; Biomol. Eng. 2001 18:95-108; Int J. Cancer 2002 100:367-374).

[0174] Multiple specificity (bifunctional) binding reagents may be designed based upon the GPNMB specific sequences of the invention (Biomol. Eng. 2001 18:31-40). For example, a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments (Clin. Exp. Immunol. 1990, 79: 315-321; J. Immunol. 199, 2148:1547-1553). Such bispecific antibodies can be generated comprising a specificity to GPNMB and a second specificity to a second molecule using techniques that are well known (Immunol Methods 1994, 4:72-81; Wright and Harris, supra.; Traunecker et al. 1992 Int. J. Cancer (Suppl.) 7:51-52). Bispecific antibodies prepared in this manner selectively kill cells expressing GPNMB.

[0175] Antibodies, in which CDR sequences differ only insubstantially from those set out in SEQ ID NOs: 4, 6, 8, 13, 15, 17, 22, 24, 26, 31, 33, 35, 40, 42, 44, 49, 51, 53, 58, 60, 62, 67, 69, 71, 76, 78, 80, 85, 87, 89, 94, 96, 98, 103, 105, 107, 112, 114, 116, 121, 123, 125, 130, 132, 134, 139, 141, 143, 148, 150, 152, 157, 159, 161, 166, 168, 170, 175, 177, 179, 184, 186, 188, 193, 195, 197, 202, 204, 206, 211, 213, 215, 220, 222, 224, 229, 231, 233, 238, 240, 242, 247, 249 and 251. And formulas: 254, 257, 261, 266, 271, 278, 282, 286, 255, 258, 262, 267, 272, 275, 279, 283, 287, 259, 263, 264, 268, 269, 273, 276, 280, 284, 288, are encompassed within the scope of this invention. Typically, an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan. Unlike in CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter the effector function such as Fc receptor binding (U.S. Pat. Nos. 5,624,821; 5,648,260; Lund et al. (1991) J. Immun. 147: 2657-2662; Morgan et al. (1995) Immunology 86: 319-324), or changing the species from which the constant region is derived.

[0176] One of skill in the art will appreciate that the derivatives and modifications described above are not all-exhaustive, and that many other modifications would be obvious to a skilled artisan in light of the teachings of the present disclosure.

Nucleic Acids, Cloning and Expression Systems

[0177] The present disclosure further provides isolated nucleic acids encoding the disclosed antibodies. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

[0178] The nucleic acids provided herein comprise a coding sequence for a CDR, a H variable domain, and/or a L variable domain disclosed herein.

[0179] The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes which comprise at least one nucleic acid encoding a CDR, a H variable domain, and/or a L variable domain disclosed here.

[0180] The disclosure further provides a host cell which comprises one or more constructs as above.

[0181] Also provided are nucleic acids encoding any CDR (CDR1, CDR2, CDR3 from either the H or L variable domain), H variable or L variable domain, as well as methods of making of the encoded products. The method comprises expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, a H variable or L variable domain, or specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

[0182] Antigen-binding fragments, H variable and/or L variable domains and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.

[0183] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art including cells suitable for producing antibodies (Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999). Briefly, suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the invention may be used to produce the disclosed antibodies. Suitable expression systems also include transgenic animals (Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999).

[0184] Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory Press, 1989). Many known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are known in the art (Current Protocols in Molecular Biology, 2.sup.nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992).

[0185] The invention also provides a host cell comprising a nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.

Immunoconjugates

[0186] In another aspect, the antibodies of the invention can be used as a targeting agent for delivery of another therapeutic or a cytotoxic agent to a cell expressing GPNMB. The method includes administering an anti-GPNMB antibody coupled to a therapeutic or a cytotoxic agent or under conditions that allow binding of the antibody to GPNMB.

[0187] Anti-GPNMB antibodies are conjugated to a therapeutic agent, such as a cytotoxic compound, such that the resulting immunoconjugate exerts a cytotoxic or cytostatic effect on a GPNMB expressing cell. Particularly suitable moieties for conjugation to antibodies are chemotherapeutic agents, prodrug converting enzymes or toxins. For example, an anti-GPNMB antibody can be conjugated to a cytotoxic agent such as a chemotherapeutic agent (see infra) or a toxin (e.g. abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin). Alternatively, anti-GPNMB antibody may be conjugated to a pro-drug converting enzyme. The pro-drug converting enzyme can be recombinantly fused to the antibody or derivative thereof or chemically conjugated thereto using known methods. Exemplary pro-drug converting enzymes are carboxypeptidase G2, .beta.-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, .beta.-lactamase, .beta.-glucosidase, nitroreductase and carboxypeptidase A.

[0188] Any agent that exerts a therapeutic effect on GPNMB expressing cells can be used as an agent for conjugation to an anti-GPNMB antibody of the invention. Useful classes of cytotoxic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, NDA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-plantin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabilites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated purimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromcins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

[0189] The therapeutic agent can be a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g. auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), cuocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

[0190] In a specific embodiment, the cytotoxic or cytostatic agent is auristatin E (dolastatin-10) or a derivative thereof (e.g. an ester formed between auristatin E and a keto acid). Other typical auristatin derivatives include AFP, MMAR, and MMAE. The synthesis and structure of auristatin E and its derivates are described in U.S. Patent Application Publication No. 20030083263; PCT/US03/24209; PCT/US02/13435; and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

[0191] In a specific embodiment anti-GPNMB antibody 1.15.1 was coupled to monomethylauristatin E via intracellular protease-sensitive valine-citrulline peptide linker (vcMMAE). Methods for making the immunoconjugate can be found in Doronina S. O. et al, 2003 Nature Biotechnology 21(7):778-794.

[0192] Techniques for conjugating therapeutic agents to proteins, and in particular, antibodies are known in the art (see, e.g. Amon et al., 1985 in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. eds., Alan R. Liss, Inc., 1985; Hellstrom et al., 1987 in Controlled Drug Delivery, Robinson et al. eds., Marcel Dekker, Inc., 2.sup.nd ed. 1987; Thorpe 1985, in Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al. eds., EDITOR, 1985; Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. eds., Academic Press 1985; and Thorpe et al., 1982, Immunol. Rev. 62:119-58).

[0193] In certain embodiments of the invention, anti-GPNMB antibodies binding to GPNMB expressing cells, are internalized and accumulate in the cell. Thereby anti-GPNMB antibody immunoconjugates accumulate in GPNMB expressing cells. Typically when the anti-GPNMB antibody immunoconjugate is internalized, the agent is preferentially active. Alternatively, anti GPNMB immunoconjugates are not internalized and the drug is effective to deplete or inhibit GPNMB expressing cells by binding to the cell membrane. The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved off the antibody (e.g. by hydrolysis or by a cleaving agent). In this case, the agent can be attached to the antibody or derivative thereof with a cleavable linker that is sensitive to cleavage in the intracellular environment of the target but is not substantially sensitive to the extracellular environment, such that the conjugate is cleaved from the antibody or derivative thereof when it is internalized by the GPNMB expressing cell (e.g. in the endosomal or, for example by virtue of pH sensitivity or protease sensitivity, in the lysosomal environment or in a caveolea).

[0194] A therapeutic agent of the immunoconjugate can be charged relative to the plasma membrane (e.g. polarized or net charge relative to the plasma membrane), thereby further minimizing the ability of the agent to cross the plasma membrane once internalized by a cell.

[0195] The anti-GPNMB antibody immunoconjugate can comprise a linker region between the therapeutic agent and the antibody. The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment. The linker can be, e.g. a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to a lysosomal or endosomal protease. Often the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins and D and plasmin, all of which are known to hydrolyze dipeptide drug derivative s resulting in the release of active drug inside target cells (see Dubowchik and Walker, 1999 Pharm. Therapeutics 83:67-123). Other linkers are described e.g. in U.S. Pat. No. 6,214,345.

[0196] Linkers can be pH-sensitive can often be hydrolizable under acidic conditions such as is found in the lysosome (see e.g. U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999 Pharm. Therapeutics 83:67-123; Neville et al., 1989 BIol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the pH of the lysosome. Linkers can be cleavable under reducing conditions (e.g. a disulfide linker) (see e.g., Thorpe et al., 1987 Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunnoconjugates: Antibody Conjugates in Radioimmagery and Therapy of Cancer, C. W. Vogel ed, Oxford U. Press, 1987; U.S. Pat. No. 4,880,935). The linker can be a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoly linker (lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-1312).

Prophylactic and Therapeutic Uses of the Present Invention

[0197] The antibodies of the invention can act as either agonists or antagonists of GPNMB, depending on the methods of their use. The antibodies can be used to prevent, diagnose, or treat medical disorders in a subject, especially in humans. Antibodies of the invention can also be used for isolating GPNMB or GPNMB-expressing cells. Furthermore, the antibodies can be used to treat a subject at risk of or susceptible to a disorder or having a disorder associated with aberrant GPNMB expression or function. Antibodies of the invention can be used to detect GPNMB in such subjects.

[0198] The present invention provides methods for treating and/or preventing a disease or disorder associated with overexpression of GPNMB and/or cell hyperproliferative disorders, particularly cancer, in a subject comprising administering an effective amount of a composition that can target cells expressing GPNMB, and inhibiting the GPNMB expression or function, and/or having therapeutic or prophylactic effects on the hyperproliferative cell disease. In one embodiment, the method of the invention comprises administering to a subject a composition comprising an immunoconjugate that comprises an antibody of the invention and a cytotoxic agent against the hyperproliferative cell disease. In another embodiment, the method of the invention comprises administering to a subject in need thereof a composition comprising a naked IgG1 antibody of the invention and one or more immunomodulators. In yet another embodiment, the method of the invention comprises administering to a subject in need thereof a composition comprising a single chain Fv antibody (anti-GPNMB) conjugated to a cytotoxic agent, or a composition comprising a bispecific antibody that have a single chain anti-GPNMB antibody component and a anti-CD3 antibody component. In a preferred embodiment, the hyperproliferative cell disease is cancer. More preferably, the cancer is melanoma, or a cancer of the CNS system, such as astrocytoma, glioblastoma, medulloblastoma, or neoplastic meningitis.

[0199] The present invention provides therapies comprising administering one of more antibodies of the invention and compositions comprising said antibodies to a subject, preferably a human subject, for preventing and/or treating a disorder characterized by or associated with aberrant expression and/or activity of GPNMB or a symptom thereof. In one embodiment, the invention provides a method of preventing or treating a disorder characterized by or associated with aberrant expression and/or activity of GPNMB or a symptom thereof, said method comprising administering to a subject in need thereof an effective amount of one or more antibodies of the invention. In certain embodiments, an effective amount of one or more immunoconjugates comprising one or more antibodies of the invention is administered to a subject in need thereof to prevent or treat a disorder characterized by or associated with aberrant expression and/or activity of GPNMB or a symptom thereof.

[0200] The invention also provides methods of preventing or treating a disorder characterized by or associated with aberrant expression and/or activity of GPNMB or a symptom thereof, said methods comprising administering to a subject in need thereof one or more of the antibodies of the invention and one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than antibodies of the invention. The prophylactic or therapeutic agents of the combination therapies of the invention can be administered sequentially or concurrently. In a specific embodiment, the combination therapies of the invention comprise an effective amount of one or more antibodies of the invention and an effective amount of at least one other therapy (e.g., prophylactic or therapeutic agent) which has a different mechanism of action than said antibodies. In certain embodiments, the combination therapies of the present invention improve the prophylactic or therapeutic effect of one or more antibodies of the invention by functioning together with the antibodies to have an additive or synergistic effect. In certain embodiments, the combination therapies of the present invention reduce the side effects associated with the therapies (e.g., prophylactic or therapeutic agents).

[0201] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject, preferably a human subject, in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

[0202] In a specific embodiment, a pharmaceutical composition comprising one or more antibodies of the invention described herein is administered to a subject, preferably a human, to prevent and/or treat a disorder characterized by or associated with aberrant expression and/or activity of GPNMB or a symptom thereof. In accordance with the invention, pharmaceutical compositions of the invention may also comprise one or more therapies (e.g., prophylactic or therapeutic agents), other than antibodies of the invention.

[0203] The antibodies of the invention may also be used to detect the presence of GPNMB in biological samples (in diagnostic methods or use as an efficacy marker). The amount of GPNMB detected may be correlated with the expression level of GPNMB, which, in turn, is correlated with the disease, tumor type, tumor burden or stage using methods known in the art (see for example recommendations of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) Pharm Res. 2003 November; 20(11):1885-900). Detection methods that employ antibodies are well known in the art and include, for example, ELISA, radioimmunoassay, immunoblot, Western blot, IHC, immunofluorescence, immunoprecipitation. The antibodies may be provided in a diagnostic kit that incorporates one or more of these techniques to detect GPNMB. Such a kit may contain other components, packaging, instructions, or other material to aid the detection of the protein. In a specific embodiment, the antibodies of the invention are conjugated to a radioactive isotope, and are injected to a subject to detect cells that overexpressing GPNMB.

[0204] Where the antibodies are intended for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme). If desired, the antibodies of the invention may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

[0205] Antibodies of the invention can be used in screening methods to identify inhibitors of GPNMB effective as therapeutics. In such a screening assay, a first binding mixture is formed by combining GPNMB and an antibody of the invention; and the amount of binding in the first binding mixture (M.sub.0) is measured. A second binding mixture is also formed by combining GPNMB, the antibody, and the compound or agent to be screened, and the amount of binding in the second binding mixture (M.sub.1) is measured. A compound to be tested may be another anti-GPNMB antibody. The amounts of binding in the first and second binding mixtures are then compared, for example, by calculating the M.sub.1/M.sub.0 ratio. The compound or agent is considered to be capable of modulating a GPNMB-associated responses if a decrease in binding in the second binding mixture as compared to the first binding mixture is observed. The formulation and optimization of binding mixtures is within the level of skill in the art, such binding mixtures may also contain buffers and salts necessary to enhance or to optimize binding, and additional control assays may be included in the screening assay of the invention. Compounds found to reduce the GPNMB-antibody binding by at least about 10% (i.e., M.sub.1/M.sub.0<0.9), preferably greater than about 30% may thus be identified and then, if desired, secondarily screened for the capacity to ameliorate a disorder in other assays or animal models as described below. The strength of the binding between GPNMB and an antibody can be measured using, for example, an enzyme-linked immunoadsorption assay (ELISA), radio-immunoassay (RIA), surface plasmon resonance-based technology (e.g., Biacore), all of which are techniques well known in the art.

[0206] The compound may then be tested in vitro as described in the Examples, infra.

Dosage and Frequency of Administration

[0207] The amount of a prophylactic or therapeutic agent or a composition of the invention which will be effective in the prevention and/or treatment of a disorder associated with or characterized by aberrant expression and/or activity of GPNMB can be determined by standard clinical methods. For example, the dosage of the composition which will be effective in the treatment and/or prevention of cancer can be determined by administering the composition to an animal model. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. Preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. Therapeutically effective dosages achieved in one animal model can be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et al. (1966) Cancer Chemother. Reports, 50(4): 219-244).

[0208] Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, gender, immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in literature and recommended in the Physician's Desk Reference (59th ed., 2005).

[0209] The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0210] For other cancer therapeutic agents administered to a patient, the typical doses of various cancer therapeutics are known in the art. Given the invention, certain preferred embodiments will encompass the administration of lower dosages in combination treatment regimens than dosages recommended for the administration of single agents.

[0211] In a specific embodiment, the dosage of an antibody or an immunoconjugate comprising an antibody of the invention administered to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB (e.g., cancer) in a patient is 30 mg/kg or less, 25 mg/kg or less, 20 mg/kg or less, 15 mg/kg or less, preferably 12 mg/kg or less, 11 mg/kg or less, 10 mg/kg or less, 9 mg/kg or less, 8 mg/kg or less, 7 mg/kg or less, 6 mg/kg or less, 5 mg/kg or less, 4 mg/kg or less, 3 mg/kg or less, 2 mg/kg or less, or 1 mg/kg or less of a patient's body weight. In another embodiment, the dosage of an antibody or an immunoconjugate of the invention administered to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB (e.g., cancer) in a patient is a unit dose of about 0.01 mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 8 mg/kg, about 0.1 mg/kg to about 7 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, preferably, about 0.1 mg/kg to about 3 mg/kg, about 0.2 mg/kg to 3 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 0.4 mg/kg to about 3 mg/kg, about 0.6 mg/kg to about 3 mg/kg, about 0.8 mg/kg to about 3 mg/kg, about 0.1 mg/kg to 2 mg/kg, about 0.1 mg/kg to 1 mg/kg. In certain embodiments, the dosage of an antibody or an immunoconjugate comprising an antibody of the invention administered to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB (e.g., cancer) in a patient is a unit dose of about 0.1 mg/kg, about 0.2 mg/kg, about 0.4 mg/kg, about 0.6 mg/kg, about 0.8 mg/kg, about 1.1 mg/kg, or about 1 mg/kg.

[0212] In certain embodiments, a subject is administered one or more doses of an effective amount of one or more antibodies or immunoconjugates of the invention to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, wherein the dose of an effective amount of said antibodies, immunoconjugates, compositions, or combination therapies reduces and/or inhibits proliferation of cancerous cells by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, at least 80 to 85%, at least 85% to 90%, at least 90% to 95%, or at least 95% to 98% relative to a control such as PBS in an in vitro and/or in vivo assay well-known in the art.

[0213] In other embodiments, a subject is administered one or more doses of an effective amount of one or more antibodies or immunoconjugates of the invention to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, wherein the dose of an effective amount achieves a serum titer of at least 0.1 .mu.g/mL, at least 0.5 .mu.g/mL, at least 1 .mu.g/mL, at least 2 .mu.g/mL, at least 5 .mu.g/mL, at least 6 .mu.g/mL, at least 10 .mu.g/mL, at least 15 .mu.g/mL, at least 20 .mu.g/mL, at least 25 .mu.g/mL, at least 50 .mu.g/mL, at least 100 .mu.g/mL, at least 125 .mu.g/mL, at least 150 .mu.g/mL, at least 175 .mu.g/mL, at least 200 .mu.g/mL, at least 225 .mu.g/mL, at least 250 .mu.g/mL, at least 275 .mu.g/mL, at least 300 .mu.g/mL, at least 325 .mu.g/mL, at least 350 .mu.g/mL, at least 375 .mu.g/mL, or at least 400 .mu.g/mL of the antibodies of the invention. In yet other embodiments, a subject is administered a dose of an effective amount of one or more antibodies or immunoconjugates of the invention to achieve a serum titer of at least 0.1 .mu.g/mL, at least 0.5 .mu.g/mL, at least 1 .mu.g/mL, at least, 2 .mu.g/mL, at least 5 .mu.g/mL, at least 6 .mu.g/mL, at least 10 .mu.g/mL, at least 15 .mu.g/mL, at least 20 .mu.g/mL, at least 25 .mu.g/mL, at least 50 .mu.g/mL, at least 100 .mu.g/mL, at least 125 .mu.g/mL, at least 150 .mu.g/mL, at least 175 .mu.g/mL, at least 200 .mu.g/mL, at least 225 .mu.g/mL, at least 250 .mu.g/mL, at least 275 .mu.g/mL, at least 300 .mu.g/mL, at least 325 .mu.g/mL, at least 350 .mu.g/mL, at least 375 .mu.g/mL, or at least 400 .mu.g/mL of the antibodies and a subsequent dose of an effective amount of one or more antibodies or immunoconjugates of the invention is administered to maintain a serum titer of at least 0.1 .mu.g/mL, at least 0.5 .mu.g/mL, at least 1 .mu.g/mL, at least, 2 .mu.g/mL, at least 5 .mu.g/mL, at least 6 .mu.g/mL, at least 10 .mu.g/mL, at least 15 .mu.g/mL, at least 20 .mu.g/mL, at least 25 .mu.g/mL, at least 50 .mu.g/mL, at least 100 .mu.g/mL, at least 125 .mu.g/mL, at least 150 .mu.g/mL, at least 175 .mu.g/mL, at least 200 .mu.g/mL, at least 225 .mu.g/mL, at least 250 .mu.g/mL, at least 275 .mu.g/mL, at least 300 .mu.g/mL, at least 325 .mu.g/mL, at least 350 .mu.g/mL, at least 375 .mu.g/mL, or at least 400 .mu.g/mL. In accordance with these embodiments, a subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subsequent doses.

[0214] In a specific embodiment, the invention provides methods of preventing and/or treating a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, said method comprising administering to a subject in need thereof a unit dose of at least 0.01 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.4 mg/kg, at least 0.6 mg/kg, at least 0.8 mg/kg, at least 1 mg/kg, or at least 1.1 mg/kg of one or more antibodies or immunoconjugates of the invention. In another embodiment, the invention provides methods of preventing and/or treating a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, said method comprising administering to a subject in need thereof a unit dose of at least 0.01 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.4 mg/kg, at least 0.6 mg/kg, at least 0.8 mg/kg, at least 1 mg/kg, or at least 1.1 mg/kg of one or more antibodies or immunoconjugates of the invention once every 7 days, preferably, once every 10 days, once every 12 days, once every 14 days, once every 16 days, once every 18 days, once every three weeks, or once a month. In a preferred embodiment, an immunoconjugate of the instant invention is administered at a unit dose of about 0.1 mg/kg, about 0.2 mg/kg, about 0.4 mg/kg, about 0.6 mg/kg, about 0.8 mg/kg, about 1.1 mg/kg, or about 1 mg/kg once every 10 to 20 days with 2 to 4 cycles.

[0215] The present invention provides methods of preventing and/or treating a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, said method comprising: (a) administering to a subject in need thereof one or more doses of a prophylactically or therapeutically effective amount of one or more antibodies or immunoconjugates of the invention; and (b) monitoring the plasma level/concentration of the said administered antibody or antibodies in said subject after administration of a certain number of doses of the said antibody or antibodies. Moreover, preferably, said certain number of doses is 1, 2, 3, 4, 5, 6, 7, or 8 doses of a prophylactically or therapeutically effective amount one or more antibodies or immunoconjugates of the invention.

[0216] In a specific embodiment, the invention provides a method of preventing and/or treating a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, said method comprising: (a) administering to a subject in need thereof a dose of at least 0.1 mg/kg (preferably at least at least 0.2 mg/kg, at least 0.4 mg/kg, at least 0.6 mg/kg, at least 0.8 mg/kg, at least 1 mg/kg, or at least 1.1 mg/kg) of one or more antibodies or immunoconjugates of the invention; and (b) administering one or more subsequent doses to said subject when the plasma level of the antibody or antibodies administered in said subject is less than 0.1 .mu.g/mL, preferably less than 0.25 .mu.g/mL, less than 0.5 .mu.g/mL, less than 0.75 .mu.g/mL, or less than 1 .mu.g/mL. In another embodiment, the invention provides a method of preventing and/or treating a disorder associated with or characterized by aberrant expression and/or activity of GPNMB, said method comprising: (a) administering to a subject in need thereof one or more doses of at least at least 0.1 mg/kg (preferably at least at least 0.2 mg/kg, at least 0.4 mg/kg, at least 0.6 mg/kg, at least 0.8 mg/kg, at least 1 mg/kg, or at least 1.1 mg/kg) of one or more antibodies of the invention; (b) monitoring the plasma level of the administered antibody or antibodies of the invention in said subject after the administration of a certain number of doses; and (c) administering a subsequent dose of the antibody or antibodies of the invention when the plasma level of the administered antibody or antibodies in said subject is less than 0.1 .mu.g/mL, preferably less than 0.25 .mu.g/mL, less than 0.5 .mu.g/mL, less than 0.75 .mu.g/mL, or less than 1 .mu.g/mL. Preferably, said certain number of doses is 1, 2, 3, 4, 5, 6, 7, or 8 doses of an effective amount of one or more antibodies or immunoconjugates of the invention.

[0217] Therapies (e.g., prophylactic or therapeutic agents), other than antibodies or immunoconjugates of the invention, which have been or are currently being used to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB can be administered in combination with one or more antibodies or immunoconjugates of the invention according to the methods of the invention to treat and/or prevent a disorder associated with or characterized by aberrant expression and/or activity of GPNMB. Preferably, the dosages of prophylactic or therapeutic agents used in combination therapies of the invention are lower than those which have been or are currently being used to prevent and/or treat a disorder associated with or characterized by aberrant expression and/or activity of GPNMB.

[0218] In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In preferred embodiments, two or more therapies are administered within the same patient visit.

[0219] In certain embodiments, one or more antibodies of the invention and one or more other therapies (e.g., prophylactic or therapeutic agents) are cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.

Pharmaceutical Compositions and Methods of Administration

[0220] The disclosure provides compositions comprising anti-GPNMB antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions typically comprise one or more antibodies of the present invention and a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.

[0221] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. The administration may, for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.

[0222] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0223] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars; polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate, and gelatin.

[0224] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the antibodies can be combined with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature; a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0225] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished, for example, through the use of lozenges, nasal sprays, inhalers, or suppositories. For example, in case of antibodies that comprise the Fc portion, compositions may be capable of transmission across mucous membranes in intestine, mouth, or lungs (e.g., via the FcRn receptor-mediated pathway as described in U.S. Pat. No. 6,030,613). For transdermal administration, the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art. For administration by inhalation, the antibodies may be delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0226] In certain embodiments, the presently disclosed antibodies are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions containing the presently disclosed antibodies can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0227] It may be advantageous to formulate oral or parenteral compositions in a dosage unit form for ease of administration and uniformity of dosage. The term "dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0228] Toxicity and therapeutic efficacy of the composition of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compositions that exhibit large therapeutic indices are preferred.

[0229] For any composition used in the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. Examples of suitable bioassays include DNA replication assays, clonogenic assays and other assays as, for example, described in the Examples. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the antibody which achieves a half-maximal inhibition of symptoms). Circulating levels in plasma may be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage lies preferably within a range of circulating concentrations with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized.

[0230] Antibodies can be modified to become immunotoxins utilizing techniques that are well known in the art (Vitetta 1993, Immunol Today 14:252; U.S. Pat. No. 5,194,594). Cyotoxic immunoconjugates are known in the art and have been used as therapeutic agents. Such immunoconjugates may for example, use maytansinoids (U.S. Pat. No. 6,441,163), tubulin polymerization inhibitor, auristatin (Mohammad et al, 1999 Int. J. Oncol 15(2):367-72; Doronina et al, 2003 Nature Biotechnology 21(7):778-784), dolastatin derivatives (Ogawa et al, 2001 Toxicol Lett. 121(2):97-106) 21(3)778-784), Mylotarg.RTM. (Wyeth Laboratories, Philadelphia, Pa.); maytansinoids (DM1), taxane or mertansine (ImmunoGen Inc.).

[0231] Immunoradiopharmaceuticals utilizing anti-GPNMB antibodies may be prepared utilizing techniques that are well known in the art (Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996); U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902). Each of the immunotoxins and radiolabeled antibody molecules selectively kill cells expressing GPNMB. Radiolabels are known in the art and have been used for diagnostic or therapeutic radioimmuno conjugates. Examples of radiolabels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.105Rh, Rhenium-186, Rhenium-188, Samarium-153, Copper-64, Scandium-47). For example, radionuclides which have been used in radioimmunoconjugate guided clinical diagnosis include, but are not limited to: .sup.123I .sup.99Tc, .sup.67Ga, as well as .sup.111In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (see Peirersz et al., 1987). These radionuclides include, for example, .sup.188Re and .sup.186Re as well as .sup.90Y, and to a lesser extent .sup.199Au and .sup.67Cu. I-(131) (see for example U.S. Pat. No. 5,460,785). Radiotherapeutic chelators and chelator conjugates are known in the art (U.S. Pat. Nos. 4,831,175, 5,099,069, 5,246,692, 5,286,850, and 5,124,471).

EXAMPLES

[0232] The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.

Example 1

Immunogen

[0233] Recombinant human GPNMB (SEQ ID NO:289), specifically the extra-cellular domain (ECD) was prepared for use as the immunogen. Generally, cDNA encoding the ECD of GPNMB with a C-terminus VS-HIS tag was transfected into HEK 293 cells, expressed and purified using cation exchange chromatography with a POROS HS 50 (Applied Biosystems, Foster City, Calif.). Sample was eluted with 1M NaCl at a pH of 5.5, followed by metal affinity chromatography (Pharmacia metal chelate 5 mL). The sample was eluted against a linear gradient from 10-500 mM imidazole over 10 CV (column volume) Dialysis occurred using 20 mM Tris/50 mM NaCl at pH 7.4 (2 L.times.2). The sample was then filtered through a 0.22 .mu.m filter.

Example 2

Immunization

[0234] A preferred method for generating fully human antibodies uses XenoMouse.RTM. strains of mice which have been engineered to contain 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus (Green et al. 1994 Nature Genetics 7:13-21; Mendez et al. 1997 Nature Genetics 15:146-156; Green and Jakobovits, 1998 J. Exp. Med. 188:483-495; U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598.) In an alternative approach, the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V.sub.H genes, one or more D.sub.H genes, one or more J.sub.H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal (Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996); U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, 6,255,458, 5,591,669, 6,023,010, 5,612,205, 5,721,367, 5,789,215, 5,643,763, 5,981,175). It is understood that the .lamda..kappa. XenoMouse.RTM. may be used to generate anti-GPNMB antibodies utilizing lambda V regions. Such antibodies are within the scope of the invention.

[0235] Immunization

[0236] GPNMB-V5His immunogen (as prepared in Example 1) was used as an antigen. Monoclonal antibodies against GPNMB were developed by sequentially immunizing XenoMouse.RTM. mice (XenoMouse.RTM. XMG2 strain), Abgenix, Inc. Fremont, Calif. XenoMouse.RTM. animals were immunized via footpad route for all injections. The total volume of each injection was 50 .mu.l per mouse, 25 .mu.l per footpad.

[0237] For cohort 1 (10 XMG2 mice), the initial immunization was with 10 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with 100 .mu.g alum gel ("Adju-Phos": aluminum phosphate gel adjuvant, Superfos BIOSECTOR.TM. a/s, distributed by E.M. Sergent Pulp and Chemical Co., Clifton, N.J., cat. #1452-250) per mouse. The subsequent five boosts were made with 5 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with 100 .mu.g alum gel in pyrogen-free D-PBS. The seventh boost consisted of 5 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with TITERMAX GOLD.RTM.(Sigma; cat. # T2684). The eighth injection consisted of 5 .mu.g of GPNMB-V5His admixed 1:1 v/v with 100 .mu.g alum gel. A final boost was made with 5 .mu.g GPNMB-V5His in pyrogen-free DPBS, without adjuvant. The XenoMouse.RTM. mice were immunized on days 0, 3, 6, 10, 14, 17, 23, and 27 for this protocol and fusions were performed on day 31. The bleed was made through Retro-Orbital Bleed procedure on day 21 after the sixth boost.

[0238] For cohort 2 (10 XMG2 mice), the initial immunization was with 10 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with 100 .mu.g alum gel per mouse. The subsequent two boosts were made with 5 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with 100 .mu.g alum gel in pyrogen-free D-PBS. The fourth boost consisted of 5 .mu.g of GPNMB-V5His admixed 1:1 (v/v) with TITERMAX GOLD.RTM.(Sigma; cat. # T2684). The following fifth to seventh injection consisted of 5 .mu.g of GPNMB-V5His admixed 1:1 v/v with 100 .mu.g alum gel. The eighth injection and final boost was made with 5 .mu.g GPNMB-V5His in pyrogen-free DPBS, without adjuvant. The XenoMouse.RTM. mice were immunized on days 0, 3, 7, 11, 14, 17, 22, 25 and 74. for this protocol and fusions were performed on day 78. The bleeds was made through Retro-Orbital Bleed procedure on day 21 after the sixth boost.

[0239] The footpad injection was performed by the following protocol using only the ventral surface of both hind limb paws. A solution was injected beneath the skin without piercing the muscle tissue by using an insulin 1/2 mL syringe with attached 28 or 30 gauge.times.1/2'' needle. The mouse to be injected was grasped by the loose fur along its neck and back so that it was immobilized and was turned over so the ventral side was accessible. The hind limb of the mouse was grasped and the needle was inserted (bevel side up) at the ankle, threading just under the skin until the needle tip reached the paw. The needle was inserted along the outside length of the hind foot carefully, to avoid the vein located towards the inner side of the foot. Once the tip of the needle reached the paw, the solution was injected slowly until resistance was felt or the designated volume had been dispensed. The needle was then withdrawn and the second hind foot injected in the same manner.

[0240] The following Table 4 provides the immunization schedule for the 2 groups of mice.

TABLE-US-00043 TABLE 4 Immunization Schedule of GPNMB Antigen: GPNMB-soluble at 043 mg/mL Target Group No. Mode of Immunization No. of mice Antigen 1st injection 2nd boost GPNMB 1 Footpad 10 GPNMB- 10 ug/mouse 5 ug/mouse soluble Alum Gel Alum Gel Day 0 Day 3 3rd boost 4th boost 5th boost 6th boost Bleed 7th boost 8th boost Fusion 5 ug/mouse 5 ug/mouse 5 ug/mouse 5 ug/mouse Day 21 5 ug/mouse 10 ug/mouse Day 31 Alum Gel Alum Gel Alum Gel Alum Gel Titermax Gold D-PBS Day 6 Day 10 Day 14 Day 17 Day 23 Day 27 Target group# Mode of Immunization # mice Antigen 1st injection 2nd boost GPNMB 2 Footpad 10 GPNMB- 10 ug/mouse 5 ug/mouse soluble Alum Gel Alum Gel Day 0 Day 3 3rd boost 4th boost 5th boost 6th boost Bleed 7th boost 8th boost 9th boost Fusion 5 ug/mouse 5 ug/mouse 5 ug/mouse 5 ug/mouse Day 21 5 ug/mouse 10 ug/mouse 10 ug/mouse Day 104 Alum Gel Titermax Gold Alum Gel Alum Gel Alum Gel D-PBS D-PBS Day 7 Day 11 Day 14 Day 17 Day 22 Day 25 Day 100

Selection of Animals for Harvest by Titer

[0241] Anti-GPNMB antibody titers in the serum from immunized XenoMouse.RTM. mice were determined by ELISA. Briefly, three sets of ELISAs were set up. GPNMB (+NMB) at 1 .mu.g/mL, GPNMB(-NMB) at 1 .mu.g/mL, and NMB at 1 .mu.g/mL were coated onto Costar Labcoat Universal Binding Polystyrene 96-well plates (Corning, Acton, Mass.) overnight at 4.degree. C. in Antigen Coating Buffer (0.1 M Carbonate Buffer, pH 9.6 NaHCO.sub.3 (MW 84) 8.4 g/L). The next day, the plates were washed three times with washing buffer (0.05% Tween 20 in 1.times.PBS) using a Biotek plate washer. The plates were then blocked with 200 ul/well blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1.times.PBS) and incubated at room temperature for 1 h. After the one-hour blocking, the plates were washed three times with washing buffer using a Biotek plate washer. Sera from either GPNMB immunized XenoMouse.RTM. mice or naive XenoMouse.RTM. animals were titrated in 0.5% BSA/PBS buffer at 1:3 dilutions in duplicate from a 1:100 initial dilution. The last well was left blank. These plates were incubated at room temperature for 2 h, and the plates were then washed three times with washing buffer using a Biotek plate washer. A goat anti-human IgG Fc-specific horseradish peroxidase (HRP, Pierce, Rockford, Ill.) conjugated antibody was added at a final concentration of 1 .mu.g/mL and incubated for 1 hour at room temperature. The plates were washed three times with washing buffer using a Biotek plate washer. After washing, the plates were developed with the addition of TMB chromogenic substrate (BioFx BSTP-0100-01) for 10-20 min or until negative control wells start to show color. Then the ELISA was stopped by the addition of Stop Solution (650 nM Stop reagent for TMB (BioFx BSTP-0100-01), reconstituted with 100 mL H.sub.2O per bottle). The specific titer of each XenoMouse.RTM. animal was determined from the optical density at 650 nm and is shown in Tables 2 and 3 below. The titer value is the reciprocal of the greatest dilution of sera with an OD reading two-fold that of background. Therefore, the higher the number, the greater was the humoral immune response to GPNMB. The results are provided in Table 5.

TABLE-US-00044 TABLE 5 XENOMOUSE .RTM. Anti-GPNMB Serum titers Group 1 mice, fusion on Day 21 after 6 inj. Reactivity to Reactivity to GPNMB Reactivity Mouse GPNMB (+GPNMB) to GPNMB ID Titers via hIgG Titers via hIgG Titers via hIgG 1-1 20,000 5,000 225 1-2 5,000 800 200 1-3 35,000 7,500 225 1-4 75,000 22,000 225 1-5 8,000 2,000 325 1-6 6,000 800 1800 1-7 22,000 7,500 225 1-8 6,000 2,000 200 1-9 7,000 2,000 75 1-10 22,000 7,500 200 1-NC1 <100 <100 <100 1-NC2 <100 <100 <100 Group 2 mice, bled on Day 21 after 6 inj. Reactivity to Reactivity to GPNMB Reactivity to Mouse GPNMB (-GPNMB) (+GPNMB) GPNMB Titers ID Titers via hIgG Titers via hIgG via hIgG 2-1 100,000 2,600 50 2-2 8,000 2,600 50 2-3 15,000 4,000 50 2-4 7,000 2,200 75 2-5 22,000 6,500 250 2-6 60,000 22,000 60 2-7 19,000 7,000 50 2-8 5,000 1,200 50 2-9 16,000 3,500 110 2-10 12,000 5,000 110 2-NC1 <100 <100 <100 2-NC2 <100 <100 <100

[0242] Pooled anti-GPNMB sera from immunized animals was also evaluated by FACS for reactivity to UACC-62, SF539, SKMEL5, U87MG, and LOX1MVI cell lines. Pooled sera were tested at 1:10, 1:100 and 1:500 compared to Anti-IL13 serum (control) and prebleeds diluted at 1:10, 1:100 (control).

Example 3

Antibodies

[0243] Hybridoma cell lines were generated from immunized mice demonstrated to have anti-GPNMB titers using standard techniques (see Mendez et al, 1997, Nat Genet. 15:146-156).

[0244] Immunized mice were sacrificed by cervical dislocation, and the lymph nodes were harvested and pooled from each cohort. The lymphoid cells were dissociated by grinding in DMEM to release the cells from the tissues, and the cells were suspended in DMEM. The cells were counted, and 0.9 mL DMEM per 100 million lymphocytes was added to the cell pellet to resuspend the cells gently but completely. Using 100 .mu.l of CD90+ magnetic beads per 100 million cells, the cells were labeled by incubating the cells with the magnetic beads at 4.degree. C. for 15 minutes. The magnetically-labeled cell suspension containing up to 10.sup.8 positive cells (or up to 2.times.10.sup.9 total cells) was loaded onto a LS+ column and the column washed with DMEM. The total effluent was collected as the CD90-negative fraction (most of these cells were expected to be B cells).

[0245] The fusion was performed by mixing washed enriched B cells from above and nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC, cat.# CRL 1580 (Kearney et al, J. Immunol. 123, 1979, 1548-1550) at a ratio of 1:1. The cell mixture was gently pelleted by centrifugation at 800 g. After complete removal of the supernatant, the cells were treated with 2-4 mL of Pronase solution (CalBiochem, cat. #53702; 0.5 mg/mL in PBS) for no more than 2 minutes. Then 3-5 mL of FBS was added to stop the enzyme activity and the suspension was adjusted to 40 mL total volume using electro cell fusion solution, ECFS (0.3 M Sucrose, Sigma, Cat#57903, 0.1 mM Magnesium Acetate, Sigma, Cat# M2545, 0.1 mM Calcium Acetate, Sigma, Cat# C4705). The supernatant was removed after centrifugation and the cells were resuspended in 40 mL ECFS. This wash step was repeated and the cells again were resuspended in ECFS to a concentration of 2.times.10.sup.6 cells/mL.

[0246] Electro-cell fusion was performed using a fusion generator, model ECM2001, Genetronic, Inc., San Diego, Calif. The fusion chamber size used was 2.0 mL, using the Abgenix, Inc. optimum instrument settings to do ECF.

[0247] After ECF, the cell suspensions were carefully removed from the fusion chamber under sterile conditions and transferred into a sterile tube containing the same volume of Hybridoma Culture Medium (DMEM (JRH Biosciences), 15% FBS (Hyclone), supplemented with L-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma) and IL-6 (Boehringer Mannheim)). The cells were incubated for 15-30 minutes at 37.degree. C., and then centrifuged at 400 g (1000 rpm [but in what rotor? Otherwise, leave out the rpm]) for five minutes. The cells were gently resuspended in a small volume of Hybridoma Selection Medium (Hybridoma Culture Medium supplemented with 0.5.times. HA (Sigma, cat. # A9666)), and the volume was adjusted appropriately with more Hybridoma Selection Medium, based on a final plating of 5.times.10.sup.6 B cells total per 96-well plate and 200 .mu.L per well. The cells were mixed gently and pipetted into 96-well plates and allowed to grow. On day 7 or 10, one-half the medium was removed, and the cells were re-fed with Hybridoma Selection Medium.

[0248] After 14 days of culture, hybridoma supernatants were screened for GPNMB specific monoclonal antibodies. In the Primary screen, the ELISA plates (Fisher, Cat. No. 12-565-136) were coated with 50 .mu.L/well of GPNMB (1 .mu.g/mL) in Coating Buffer (0.1 M Carbonate Buffer, pH 9.6, NaHCO.sub.3 8.4 g/L), then incubated at 4.degree. C. overnight. After incubation, the plates were washed with Washing Buffer (0.05% Tween 20 in PBS) three times. 200 .mu.L/well Blocking Buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1.times.PBS) were added and the plates were incubated at room temperature for 1 h. After incubation, the plates were washed with Washing Buffer three times. Aliquots (50 .mu.L/well) of hybridoma supernatants and positive and negative controls were added, and the plates were incubated at room temperature for 2 h. The positive control used throughout was serum from the relevant GPNMB immunized XenoMouse.RTM. mouse and the negative control was serum from the KLH-immunized relevant strain of XenoMouse.RTM. mouse. After incubation, the plates were washed three times with Washing Buffer. 100 .mu.L/well of detection antibody goat anti-huIgGfc-HRP (Caltag, Cat. No. H10507, using concentration was 1:2000 dilution) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed three times with Washing Buffer. 100 .mu.l/well of TMB (BioFX Lab. Cat. No. TMSK-0100-01) was added, and the plates were allowed to develop for about 10 minutes (until negative control wells barely started to show color). 50 .mu.l/well stop solution (TMB Stop Solution (BioFX Lab. Cat. No. STPR-0100-01) was then added and the plates were read on an ELISA plate reader at a wavelength of 450 nm.

[0249] The old culture supernatants from the positive hybridoma cells growth wells based on primary screen were removed completely and the IL-1b positive hybridoma cells were suspended with fresh hybridoma culture medium and were transferred to 24-well plates. After 2 days in culture, these supernatants were ready for a secondary confirmation screen. In the secondary confirmation screen, the positives in the first screening were screened in GPNMB binding ELISA described as above, and two sets of detective system for the secondary confirmation ELISA, one set for hIgG detection, one set for human Ig kappa light chain detection (goat anti-hIg kappa-HRP, Southern Biotechnology, Cat. No. 2060-05) in order to demonstrate fully human composition for both heavy and light chains. The two sets of ELISA procedures were identical to the descriptions above except the three different detection antibodies were used separately. All positive hits from the secondary confirmation ELISA assay were counter screened for binding to immunogen by .quadrature.ELISA in order to exclude those that cross-react with IL-1a. The ELISA plates (Fisher, Cat. No. 12-565-136) were coated with 50 .mu.L/well of irrelevant V5His-fusion protein, 1 ug/mL in Coating Buffer (0.1 M Carbonate Buffer, pH 9.6, NaHCO.sub.3 8.4 g/L), then incubated at 4.degree. C. overnight. The remaining procedures were identical to the descriptions above. There are 33 fully human GPNMB specific monoclonal antibodies that were generated.

[0250] Hybridoma supernatants were screened for binding to GPNMB by ELISA as described above in Example 2. Results are shown in Table 6.

TABLE-US-00045 TABLE 6 Hybridoma anti-GPNMB activity. 3 1 333 111 37 12.3 .mu.g/mL .mu.g/mL ng/mL ng/mL ng/mL ng/mL Avg Avg Avg Avg Avg Avg OD OD OD OD OD OD 1.2.2 0.763 0.499 0.356 0.199 0.094 0.049 1.7.3 1.003 0.871 0.760 0.451 0.239 0.094 1.15.1 1.159 1.051 0.902 0.701 0.381 0.168 1.16.2 0.036 0.015 0.010 0.008 0.008 0.007 2-3 1.282 1.204 0.963 0.713 0.359 0.179 2-6 1.254 1.295 1.092 0.875 0.443 0.183 2-7 0.827 0.719 0.680 0.494 0.308 0.156 2-8 0.921 0.635 0.229 0.109 0.056 0.028 2-10 1.095 1.066 0.849 0.583 0.272 0.132 2-15 0.601 0.568 0.578 0.395 0.246 0.127 2-16 0.359 0.173 0.068 0.032 0.017 0.011 2-17 0.053 0.019 0.010 0.009 0.008 0.011 2-22 0.714 0.707 0.538 0.355 0.171 0.068 2-24 0.060 0.042 0.028 0.023 0.016 0.017 Isotype 0.009 0.008 0.009 0.009 0.009 0.011 control Irrelevant 0.009 0.008 0.012 0.013 Antibody Secondary 0.011 Ab control Anti-V5 3.066 Ab control

[0251] Certain Hybridoma cell supernatants (29) were analyzed for binding to GPNMB by BiaCore.RTM. 2000 biosensor equipped with a research-grade CM5 sensor chip. A 1:25 dilution of cell supernatant was passed over a protein A surface for 5 min followed by washing the surface for 10 mins. Subsequently, GPNMB was injected for 90 sec. over the surface at a concentration of 880 nM followed by dissociation. Double-referenced binding data were obtained by subtracting the signal from a control flow cell and subtracting the baseline drift of a buffer injected just prior to the antigen injection. GPNMB binding data for each mAb was normalized for the amount of mAb captured on each surface. Normalized, drift corrected responses were also measured. The sensorgrams were fit to a simple 1:1 kinetic model. The results are shown in Table 7. Sixteen of the cell supernatants contained mAb that significantly bound to GPNMB and three Mabs, 15.1, 15.2, and 15.3 showed strong binding to GPNMB.

TABLE-US-00046 TABLE 7 Expression Sample Kd (nM) ka (M-1s-1) kd (s-1) Level 15.1 52 16524 8.55E-04 medium 15.3 59 13417 7.97E-04 medium 15.2 61 12635 7.70E-04 high 2.2 96 9257 8.90E-04 medium 10.2 118 3955 4.66E-04 low 7.3 121 9648 1.17E-03 medium 7.1 122 11842 1.44E-03 medium 7.2 141 9356 1.32E-03 high 10.3 147 3626 5.32E-04 low 10.1 209 4235 8.85E-04 low 8.2 242 7555 1.83E-03 medium 8.3 264 6551 1.73E-03 low 8.1 329 6830 2.25E-03 medium 12.3 407 1549 6.31E-04 medium 12.2 435 1280 5.57E-04 medium 12.1 630 1587 1.00E-03 low 1.1 >1000 <1500 nd high 1.2 >1000 <1500 nd high 1.3 >1000 <1500 nd medium 2.1 >1000 <1500 nd medium 5.1 >1000 <1500 nd medium 5.2 >1000 <1500 nd medium 5.3 >1000 <1500 nd medium 9.1 >1000 <1500 nd medium 9.2 >1000 <1500 nd low 9.3 >1000 <1500 nd low 11.1 >1000 <1500 nd low 11.2 >1000 <1500 nd low 11.3 >1000 <1500 nd low

Example 4

Binning of Antibodies

[0252] Certain antibodies, described herein were binned in accordance with the protocol described in U.S. Patent Application Publication No. 20030157730. MxhIgG conjugated beads are prepared for coupling to primary antibody. The volume of supernatant needed is calculated using the following formula: (n+10).times.50 .mu.L (where n=total number of samples on plate). Where the concentration is known, 0.5 .mu.g/mL is used. Bead stock is gently vortexed, then diluted in supernatant to a concentration of 2500 of each bead per well or 0.5.times.10.sup.5/mL and incubated on a shaker in the dark at RT overnight, or 2 hours if at a known concentration of 0.5 .mu.g/mL. Following aspiration, 50 .mu.L of each bead is added to each well of filter plate, then washed once by adding 100 .mu.L/well wash buffer and aspirating. Antigen and controls are added to filter plate 50 .mu.L/well then covered and allowed to incubate in the dark for 1 hour on shaker. Following a wash step, a secondary unknown antibody is added at 50 .mu.L/well using the same dilution (or concentration if known) as is used for the primary antibody. The plates are then incubated in the dark for 2 hours at RT on shaker followed by a wash step. Next, 50 .mu.L/well biotinylated mxhIgG diluted 1:500 is added and allowed to incubate in the dark for 1 hour on shaker at RT. Following a wash step, 50 .mu.L/well Streptavidin-PE is added at 1:1000 and allowed to incubate in the dark for 15 minutes on shaker at RT. Following a wash step, each well is resuspended in 80 .mu.L blocking buffer and read using Luminex. Results show that the monoclonal antibodies belong to distinct bins. Competitive binding by antibodies from different bins supports antibody specificity for similar or adjacent epitopes. Non competitive binding supports antibody specificity for unique epitopes.

[0253] Three bins were created to further test the binding of six anti-GPNMB antibodies. Bin 1 included GPNMB antibodies (1.2.1), (1.10.1), and (2.22.1). Bin 2 included GPNMB antibodies (2.3.1) and (1.15.1), and Bin 3 included GPNMB antibody (2.10.1). The results of the binning assays are provided below in Tables 8 and 9.

TABLE-US-00047 TABLE 8 BB 1.1 1.2 1.3 1.5 1.7 1.8 1.9 1.11 1.12 1.13 1.15 xV5 BB 0 16 58 24 6 25 14 9 8 9 7 15 32 1.1 -16 0 57 16 -29 34 9 -35 -9 -7 -24 35 28 1.2 -42 -16 0 -60 -89 -49 -81 -75 -73 -65 -81 -43 45 1.3 -11 -33 8 0 -75 -40 -49 171 -29 -33 -67 -73 -15 1.5 25 35 64 60 0 20 10 24 17 27 12 -8 61 1.7 -1 76 65 20 -8 0 -8 4 4 6 -3 -3 95 1.8 -7 29 45 35 -3 -7 0 4 -1 0 -6 3 52 1.9 -5 18 47 -7 -10 3 4 0 4 5 -5 -1 17 1.11 18 40 60 29 -11 1 15 16 0 8 5 -23 48 1.12 -10 26 43 27 -5 3 -12 -4 -12 0 -9 -13 57 1.13 1 30 40 27 2 9 2 10 11 17 0 -13 59 1.15 -19 91 79 71 15 21 8 12 10 15 13 0 89 xV5 41 134 239 46 5 443 230 -1 70 257 24 535 0 I II III IV V VI VII VIII IX 1.1 1.2 1.3 1.5 1.7 1.8 1.9 1.15 xV5 1.13 1.11 1.12

TABLE-US-00048 TABLE 9 1.1 1.2 1.3 1.5 1.7 1.8 1.9 1.11 1.12 1.13 1.15 xV5 BB 1.1 0 72 39 -36 49 8 -14 -3 18 -14 35 28 -2 1.2 10 0 -60 -103 -46 -64 -76 -71 -69 -83 -74 44 -46 1.3 -49 -9 0 -111 -88 -78 281 -66 -57 -93 -115 -89 -33 1.5 61 106 77 0 13 28 17 20 40 2 -3 87 19 1.7 94 77 51 -25 0 -9 -3 12 4 -4 -17 96 17 1.8 42 71 74 -24 2 0 -9 1 -1 -12 -5 61 4 1.9 14 74 28 -24 6 4 0 3 5 -13 8 16 -17 1.11 59 66 77 -20 3 -5 13 0 11 -9 -5 92 21 1.12 84 67 61 -36 -12 -8 -6 -4 0 -16 -34 95 12 1.13 74 93 49 -12 22 12 23 21 19 0 20 98 55 1.15 127 90 51 -9 17 12 19 19 21 5 0 125 59 xV5 189 330 22 14 611 376 -17 113 445 44 750 0 100 BB 25 73 65 3 34 23 14 19 22 13 39 44 0 I II III IV V VI VII VIII Cut-off = 100 1.1 1.2 1.3 1.5 1.7 1.9 1.15 xV5 1.8 1.11 1.12 1.13 I II III IV V VI VII VIII IX Cut-off = 90 1.1 1.2 1.3 1.5 1.7 1.8 1.9 1.15 xV5 1.13 1.11 1.12

Example 5

GPNMB Immunohistochemistry (IHC) Analysis

[0254] Anti-GPNMB monoclonal antibodies were evaluated for reactivity with frozen and fixed tissue specimens. Tissue sections (5 .mu.m) were cut from formalin fixed and paraffin embedded tissue samples and were rehydrated through incubations in xylene and a graded ethanol series terminating in PBS. Endogenous peroxidase activity was quenched in a 3% solution of hydrogen peroxide in methanol.

[0255] Tissue sections were blocked in blocking buffer (5% BSA (Sigma), 1% goat serum (Jackson Immunolabs, West Grove, Pa.) in PBS) for 1 hour. Primary and secondary antibodies were precomplexed in 5% BSA and 1% goat serum in PBS for 1 hour at 37.degree. C. at a molar ratio of approximately 10:1 of anti-GPNMB or control IgG to secondary biotinylated goat anti-human IgG (Jackson Immunolabs). Complexes were blocked with a 1:2000 dilution of human serum and incubated again for 1 hour at 37.degree. C. Tissue sections were incubated with anti-GPNMB antibody or isotype control antibody complexes diluted in blocking buffer for 1 hour. Sections were washed in 3 changes of PBS for 5 to 10 minutes each and incubated with a 1:200 dilution of streptavidin conjugated horseradish peroxidase (Jackson Immunolabs) in blocking buffer for 30 minutes and then washed as before. Antibody was detected using DAB reagent (Vector labs). Sections were counterstained in hematoxylin (Fisher Scientific) and dehydrated through alcohol and xylene and coverslipped with permount (Fisher Scientific).

[0256] Anti-GPNMB Mabs 2.22.1 and 2.22.2 were used to stain normal and tumor human tissue microarrays (IMPATH, Los Angeles, Calif.). Positive staining was seen in lung, ovarian, renal, esophagus, and head & neck carcinomas, squamous cell carcinoma, melanomas and normal skin specimens. Melanoma and lung carcinomas showed the highest staining intensities with subcellular staining located in the membrane and cytoplasm. Anti-GPNMB Mab 2.10.2 also stained primary melanoma.

[0257] Anti-GPNMB antibody staining of melanoma tissue microarray showed a large proportion of melanoma cases to be positively stained as shown in Tables 10 and 11.

TABLE-US-00049 TABLE 10 anti-GPNMB Mab Melanoma Staining Intensity Staining Intensity* # of Samples % 0 10 17 1 1 2 1-2 2 3 1-3 11 19 2 9 15 2-3 17 29 3 9 15 Total n = 59 100 On a scale of 0 (no staining) to 3 (strong staining)

TABLE-US-00050 TABLE 11 anti-GPNMB Mab Staining Frequency % Tumor Reactivity* # Samples % 0-24 18 31 25-49 6 10 50-74 7 12 75-100 28 47 n = 59 100% *% tumor cells exhibiting positive staining

[0258] Anti-GPNMB antibody stained 10 of 14 lung squamous cell carcinoma (SCC) samples in a general oncology tissue microarray and 24 of 60 in a SCC specific array were positive.

Example 6

FACS Analysis of Anti-GPNMB Antibody Binding to Melanoma Cell Lines

[0259] The specificity of anti-GPNMB antibodies to cell membrane-bound GPNMB protein expressed by melanoma cancer cell line, UACC-62 was analyzed by FACS analysis. A renal cancer cell line, TK10, which does not express GPNMB antigen was used as a negative control. Isotype matched antibody pK16.3 was used as a negative control. Cells were washed twice with PBS (Ca and Mg free), incubated with Versene at 37.degree. C. until cells detached, counted and aliquoted at 1 million cells per assay tube. Cells were then washed twice and resuspended in ice-cold FACS buffer (0.01M HEPES, 0.15M NaCl, 0.1% NaN.sub.3 and 4% FBS). Primary antibody at 1 .mu.g/mL was added to the cells. Cells were incubated on ice for 30 min, washed 2-3 times and resuspended in 1 mL of ice-cold FACS buffer. R-PE-conjugated goat anti-human antibody (Jackson ImmunoResearch Laboratory) at 1:100 dilution was added and cells were incubated on ice for 30 min. After washing 3 times with 1 mL of ice-cold FACS buffer, cells were fixed with 0.5-1 mL of 1% formaldehyde in PBS and analyzed by flow cytometry.

[0260] Results expressed as Geo Mean Ratios are summarized in Table 12 and show UACC-62 cells but not TK10 cells highly express CR011 protein on the cell surface which was detected by 2.10.2; 2.22.1 and 1.15.1 antibodies.

TABLE-US-00051 TABLE 12 Geo Mean Ratio of anti-GPNMB Staining (relative to pK16) A. Antibody B. UACC-62 Cells C. TK10 Cells D. CR011.2.10.2 E. 2.60 F. 1.10 G. CR011.2.22.1 H. 4.46 I. 1.10 J. CR011.1.2.2 K. 1.24 L. 0.98 M. CR011.1.15.1 N. 7.89 O. 0.96 P. CR011.2.6.2 Q. 1.90 R. 1.70

[0261] To examine the relative GPNMB antigen expression among melanoma cell lines, MAb 1.15.1 antibody was used to survey a panel of 15 melanoma cell lines by FACS analysis. As shown in Table 13, 80% (12/15) of cell lines showed GPNMB antigen expression. Cell line SK-Mel-2 demonstrated the highest Geo Mean ratio among the cell lines tested.

TABLE-US-00052 TABLE 13 Geo Mean Ratio of anti GPNMB Staining of Melanoma Cell Lines Geo Mean Ratio Cell Line (relative to isotype) SK-Mel 2 16.5 M14 16.1 MEWO 14.1 WM-266-4 13.6 HEMNLP 10.2 G361 8 HT144 7.4 UACC-257 7 RPMI-7951 6 SK-Mel 5 5.7 UACC-62 5.5 A2058 4.1 SK-Mel 24 1.9 WM115 1.3 LOXIMVI 1

Example 7

FACS Analysis of Anti-GPNMB MAb Binding to Lymphoma and Leukemia

[0262] To determine the relative expression of GPNMB on the surface of hematopoietic malignant cells, cell lines derived from various lymphomas and leukemias were incubated with anti-GPNMB antibody and analyzed by FACS. Lymphoma or leukemia derived cells were washed twice with ice-cold FACS buffer and resuspended at 1 million cells per assay tube. MAb 1.15.1 antibody at 1 .mu.g/mL was added to cells and cells were incubated on ice for 30 min. Cells were then washed 2-3 times and resuspended in 1 mL of ice-cold FACS buffer. R-PE-conjugated goat anti-human antibody at 1:100 dilution was added and cells were incubated on ice for 30 min Cells were washed 3 times with 1 mL of ice-cold FACS buffer, fixed with 0.5-1 mL of 1% formaldehyde in PBS and analyzed by Flow Cytometry.

[0263] Approximately half of the cell lines examined, which were derived from both myeloid and lymphoid lineages, showed GPNMB cell surface expression (Table 14). Cell line U937 demonstrated the highest Geo Mean ratio among the cell lines tested.

TABLE-US-00053 TABLE 14 Geo Mean Ratio of anti-GPNMB Staining of Lymphoma and Leukemia Cells Cell line Geo Mean Ratio U937 (histiocytic lymphoma, monocytic) 17.3 Jurkat (acute T-cell leukemia) 14.7 SR (anaplastic large T cell lymphoma, ALCL) 7.1 KG-1 (acute myelogenous leukemia) 6.9 MOLT-4 (acute T cell lymphoblastic leukemia) 6.2 THP-1 (acute monocytic leukemia) 6.1 MV4-11 (myelomonocytic leukemia) 1.9 AML-193 (acute monocytic leukemia) 1.8 HUT-78 (T cell lymphoma) 1.5 CCRF-CEM (acute T cell lymphoblastic leukemia) 10.9 Karpas 299 (ALCL) 10.7 SU-DHL-1 (ALCL) 4.8 SU-DHL-4 (B cell lymphoma) 1.8 ML-2 (acute myelomonocytic leukemia) 2.1 HH (cutaneous T-cell leukemia) 1 SUP-M2 (ALCL) 4.8 PL-21 (acute myeloid leukemia) 12 DEL (ALCL) 7.9 SIG-M5 (acute monocytic leukemia) 2.9 K562 (Chronic myelogenous leukemia) 2.8 KG1a (acute myelogenous leukemia) 2.7 HL-60 (acute promyelocytic leukemia) 2.3 WSU-NH2 (B cell lymphoma) 1 EOL-1 (acute myeloid leukemia) 1 HUT-102 (T cell lymphoma) 1

Example 8

Detection of GPNMB Protein by IP and Western Blot Analysis

[0264] Cells were washed twice with PBS (Ca and Mg free), incubated with Versene at 37.degree. C. until cells detached, counted, collected and lysed in lysis buffer (0.15M NaCl, 0.02M Tris HCl, 10% glycerol, 1% NP-40, 0.01M EDTA and protease inhibitors containing pancreas extract, pronase, thermolysin, chymotrypsin and papain (Roche, Germany) for 30 min on ice. Supernatants were collected and protein concentrations were determined by BCA protein assay kit (Pierce, USA). Primary antibody was added to the cell lysates and incubated on ice for 3 hr followed by addition of Protein-G agarose (Amersham, USA) for 2 hr Immunoprecipitated proteins were washed, boiled in sample buffer and resolved by 4-20% gels. For immunoblotting, proteins were transferred to PVDF membranes (Invitrogen, USA) and probed with anti-GPNMB antibody (0.5 .mu.g/mL) followed by HRP-conjugated goat anti-human antibody (Jackson ImmunoResearch Laboratory) at 1:4000 dilution. The immunocomplexes were detected with ECL Western blotting detection reagents (Amersham, USA).

[0265] Western blot analysis showed anti-GPNMB antibodies immunoprecipitated GPNMB protein expressed in cell lysates of UACC-62, SK-Mel5 and SK-Mel2 cell lines. The results are in concurrence with the cell surface expression determined by FACS analysis.

Example 9

Anti GPNMB Antibody Mediated Indirect Cell Killing

[0266] UACC-62, a GPNMB antigen expressing cell line, and TK10, a non-expressing cell line were plated onto flat bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J., USA) at a density of 3000 cells per well. Once the cells reached .about.25% confluency, 100 ng/well of secondary antibody-toxin conjugate (goat anti-human IgG-saporin; Advanced Targeting Systems, San Diego, USA, HUM-ZAP; cat. # IT-22) was added. Anti-GPNMB MAbs 2.10.2, 2.22.1, 1.15.1 or isotype control mAb (pK16.3) were added to each well at a final concentration of 10 or 50 ng/mL. An anti-EGFR monoclonal antibody (MS-269-PABX, NeoMarkers, Fremont, Calif., USA) was used as a positive primary antibody control. Chemotherapy reagent 5-FU at 600 uM was used as a positive reagent control. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37.degree. C. Plates were examined daily and between 8-10 days, all plates were Giemsa stained and colonies were counted.

[0267] The percent viability of GPNMB positive UACC-62 after treatment is shown in FIG. 2. Chemotherapy reagent 5-FU induced a complete killing whereas addition of saporin toxin-conjugated secondary antibody alone or in combination with isotype control pK16.3 antibody had no effect on cell growth for both cell lines. Both UACC-62 and TK10 cell lines express EGFR protein and addition of EGFR specific antibody at 50 ng/mL and secondary antibody toxin conjugate resulted in a complete killing of UACC-62 and TK10 cells. At the same dose, all three GPNMB specific antibodies, 2.10.2, 2.22.1 and 1.15.1 induced over 70% killing of UACC-62 cells. Anti-GPNMB antibodies 2.10.2 and 2.22.1 induced less than 5% and 1.15.1 less than 24% cell death in GPNMB negative TK10 cells.

Example 10

Cell Killing by Auristatin-E (AE) Conjugated Anti-GPNMB Antibodies

[0268] UACC-62 and TK10 cells were plated onto flat bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J., USA). On day 2 or cells reach .about.25% confluency, various concentrations (1 to 1000 ng/mL) of unconjugated and Auristatin E-conjugated antibodies (Seattle Genetics, Bothell, Wash., USA), including isotype control, EGFR (NeoMarkers MS-269-PABX, Fremont, Calif., USA), 2.22.1 or 2.10.2, were added to cells. MAb 2.3.1 was chosen for the isotype control in this study because it does not bind to GPNMB expressing cells as demonstrated by FACS analysis. A monoclonal antibody generated against the EGF receptor was used to demonstrate specific killing mediated by AE-conjugated antibody. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37.degree. C. Plates were examined daily. On days 8-10, all plates were Giemsa stained and colonies on the plates were counted.

[0269] The percent viability in GPNMB positive UACC-62 cells and negative TK10 cells is presented in FIGS. 4 and 5, respectively. The results indicate that unconjugated and AE-conjugated 2.6.2 immunoconjugate had no effect on growth of both UACC-62 and TK10 cells. However, both UACC-62 and TK10 cell lines were susceptible to AE-EGFR immunoconjugate mediated cell killing in a dose-dependent fashion with over 95% cell death at 1000 ng/mL. At the same dose, both 2.22.1-AE and 2.10.2-AE immunoconjugates induced approximately 75% cell death of UACC-62 cells when compared to the isotype control. The cell killing response was dose dependent. GPNMB negative TK10 cell survival was not affected by 2.22.1-AE nor 2.10.2-AE immunoconjugates at the same dose range. These results demonstrate the specific and cytotoxic effects of AE conjugated anti-GPNMB antibodies on antigen expressing cells.

Example 11

Melanoma Cells Susceptible to MAb1.15.1-AE Immunoconjugate Killing

[0270] Melanoma cell lines were plated onto flat bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J., USA). On day 2 or when cells reach .about.25% confluency, various concentrations of unconjugated and Auristatin E-conjugated 1.15.1 were added to cells. MAb 2.6.2-AE was also used as a conjugated isotype control in this study. On day 5, the cells were trypsinized, transferred to 6-well tissue culture plates and incubated at 37.degree. C. Plates were examined daily. On days 8-10, all plates were Giemsa stained and colonies on the plates were counted.

[0271] The IC.sub.50 of 1.15.1-AE mediated killing on GPNMB positive and negative cells are presented in Table 15. Unconjugated 1.15.1 and AE-conjugated 2.6.2 had no effect on growth of all the melanoma cell lines tested. However, cell lines SK-Mel2, WM-266-4, G361, UACC-257, UACC-62, RPMI-7951 and SK-Mel5 were susceptible to 1.15.1-AE mediated killing in a dose-dependent fashion. SK-Mel2 demonstrated the lowest IC.sub.50 in this study (Table 15). These results show the specific and cytotoxic effects of AE conjugated 1.15.1 on most of GPNMB expressing melanoma cells.

TABLE-US-00054 TABLE 15 Geo Mean Ratios and IC.sub.50 Values of 1.15.1-AE Killing of Melanoma Cells Melanoma Geo Mean Ratio Clonogenic Assay with 1.15.1-AE Cell Line (relative to isotype) IC50 in ng/mL (pM) SK-Mel 2 16.5 111 (750) M14 16.1 Inconclusive MEWO 14.1 Inconclusive WM-266-4 13.6 345 (2300) HEMNLP 10.2 Inconclusive G361 8 1053 (6500) HT144 7.4 Inconclusive UACC-257 7 825 (5500) RPMI-7951 6 972 (6000) SK-Mel 5 5.7 237 (1600) UACC-62 5.5 697 (4300) A2058 4.1 No effect SK-Mel 24 1.9 No data WM115 1.3 No data LOXIMVI 1 No effect

Example 12

MAb 1.15.1-AE Killing of Lymphoma and Leukemia Cell Lines

[0272] Lymphoma or leukemia cell lines were mixed with methylcellulose base media (R&D Systems, USA) and in various concentrations of unconjugated and Auristatin E-conjugated 1.15.1 antibody before plating onto 6-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J., USA). MAb 2.6.2-AE was also included as a conjugated isotype control in this study because it does not bind to GPNMB expressing cells. Plates were incubated at 37.degree. C. and examined daily. On days 14-18, colonies on the plates were counted.

[0273] The IC.sub.50 of 1.15.1-AE induced cell killing on antigen expressing cells is presented in Table 16. Unconjugated 1.15.1 and AE-conjugated 2.6.2 immunoconjugate had no effect on growth of all antigen positive hematopoietic cell lines. However, as presented in Table 16, cell lines U937, SR and THP-1 derived from either myeloid or lymphoid lineage were susceptible to 1.15.1-AE mediated killing in a dose-dependent manner with IC.sub.50 values ranging from 207 ng/mL (1.4 nM) to 340 ng/mL (2.4 nM). These results show the specific and cytotoxic effects of 1.15.1-AE immunoconjugate on GPNMB antigen expressing hematopoietic malignant cell lines.

TABLE-US-00055 TABLE 16 Geo Mean Ratios and IC.sub.50 Values of 1.15.1-AE Killing of Lymphoma and Leukemia Cells Clonogenic Assay with 1.15.1-AE Cell line Geo Mean Ratio IC50 in ng/mL (pM) U937 (histiocytic lymphoma, monocytic) 17.3 340 (2400) Jurkat (acute T-cell leukemia) 14.7 No effect (repeating) SR (anaplastic large T cell lymphoma, ALCL) 7.1 296 (2000) KG-1 (acute myelogenous leukemia) 6.9 No growth MOLT-4 (acute T cell lymphoblastic leukemia) 6.2 No effect (repeating) THP-1 (acute monocytic leukemia) 6.1 207 (1400) MV4-11 (myelomonocytic leukemia) 1.9 ND AML-193 (acute monocytic leukemia) 1.8 ND HUT-78 (T cell lymphoma) 1.5 ND CCRF-CEM (acute T cell lymphoblastic leukemia) 10.9 No growth Karpas 299 (ALCL) 10.7 Inconclusive SU-DHL-1 (ALCL) 4.8 No effect SU-DHL-4 (B cell lymphoma) 1.8 ND ML-2 (acute myelomonocytic leukemia) 2.1 ND HH (cutaneous T-cell leukemia) 1 ND SUP-M2 (ALCL) 4.8 No growth PL-21 (acute myeloid leukemia) 12 No effect DEL (ALCL) 7.9 No effect SIG-M5 (acute monocytic leukemia) 2.9 ND K562 (Chronic myelogenous leukemia) 2.8 ND KG1a (acute myelogenous leukemia) 2.7 ND HL-60 (acute promyelocytic leukemia) 2.3 ND WSU-NH2 (B cell lymphoma) 1 ND EOL-1 (acute myeloid leukemia) 1 ND HUT-102 (T cell lymphoma) 1 ND *ND: Not done

Example 13

CR011-vcMMAE Inhibits the Growth of Human SK-MEL-2 Melanoma Xenografts Leading to Complete Regression of Established Melanoma Tumors in Athymic Mice (Study N-386)

[0274] Study N-386 was performed to assess the potency and therapeutic efficacy of the antibody-drug conjugate, CR011-vcMMAE, against the established human SK-MEL-2 melanoma xenograft in athymic mice.

Materials and Methods:

[0275] Test Animals: Five- to 6-week old athymic mice (CD-1 nu/nu females), used for human tumor xenografts, were obtained from Harlan Laboratories (Indianapolis, Ind.). Animals were housed in specific pathogen-free conditions, according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Test animals were provided pelleted food and water ad libitum and kept in a room with conditioned ventilation (HVAC), temperature (22.degree..+-.2.degree. C.), relative humidity (55%.+-.15%), and photoperiod (12 hr). All studies were carried out with approved institutional animal care and use protocols.

[0276] Human Melanoma Xenograft Models. The tumor inhibitory activity of the CR011-MMAE immunoconjugate was measured in an anti-tumor xenograft model using athymic mice, according to published methods (see Geran et al., Cancer Chemother. Rep. 3:1-104 (1972)). Briefly, test animals were implanted subcutaneously by trocar with small fragments of a human melanoma (60-125 mg) excised from athymic mouse tumor donors. When tumors became established (10-20 days), the animals were pair-matched into groups (n=6 mice/group), and treatment was administered by intravenous injection (tail vein).

[0277] The SK-MEL-2 human melanoma (ATCC #HTB-68) was derived from a metastatic site (skin of thigh) of a 60 year old Caucasian male with malignant melanoma, and the SK-MEL-5 human melanoma (ATCC #HTB-70) was derived from a metastatic site (axillary lymph node) of a 24 year old Caucasian female with malignant melanoma (see Fogh et al., J. Natl. Cancer Inst. 59: 221-226 (1977)). Both cell lines were obtained from the American Type Culture Collection.

[0278] The effects of treatment were monitored by repetitive tumor measurements across 2 diameters with Vernier calipers; tumor size (in mg) was calculated using a standard formula, (W.sup.2.times.L)/2, assuming a specific gravity of 1.0. Tumor size and body weights were assessed twice weekly. Mice were examined daily, however, and moribund animals were humanely euthanized if clinical indications of excessive pain or distress were noted (i.e., prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures, and/or hemorrhages). Animals with tumors exceeding 2,000 mg were removed from the study and euthanized humanely.

[0279] Xenograft studies in the athymic mouse have been shown to effectively demonstrate anti-tumor effects for a variety of agents which have been shown subsequently to have activity against clinical cancer (Johnson et al., Br J Cancer 84:1424-1431 (2001)).

Results:

[0280] Anti-Tumor Effects In Vivo vs. SK-MEL-2 Melanoma. Based on the potency and cytotoxicity of CR011-vcMMAE against GPNMB-expressing cells in vitro, the anti-tumor effects were examined in vivo.

[0281] The effects of intravenous CR011-vcMMAE treatment on the growth of subcutaneous human SK-MEL-2 melanoma are shown in FIG. 1. After SK-MEL-2 tumor fragments were implanted and tumors became established (day 17, 61 mg), treatment commenced with intravenous administration of: CR011-vcMMAE (0.625-20 mg/kg i.v., every 4 days for a total of 4 treatments (i.e., q4 d.times.4); saline and phosphate-buffered saline controls (i.v., q4 d.times.4); and two known anti-tumor reference agents, vinblastine sulfate (i.v., 1.7 mg/kg, q4 d.times.4) and paclitaxel (i.v., 24 mg/kg, q2 d.times.4). The reference agents were administered at the maximum tolerated dose (MTD) determined in prior studies.

[0282] Tumors in animals treated with saline or PBS grew progressively until the tumor mass reached 2,000 mg at which time the animals were removed from the study and euthanized humanely. SK-MEL-2 tumors have a high "take" rate in immunocompromised hosts (97%) and a low rate of spontaneous regression (3%) (Dykes et al., Development of human tumor xenograft models for in vivo evaluation of new antitumor drugs, in Immunodeficient mice in Oncology, vol. 42 (Fiebig H H and Berger D Pe eds) pp 1-22, Contrib. Oncol. Basel, Karger (1992)).

[0283] Vinblastine produced a very slight, but not significant, anti-tumor effect (P.ltoreq.0.20); in this and other tumor models (e.g., SK-MEL-5) vinblastine produces noticeable tumor growth inhibition, but which is only occasionally significant. Paclitaxel, however, showed significant tumor growth inhibition and tumor stasis (i.e., 100% growth inhibition) for approximately 2 weeks after treatment commenced (P.ltoreq.0.0077).

[0284] The anti-tumor effects of CR011-vcMMAE administered i.v. to SK-MEL-2-bearing mice were remarkable. At 20, 10, 5 or 2.5 mg/kg tumors rapidly diminished in size for the majority of the test animals; significant treatment effects were noted as early as 4 days after treatment commenced (P.ltoreq.0.014). Tumors that regressed completely did not re-grow during the observation period (>200 days).

[0285] The animals in this study showed no abnormal treatment effects on gross examination. Twice weekly body weight determinations showed no observable or statistically significant effects of treatment with CR011-vcMMAE on body weight or weight gain.

Conclusions:

[0286] CR011-vcMMAE produces substantial, dose-dependent and reproducible anti-tumor effects that begin as tumor growth inhibition but soon lead to complete regression of established human melanoma xenografts; the regressions are long-lived and re-growth of tumors after successful therapy has not been observed.

Example 14

Sequencing of Antibodies and their Corresponding DNA

[0287] Sequences of human GPNMB mAbs-derived heavy and kappa chain transcripts from hybridomas were obtained by direct sequencing of PCR products generated from poly(A.sup.+) RNA. PCR products were also cloned into pCRII using a TA cloning kit (Invitrogen) and both strands were sequenced using Prism dye-terminator sequencing kits and an ABI 377 sequencing instrument. Each PCR reaction used a mixture of 5' sense primers which are provided in Table 17 below.

TABLE-US-00056 TABLE 17 Primers Used VH cacc ATG GAC TGG(C) ACC TGG AGG ATC SEQ ID NO: 290 VH cacc ATG GAC TGG ACC TGG AGA(C) ATC SEQ ID NO: 291 VH cacc ATG GAC TGG ACC TGG AGG GTC SEQ ID NO: 292 VH cacc ATG GAC TGG ATT TGG AGG ATC SEQ ID NO: 293 VH cacc ATG GAC ACA CTT TGC TC(A)C AC SEQ ID NO: 294 VH cacc ATG GAA(G) TTG GGG CTG AGC TGG SEQ ID NO: 295 VH cacc ATG GAG TTG(T) GGA CTG AGC TGG SEQ ID NO: 296 VH cacc ATG GAG TTT GGG CTG(T) AGC TGG SEQ ID NO: 297 VH cacc ATG GAA CTG GGG CTC CGC TGG SEQ ID NO: 298 VH cacc ATG GAG TTG GGG CTG TGC TGG SEQ ID NO: 299 VH cacc ATG GAG TTT TGG CTG AGC TGG SEQ ID NO: 300 VH cacc ATG ACG GAG TTT GGG CTG AGC SEQ ID NO: 301 VH cacc ATG AAA(G) CAC CTG TGG TTC TTC SEQ ID NO: 302 VH cacc ATG AAA CAT CTG TGG TTC TTC SEQ ID NO: 303 VH cacc ATG GGG TCA ACC GCC ATC CTC SEQ ID NO: 304 VH cacc ATG TCT GTC TCC TTC CTC ATC TTC SEQ ID NO: 305 VK ATG GGG TCC CAG GTT CAC CTC SEQ ID NO: 306 VK ATG TTG CCA TCA CAA CTC ATT G SEQ ID NO: 307

[0288] All sequences were analyzed by alignments to the "V BASE sequence directory" (Tomlinson et al., MRC Centre for Protein Engineering, Cambridge, UK) using MACVECTOR.RTM. and GENEWORKS.TM. software programs.

Example 15

Structural Analysis of Anti-GPNMB Antibodies

[0289] The variable heavy chains and the variable light chains for the antibodies shown in Table 17 were sequenced to determine their DNA and protein sequences.

Antibody-1.10.2

TABLE-US-00057

[0290] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 1) 5'AGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCAGTAATTACTAC TGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTA TTTCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAG TCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGC TCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATAGGGG CTGGGCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 2) 5'QVQLQESGPGLVKPSETLSLTCTVS GDSISNYYWS WIRQPPGKGLE WIG YFYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR DRGWADY WGQGTLVTVSSA 3'

TABLE-US-00058 TABLE 18 1.10.2 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID NO: FRI QVQLQESGPGLVKPSETLSL 1-25 SEQ ID NO: 3 TCTVS CDR1 GDSISNYYWS 26-35 SEQ ID NO: 4 FR2 WIRQPPGKGLEWIG 36-49 SEQ ID NO: 5 CDR2 YFYYSGSTNYNPSLKS 50-65 SEQ ID NO: 6 FR3 RVTISVDTSKNQFSLKLSSV 66-97 SEQ ID NO: 7 TAADTAVYYCAR CDR3 DRGWADY 98-104 SEQ ID NO: 8 FR4 WGQGTLVTVSSA 105-116 SEQ ID NO: 9 *AA Residues of SEQ ID NO: 2

TABLE-US-00059 Light chain variable region Nucleotide sequence (SEQ ID NO: 10) 5'GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGG GAAAGGGCCACCCTCTCCTGCAGAACCAGTCAGAGTATTAGCAGCAGCTA TTTAGCCTGGTACCAGCAGAAACCTGGCCAGGTTCCCAGGCTCCTCATCT ATGGTGCTTCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAG TGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAA GATTTTGCAGTGTATTATTGTCAGCAGTATGGTAGCTCGATCACCTTCG GCCAAGGGACACGACTGGAGATTAAACG A 3' Amino acid sequence (SEQ ID NO: 11) 5'EIVLTQSPGTLSLSPGERATLSC RTSQSISSSYLA WYQQKPGQVPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSIT FGQGTRLEIKR 3'

TABLE-US-00060 TABLE 19 1.10.2 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI EIVLTQSPGTLSLSPGERAT 1-23 SEQ ID NO: 12 LSC CDR1 RTSQSISSSYLA 24-35 SEQ ID NO: 13 FR2 WYQQKPGQVPRLLIY 36-50 SEQ ID NO: 14 CDR2 GASSRAT 51-57 SEQ ID NO: 15 FR3 GIPDRFSGSGSGTDFTL 58-89 SEQ ID NO: 16 TISRLEPEDFAVYYC CDR3 QQYGSSIT 90-97 SEQ ID NO: 17 FR4 FGQGTRLEIKR 98-108 SEQ ID NO: 18 *AA Residues of SEQ ID NO: 11

Antibody-1.15.1

TABLE-US-00061

[0291] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 19) 5'CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCA CAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTTT TAATTACTACTGGAGCTGGATCCGCCACCACCCAGGGAAGGGCCTGGAGT GGATTGGGTACATCTATTACAGTGGGAGCACCTACTCCAACCCGTCCCTC AAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCT GACGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGA GAGGGTATAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 20) 5'QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGK GLEWIGYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVY YCARGYNWNYFDYWGQGTLVTVSSA 3'

TABLE-US-00062 TABLE 20 1.15.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLQESGPGLVKPSQTLSLT 1-30 SEQ ID NO: 21 CTVSGGSIS CDR1 SFNYYWS 31-37 SEQ ID NO: 22 FR2 WIRHHPGKGLEWIG 38-51 SEQ ID NO: 23 CDR2 YIYYSGSTYSNPSLKS 52-67 SEQ ID NO: 24 FR3 RVTISVDTSKNQFSLTLSSVT 68-99 SEQ ID NO: 25 AADTAVYYCAR CDR3 GYNWNYFDY 100-108 SEQ ID NO: 26 FR4 WGQGTLVTVSSA 109-120 SEQ ID NO: 27 *AA Residues of SEQ ID NO: 20

TABLE-US-00063 Light chain variable region Nucleotide sequence (SEQ ID NO: 28) 5'GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCA GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGACAACAA CTTAGTCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCT ATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGT GGGTCTGGGACAGAGTTCACTCTCACCATCAGTAGTCTGCAGTCTGAAGA TTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGTGGACGT TCGGCCAAGGGACCAAGGTGGAAATCAAACGA 3' Amino acid sequence (SEQ ID NO: 29) 5'EIVMTQSPATLSVSPGERATLSCRASQSVDNNLVWYQQKPGQAPR LLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP PWTFGQGTKVEIKR 3'

TABLE-US-00064 TABLE 21 1.15.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI EIVMTQSPATLSVSPGERAT 1-23 SEQ ID NO: 30 LSC CDR1 RASQSVDNNLV 24-34 SEQ ID NO: 31 FR2 WYQQKPGQAPRLLIY 35-49 SEQ ID NO: 32 CDR2 GASTRAT 50-56 SEQ ID NO: 33 FR3 GIPARFSGSGSGTEFTLTISS 57-88 SEQ ID NO: 34 LQSEDFAVYYC CDR3 QQYNNWPPWT 89-98 SEQ ID NO: 35 FR4 FGQGTKVEIKR 99-109 SEQ ID NO: 36 *AA Residues of SEQ ID NO: 29

Antibody-1.2.2

TABLE-US-00065

[0292] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 37) 5' ATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCAC ACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCGCTG GTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAG TGGCTTGCACTCATTTATTGGAATGATGATAAGCGCTACAGCCCATCTCT GAGGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCC TTACAATTACCAACATGGACCCTGTGGACACAGCCACATATTATTGTGCA CACAGTCACTATGATTACGATTGGGGGAGTTACTTTGACTACTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 38) 5'ITLKESGPTLVKPTQTLTLTCTFS GFSLSAGGVGVG WIRQPPG KALEWLA LIYWNDDKRYSPSLRS RLTITKDTSKNQVVLTITNMDPVDT ATYYCAH SHYDYDWGSYFDY WGQGTLVTVSSA 3'

TABLE-US-00066 TABLE 22 1.2.2 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI ITLKESGPTLVKPTQTLTLT 1-24 SEQ ID NO: 39 CTFS CDR1 GFSLSAGGVGVG 25-36 SEQ ID NO: 40 FR2 WIRQPPGKALEWLA 37-50 SEQ ID NO: 41 CDR2 LIYWNDDKRYSPSLRS 51-66 SEQ ID NO: 42 FR3 RLTITKDTSKNQVVLTITNM 67-98 SEQ ID NO: 43 DPVDTATYYCAH CDR3 SHYDYDWGSYFDY 99-111 SEQ ID NO: 44 FR4 WGQGTLVTVSSA 112-123 SEQ ID NO: 45 *AA Residues of SEQ ID NO: 38

TABLE-US-00067 Light chain variable region Nucleotide sequence (SEQ ID NO: 46) 5' GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCAC CCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGG ATAGTGATGATGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGGA CAGTCTCCACAGCTCCTGATCTATACGCTTTCCTATCGGGCCTCTGGAGT CCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAACA TCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACGT ATAGAGTTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACG A 3' Amino acid sequence (SEQ ID NO: 47) 5' DIVMTQTPLSLPVTPGEPASISC RSSQSLLDSDDGNTYLD WYLQKPGQSPQLLIY TLSYRASGVPDRFSGSGSGTDFTLNISRVEAED VGVYYC MQRIEFPIT FGQGTRLEIKR 3'

TABLE-US-00068 TABLE 23 1.2.2 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQTPLSLPVTPGEPAS 1-23 SEQ ID NO: 48 ISC CDR1 RSSQSLLDSDDGNTYLD 24-40 SEQ ID NO: 49 FR2 WYLQKPGQSPQLLIY 41-55 SEQ ID NO: 50 CDR2 TLSYRAS 56-62 SEQ ID NO: 51 FR3 GVPDRFSGSGSGTDFTLNISR 63-94 SEQ ID NO: 52 VEAEDVGVYYC CDR3 MQRIEFPIT 95-103 SEQ ID NO: 53 FR4 FGQGTRLEIKR 104-114 SEQ ID NO: 54 *AA Residues of SEQ ID NO: 47

Antibody-1.7.1

TABLE-US-00069

[0293] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 55) 5' CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC TTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCA GTGCTAATTACTACTGGACCTGGATCCGCCAGCACCCAGGGAAGGGCCTG GAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTGCAACCCGTC CCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAACCAGTTCT CCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT GCGAGAGGGTATAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 56) 5' QVQLQESGPGLVKPSQTLSLTCTVS GGSISSANYYWT WI RQHPGKGLEWIG YIYYSGSTYCNPSLKS RVIISVDTSKNQFSLKLSSV TAADTAVYYCAR GYNWNYFDY WGQGTLVTVSSA 3'

TABLE-US-00070 TABLE 24 1.7.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLQESGPGLVKPSQTLSLT 1-25 SEQ ID NO: 57 CTV CDR1 GGSISSANYYWT 26-37 SEQ ID NO: 58 FR2 WIRQHPGKGLEWIG 38-51 SEQ ID NO: 59 CDR2 YIYYSGSTYCNPSLKS 52-67 SEQ ID NO: 60 FR3 RVIISVDTSKNQFSLKLSSVT 68-99 SEQ ID NO: 61 AADTAVYYCAR CDR3 GYNWNYFDY 100-108 SEQ ID NO: 62 FR4 WGQGTLVTVSSA 109-120 SEQ ID NO: 63 *AA Residues of SEQ ID NO: 56

TABLE-US-00071 Light chain variable region Nucleotide sequence (SEQ ID NO: 64) 5'GATATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGG GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTT AGCCTGGTACCAGGAGAGACCTGGCCAGGCTCCCAGACTCCTCATCTATG GTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGG TCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTT TGCAGTTTATTACTGTCAGCAGTATAATAAGTGGCCTCCGTGGACGTTCG GCCAAGGGACCAAGGTGGAAATCGAACGAACT 3' Amino acid sequence (SEQ ID NO: 65) 5'DIVMTQSPATLSVSPGERATLSC RASQSVSSNLA WYQERPGQAPRL LIY GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNKW PPWT FGQGTKVEIER 3'

TABLE-US-00072 TABLE 25 1.7.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQSPATLSVSPGERAT 1-23 SEQ ID NO: 66 LSC CDR1 RASQSVSSNLA 24-34 SEQ ID NO: 67 FR2 WYQERPGQAPRLLIY 35-49 SEQ ID NO: 68 CDR2 GASTRAT 50-56 SEQ ID NO: 69 FR3 GIPARFSGSGSGTEFTLTISS 57-88 SEQ ID NO: 70 LQSEDFAVYYC CDR3 QQYNKWPPWT 89-98 SEQ ID NO: 71 FR4 FGQGTKVEIER 99-109 SEQ ID NO: 72 *AA Residues of SEQ ID NO: 65

Antibody-2.10.2

TABLE-US-00073

[0294] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 73) 5' CAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCGCCTTCAGTAGCTATG GCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCA GTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGG CCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAA TGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAT CTAGTGGTTCGGGGAATTAGGGGGTACTACTACTACTTCGGTATGGACGT CTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 74) 5' QLVESGGGVVQPGRSLRLSCAAS GFAFSSYGMH WVRQAP GKGLEWVA VISYDGNNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCAR DLVVRGIRGYYYYFGMDV WGQGTTVTVSSA 3'

TABLE-US-00074 TABLE 26 2.10.2 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QLVESGGGVVQPGRSLRLSC 1-23 SEQ ID NO: 75 AAS CDR1 GFAFSSYGMH 24-33 SEQ ID NO: 76 FR2 WVRQAPGKGLEWVA 34-47 SEQ ID NO: 77 CDR2 VISYDGNNKYYADSVKG 48-64 SEQ ID NO: 78 FR3 RFTISRDNSKNTLYLQMNSLR 65-96 SEQ ID NO: 79 AEDTAVYYCAR CDR3 DLVVRGIRGYYYYFGMDV 97-114 SEQ ID NO: 80 FR4 WGQGTTVTVSSA 115-126 SEQ ID NO: 81 *AA Residues of SEQ ID NO: 74

TABLE-US-00075 Light chain variable region Nucleotide sequence (SEQ ID NO: 82) 5' GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG AGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTA ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCA CAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAG GTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAG TGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAACT CCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGA 3' Amino acid sequence (SEQ ID NO: 83) 5' DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKP GQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQGLQTPIT FGQGTRLEIKR 3'

TABLE-US-00076 TABLE 27 2.10.2 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQSPLSLPVTPGEPAS 1-23 SEQ ID NO: 84 ISC CDR1 RSSQSLLHSNGYNYLD 24-39 SEQ ID NO: 85 FR2 WYLQKPGQSPQLLIY 40-54 SEQ ID NO: 86 CDR2 LGSNRAS 55-61 SEQ ID NO: 87 FR3 GVPDRFSGSGSGTDFTLKISR 62-93 SEQ ID NO: 88 VEAEDVGVYYC CDR3 MQGLQTPIT 94-102 SEQ ID NO: 89 FR4 FGQGTRLEIKR 103-113 SEQ ID NO: 90 *AA Residues of SEQ ID NO: 83

Antibody-2.15.1

TABLE-US-00077

[0295] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 91) 5' CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATG GCATTCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCA GTTATATGGTTTGATGGACGTAATAAATACTATGCAGACTCCGTGAAGGG CCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAA TGAACAGCCTGAGAGCCGAGGACGCGGCTGTGTATTACTGTGCGAGAGAT CCCTTTGACTATGGTGACTCCTTCTTTGACTACTGGGGCCAGGGCACCCT GGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 92) 5' QVQLVESGGGVVQPGRSLRLSCAAS GFTFSNYGIH WVRQAPGKGL EWVA VIWFDGRNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDAAV YYCAR DPFDYGDSFFDY WGQGTLVTVSSA 3'

TABLE-US-00078 TABLE 28 2.15.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLVESGGGVVQPGRSLRLS 1-25 SEQ ID NO: 93 CAAS CDR1 GFTFSNYGIH 26-35 SEQ ID NO: 94 FR2 WVRQAPGKGLEWVA 36-49 SEQ ID NO: 95 CDR2 VIWFDGRNKYYADSVKG 50-66 SEQ ID NO: 96 FR3 RFTISRDNSKNTLYLQMNSLR 67-98 SEQ ID NO: 97 AEDAAVYYCAR CDR3 DPFDYGDSFFDY 99-110 SEQ ID NO: 98 FR4 WGQGTLVTVSSA 111-122 SEQ ID NO: 99 *AA Residues of SEQ ID NO: 92

TABLE-US-00079 Light chain variable region Nucleotide sequence (SEQ ID NO: 100) 55' CTGACTCAGTCTCCATCCTCCCTGTCTGCATCTGTAAGAGACAGAG TCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTGG TATCAGCAGAAACCAGGGAAAGTTCCTAATCTCCTGATCTATGCTGCATC CACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACT TATTACTGTCAAAAGTATAACAGTGCCCCGCTCACTTTCGGCGGAGGGAC CAAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 101) 5' LTQSPSSLSASVRDRVTITC RASQDISNYLA WYQQKPGKVPNLLI Y AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QKYNSAP LT FGGGTKVEIKR 3'

TABLE-US-00080 TABLE 29 2.15.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI LTQSPSSLSASVRDRVTI 1-20 SEQ ID NO: 102 TC CDR1 RASQDISNYLA 21-31 SEQ ID NO: 103 FR2 WYQQKPGKVPNLLIY 32-46 SEQ ID NO: 104 CDR2 AASTLQ 47-52 SEQ ID NO: 105 FR3 GVPSRFSGSGSGTDFTLTI 53-84 SEQ ID NO: 106 SSLQPEDVATYYC CDR3 QKYNSAPLT 85-93 SEQ ID NO: 107 FR4 FGGGTKVEIKR 94-104 SEQ ID NO: 108 *AA Residues of SEQ ID NO: 101

Antibody-2.16.1

TABLE-US-00081

[0296] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 109) 5' CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCC TGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTG ACTACTACATGACCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG GTTTCATACATTAGTATTAGTGGTAGTATCACACACTACGCAGACTCAGT GAAGGGCCGATTCACCATGTCCAGGGACAACGCCAAGAACTCACTGTATC TGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCG AGAGACGGAGCAGCAGCTGGTACGGATGCTTTTGATATCTGGGGCCACGG GACAAAGGTCACCGTCTCTTCAGCC 3' Amino acid sequence (SEQ ID NO: 110) 5' QVQLVESGGGLVKPGGSLRLSCAAS GFTFSDYYMT WIRQ APGKGLEWVS YISISGSITHYADSVKG RFTMSRDNAKNSLYLQMNSLR AEDTAVYYCAR DGAAAGTDAFDI WGHGTKVTVSSA 3'

TABLE-US-00082 TABLE 30 2.16.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLVESGGGLVKPGGSLR 1-25 SEQ ID NO: 111 LSCAAS CDR1 GFTFSDYYMT 26-35 SEQ ID NO: 112 FR2 WIRQAPGKGLEWVS 36-49 SEQ ID NO: 113 CDR2 YISISGSITHYADSVKG 50-66 SEQ ID NO: 114 FR3 RFTMSRDNAKNSLYLQMN 67-98 SEQ ID NO: 115 SLRAEDTAVYYCAR CDR3 DGAAAGTDAFDI 99-110 SEQ ID NO: 116 FR4 WGHGTKVTVSSA 111-122 SEQ ID NO: 117 *AA Residues of SEQ ID NO: 110

TABLE-US-00083 Light chain variable region Nucleotide sequence (SEQ ID NO: 118) 5'GAGATAGTGATGACGCAGTCTCCAGCCACCCTATCTGTGTCTCCAGGG AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAATGTTAGCAGCAACTT GGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTG GTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGG TCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTACAGTCTGAAGATTT TGCAGTTTATTACTGTCAGCAGTATCATTACTGGCCCACTTTCGGCCCTG GGACCAAAGTGGATATCAAACGA 3' Amino acid sequence (SEQ ID NO: 119) 5'EIVMTQSPATLSVSPGDRATLSC RASQNVSSNLA WYQQKPGQAPRLLIF GASTRAT GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYHYWPT FGPGTKVDIKR 3'

TABLE-US-00084 TABLE 31 2.16.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI EIVMTQSPATLSVSPGDRA 1-23 SEQ ID NO: 120 TLSC CDR1 RASQNVSSNLA 24-34 SEQ ID NO: 121 FR2 WYQQKPGQAPRLLIF 35-49 SEQ ID NO: 122 CDR2 GASTRAT 50-56 SEQ ID NO: 123 FR3 GIPARFSGSGSGTEFTLTI 57-88 SEQ ID NO: 124 SSLQSEDFAVYYC CDR3 QQYHYWPT 89-96 SEQ ID NO: 125 FR4 FGPGTKVDIKR 97-107 SEQ ID NO: 126 *AA Residues of SEQ ID NO: 119

Antibody-2.17.1

TABLE-US-00085

[0297] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 127) 5' CAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT GAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTTCTATATGC ACTGGGTGCGACAGACCCCTGGACAAGGGCTTGAGTGGATGGGATGGATC AACCCTAACAGTGGTGGCACATATTATGTACAGAAGTTTCAGGGCAGGGT CACCATGACCAGGGACACGTCCATCAGCACAGTCTACATGGAGCTGAGCA GGTTGAGATCTGACGACACGGCCGTATATTACTGTGCGAGAGATGGGTAT AGCAGTGGAGAGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 128) 5' QLVQSGAEVKKPGASVKVSCKAS GYTFTGFYMH WVRQTPGQGLEW MG WINPNSGGTYYVQKFQG RVTMTRDTSISTVYMELSRLRSDDTAVYY CAR DGYSSGEDWFDP WGQGTLVTVSSA 3'

TABLE-US-00086 TABLE 32 2.17.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QLVQSGAEVKKPGASVKVS 1-23 SEQ ID NO: 129 CKAS CDR1 GYTFTGFYMH 24-33 SEQ ID NO: 130 FR2 WVRQTPGQGLEWMG 34-47 SEQ ID NO: 131 CDR2 WINPNSGGTYYVQKFQG 48-64 SEQ ID NO: 132 FR3 RVTMTRDTSISTVYMELSR 65-96 SEQ ID NO: 133 LRSDDTAVYYCAR CDR3 DGYSSGEDWFDP 97-108 SEQ ID NO: 134 FR4 WGQGTLVTVSSA 109-120 SEQ ID NO: 135 *AA Residues of SEQ ID NO: 128

TABLE-US-00087 Light chain variable region Nucleotide sequence (SEQ ID NO: 136) 5'GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGA CAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTGG TGGAAAGACCTATTTGTATTGGTACCTGCAGAGGCCAGGCCAGCCTCCAC AGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGG TTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACACCTTC CGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 137) 5'DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSGGKTYLY WYLQRPGQPPQLLIY EVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIHLPLT FGGGTKVEIKR 3'

TABLE-US-00088 TABLE 33 2.17.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQTPLSLSVTPGQPA 1-23 SEQ ID NO: 138 SISC CDR1 KSSQSLLHSGGKTYLY 24-39 SEQ ID NO: 139 FR2 WYLQRPGQPPQLLIY 40-54 SEQ ID NO: 140 CDR2 EVSNRFS 55-61 SEQ ID NO: 141 FR3 GVPDRFSGSGSGTDFTLKI 62-93 SEQ ID NO: 142 SRVEAEDVGVYYC CDR3 MQSIHLPLT 94-102 SEQ ID NO: 143 FR4 FGGGTKVEIKR 103-113 SEQ ID NO: 144 *AA Residues of SEQ ID NO: 137

Antibody-2.21.1

TABLE-US-00089

[0298] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 145) 5' CAGGTGCAGCTGGAGCAGTCGGGGGGAGGCCTGGTCAAGCCTGGGGG GTCCCTGAGATTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATA GCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA TTCATTAGTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAA TGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAG GACTGGGTGGGAGCTACCTTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 146) 5' QVQLEQSGGGLVKPGGSLRFSCAAS GFTFSSYSMN WVRQAPGKGLEWVS FISSSSSYIYY ADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EDWVGATFDY WGQGTLVTVSSA 3'

TABLE-US-00090 TABLE 34 2.21.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLEQSGGGLVKPGGS 1-25 SEQ ID NO: 147 LRFSCAAS CDR1 GFTFSSYSMN 26-35 SEQ ID NO: 148 FR2 WVRQAPGKGLEWVS 36-49 SEQ ID NO: 149 CDR2 FISSSSSYIYYADSVKG 50-66 SEQ ID NO: 150 FR3 RFTISRDNAKNSLYLQMNS 67-98 SEQ ID NO: 151 LRAEDTAVYYCAR CDR3 EDWVGATFDY 99-108 SEQ ID NO: 152 FR4 WGQGTLVTVSSA 109-120 SEQ ID NO: 153 *AA Residues of SEQ ID NO: 146

TABLE-US-00091 Light chain variable region Nucleotide sequence (SEQ ID NO: 154) 5' GACATTCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG AGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGGAATTATT TAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTAT GCTGCTTCCGCTTTGAAATTAGGGGTCCCATCTCGGTTCAGTGGCAGTGG ATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATG TTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCGATCACCTTCGGC CAAGGGACACGACTGGACATTAAACGA 3' Amino acid sequence (SEQ ID NO: 155) 5' DIQLTQSPSSLSASVGDRVTITC RASQGIRNYLA WYQQKPGKVPKLLIY AASALKL GVPS RFSGSGSGTDFTLTISSLQPEDVATYYC QKYNSAPIT FGQGTRLDIKR 3'

TABLE-US-00092 TABLE 35 2.21.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIQLTQSPSSLSASVGD 1-23 SEQ ID NO: 156 RVTITC CDR1 RASQGIRNYLA 24-34 SEQ ID NO: 157 FR2 WYQQKPGKVPKLLIY 35-49 SEQ ID NO: 158 CDR2 AASALKL 50-56 SEQ ID NO: 159 FR3 GVPSRFSGSGSGTDFTLTI 57-88 SEQ ID NO: 160 SSLQPEDVATYYC CDR3 QKYNSAPIT 89-97 SEQ ID NO: 161 FR4 FGQGTRLDIKR 98-108 SEQ ID NO: 162 *AA Residues of SEQ ID NO: 155

Antibody-2.22.1

TABLE-US-00093

[0299] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 163) 5' CAGGTGCAGCTGGAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCACA GAACCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTG GTTATTTCTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGG ATTGGGTACATCTATTACAGTGGGAACACCTACTACAACCCGTCCCTCAA GAGTCGAGTTACCATATCAGTTGACACGTCTAAGAACCAGTTCTCCCTGA AACTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGA GACTATTACTATGATACTAGTGGTTTTTCCTACCGTTACGACTGGTACTA CGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 164) 5' QVQLEQSGPGLVKPSQNLSLTCTVS GGSISSGGYFWS WIRQHPGKGLEWIG YIYYSGNTY YNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR DYYYDTSGFSYRYDWYYGMDV WGQGTTVTVSSA 3'

TABLE-US-00094 TABLE 36 2.22.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLEQSGPGLVKPSQN 1-25 SEQ ID NO: 165 LSLTCTVS CDR1 GGSISSGGYFWS 26-37 SEQ ID NO: 166 FR2 WIRQHPGKGLEWIG 38-51 SEQ ID NO: 167 CDR2 YIYYSGNTYYNPSLKS 52-67 SEQ ID NO: 168 FR3 RVTISVDTSKNQFSLKL 68-99 SEQ ID NO: 169 SSVTAADTAVYYCAR CDR3 DYYYDTSGFSYRYDWYYG 100-120 SEQ ID NO: 170 MDV FR4 WGQGTTVTVSSA 121-132 SEQ ID NO: 171 *AA Residues of SEQ ID NO: 164

TABLE-US-00095 Light chain variable region Nucleotide sequence (SEQ ID NO: 172) 5' GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATT TAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTAT GCTGCATCCAGTTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGG ATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATT TTGCAACTTATTACTGTCTACAACATAATACTTACCCGGCGTTCGGCCAA GGGACCAAGGTGGAAATCAAACGA 3' Amino acid sequence (SEQ ID NO: 173) 5' DIQLTQSPSSLSASVGDRVTITC RASQGIRNDLG WYQQKPGKAPKRLIY AASSLQNGVPS RFSGSGSGTEFTLTISSLQPEDFATYYC LQHNTYPA FGQGTKVEIKR 3'

TABLE-US-00096 TABLE 37 2.22.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIQLTQSPSSLSASVGDRV 1-23 SEQ ID NO: 174 TITC CDR1 RASQGIRNDLG 24-34 SEQ ID NO: 175 FR2 WYQQKPGKAPKRLIY 35-49 SEQ ID NO: 176 CDR2 AASSLQN 50-56 SEQ ID NO: 177 FR3 GVPSRFSGSGSGTEFTLTI 57-88 SEQ ID NO: 178 SSLQPEDFATYYC CDR3 LQHNTYPA 89-97 SEQ ID NO: 179 FR4 FGQGTKVEIKR 98-108 SEQ ID NO: 180 *AA Residues of SEQ ID NO: 173

Antibody-2.24.1

TABLE-US-00097

[0300] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 181) 5' CAGCTGGTGCAGTCTGGAGCAGAAGTGAAAAAGCCCGGGGA GTCTCTGAAGATCTCCTGTCAGGGTTCTGGATACATCTTTACCAACTACT GGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGG GTCATCTATCCTGATGACTCTGATACCAGATACAGCCCGTCCTTCCAAGG CCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGT GGAGCAGCCTGAAGGCCTCGGACACCGCCATATATTACTGTGCGAGACAA AAATGGCTACAACACCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 182) 5' QLVQSGAEVKKPGESLKISCQGS GYIFTNYWIG WVRQMP GKGLEWMG VIYPDDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKAS DTAIYYCAR QKWLQHPFDY WGQGTLVTVSSA 3'

TABLE-US-00098 TABLE 38 2.24.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QLVQSGAEVKKPGESLKIS 1-23 SEQ ID NO: 183 CQGS CDR1 GYIFTNYWIG 24-33 SEQ ID NO: 184 FR2 WVRQMPGKGLEWMG 34-47 SEQ ID NO: 185 CDR2 VIYPDDSDTRYSPSFQG 48-64 SEQ ID NO: 186 FR3 QVTISADKSISTAYLQW 65-96 SEQ ID NO: 187 SSLKASDTAIYYCAR CDR3 QKWLQHPFDY 97-106 SEQ ID NO: 188 FR4 WGQGTLVTVSSA 107-118 SEQ ID NO: 189 *AA Residues of SEQ ID NO: 182

TABLE-US-00099 Light chain variable region Nucleotide sequence (SEQ ID NO: 190) 5' GAAATTGTGTTGACGCAGTCACCAGGCACCCTGTCTTTGTC TCCAGGGGAAAGAGTCACCCTCTCATGCAGGGCCAGTCAGAGTGTTAGCA GCAGATACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTC CTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAG TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGC CTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATGGTAGCTCACCTCGG ACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGA 3' Amino acid sequence (SEQ ID NO: 191) 5' EIVLTQSPGTLSLSPGERVTLSC RASQSVSSRYLA WYQQ KPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVY YC QQYGSSPRT FGQGTKVEIKR 3'

TABLE-US-00100 TABLE 39 2.24.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI EIVLTQSPGTLSLSPGER 1-23 SEQ ID NO: 192 VTLSC CDR1 RASQSVSSRYLA 24-35 SEQ ID NO: 193 FR2 WYQQKPGQAPRLLIY 36-50 SEQ ID NO: 194 CDR2 GASSRAT 51-57 SEQ ID NO: 195 FR3 GIPDRFSGSGSGTDFTL 58-88 SEQ ID NO: 196 TISRLEPEDFAVYY CDR3 QQYGSSPRT 89-97 SEQ ID NO: 197 FR4 FGQGTKVEIKR 98-109 SEQ ID NO: 198 *AA Residues of SEQ ID NO: 191

Antibody-2.3.1

TABLE-US-00101

[0301] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 199) 5'CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCC TCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTA TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAT GGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGAC AGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCT GAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATT TCTTTGGTTCGGGGAGTCTCCTCTACTTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 200) 5'QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPNSGGTNYAQKFQDRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR DFFGSGSLLYFDYWGQGTLVTVSSA 3'

TABLE-US-00102 TABLE 40 2.3.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLVQSGAEVKKPGASV 1-25 SEQ ID NO: 201 KVSCKAS CDR1 GYTFTGYYMH 26-35 SEQ ID NO: 202 FR2 WVRQAPGQGLEWMG 36-49 SEQ ID NO: 203 CDR2 WINPNSGGTNYAQKFQD 50-66 SEQ ID NO: 204 FR3 RVTMTRDTSISTAYMELSR 67-98 SEQ ID NO: 205 LRSDDTAVYYCAR CDR3 DFFGSGSLLYFDY 99-111 SEQ ID NO: 206 FR4 WGQGTLVTVSSA 112-123 SEQ ID NO: 207 *AA Residues of SEQ ID NO: 200

TABLE-US-00103 Light chain variable region Nucleotide sequence (SEQ ID NO: 208) 5'GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGA CAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTGG TGGAAAGACCTATTTGTATTGGTACCTGCAGAGGCCAGGCCAGCCTCCAC AGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGG TTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACACCTTC CGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 209) 5'DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSGGKTYLY WYLQRPGQPPQLLIY EVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIHLPLT FGGGTKVEIKR 3'

TABLE-US-00104 TABLE 41 2.3.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQTPLSLSVTPGQPA 1-23 SEQ ID NO: 210 SISC CDR1 KSSQSLLHSGGKTYLY 24-39 SEQ ID NO: 211 FR2 WYLQRPGQPPQLLIY 40-54 SEQ ID NO: 212 CDR2 EVSNRFS 55-61 SEQ ID NO: 213 FR3 GVPDRFSGSGSGTDFTL 62-93 SEQ ID NO: 214 KISRVEAEDVGVYYC CDR3 MQSIHLPLT 94-102 SEQ ID NO: 215 FR4 FGGGTKVEIKR 103-113 SEQ ID NO: 216 *AA Residues of SEQ ID NO: 209

Antibody-2.6.1

TABLE-US-00105

[0302] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 309) 5'CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTA CTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGG GATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAG GACAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGA GCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAG ATTTCTTTGGTTCGGGGAGTCTCCTCTACTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 310) 5'QVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYYMH WVRQAPGQ GLEWMG WINPNSGGTNYAQKFQDRVTMTRDTSISTAYMELSRLRSDDTA VYYCAR DFFGSGSLLYFDY WGQGTLVTVSSA 3'

TABLE-US-00106 TABLE 42 2.6.1 Heavy chain V region domains. AA RES- SEQ REGION SEQUENCE IDUES* ID FRI QVQLVQSGAEVKKPGASVKVSCKAS 1-25 311 CDR1 GYTFTGYYMH 26-35 312 FR2 WVRQAPGQGLEWMG 36-49 313 CDR2 WINPNSGGTNYAQKFQD 50-66 314 FR3 RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 67-98 315 CDR3 DFFGSGSLLYFDY 99-112 316 FR4 WGQGTLVTVSSA 113-124 317 *AA Residues of SEQ ID NO: 310

TABLE-US-00107 Light chain variable region Nucleotide sequence (SEQ ID NO: 318) 5'GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGA CAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTGG TGGAAAGACCTATTTGTATTGGTACCTGCAGAGGCCAGGCCAGCCTCCAC AGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGG TTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACACCTTC CGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 319) 5'DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSGGKTYLY WYLQRPGQPPQLLIY EVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIHLPLT FGGGTKVEIKR 3'

TABLE-US-00108 TABLE 43 2.6.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQTPLSLSVTPGQPASISC 1-23 320 CDR1 KSSQSLLHSGGKTYLY 24-39 321 FR2 WYLQRPGQPPQLLIY 40-54 322 CDR2 EVSNRFS 55-61 323 FR3 GVPDRFSGSGSGTDFTLKISRVEAEDV 62-93 324 GVYYC CDR3 MQSIHLPLT 94-102 325 FR4 FGGGTKVEIKR 103-113 326 *AA Residues of SEQ ID NO: 319

Antibody-2.7.1

TABLE-US-00109

[0303] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 217) 5' CAGGTGCAGCTGGAGCAGTCGGGGGGAGGCGTGGTCCAGCC TGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAATA ACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGG GTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCG AAAGATGAGGAATACTACTATGTTTCGGGGCTTGACTACTGGGGCCAGGG AACCCTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 218) 5' QVQLEQSGGGVVQPGRSLRLSCAAS GFTFNNYGMH WVRQ APGKGLEWVA VIWYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLR AEDTAVYYCAK DEEYYYVSGLDY WGQGTLVTVSSA 3'

TABLE-US-00110 TABLE 44 2.7.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QVQLEQSGGGVVQPGRS 1-25 SEQ ID NO: 219 LRLSCAAS CDR1 GFTFNNYGMH 26-35 SEQ ID NO: 220 FR2 WVRQAPGKGLEWVA 36-49 SEQ ID NO: 221 CDR2 VIWYDGSNKYYADSVKG 50-66 SEQ ID NO: 222 FR3 RFTISRDNSKNTLYLQMN 67-98 SEQ ID NO: 223 SLRAEDTAVYYCAK CDR3 DEEYYYVSGLDY 99-110 SEQ ID NO: 224 FR4 WGQGTLVTVSSA 111-122 SEQ ID NO: 225 *AA Residues of SEQ ID NO: 218

TABLE-US-00111 Light chain variable region Nucleotide sequence (SEQ ID NO: 226) 5' CTGACTCAGTCTCCATCCTCCCTGTCTGCATCTGTAAGAGACAGAGT CACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTGGT ATCAGCAGAAACCAGGGAAAGTTCCTAATCTCCTGATCTATGCTGCATCC ACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGAC AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTT ATTACTGTCAAAAGTATAACAGTGCCCCGCTCACTTTCGGCGGAGGGACC AAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 227) 5' LTQSPSSLSASVRDRVTITC RASQDISNYLA WYQQKPGKVPNLLIY AASTLQS GVPSRFS GSGSGTDFTLTISSLQPEDVATYYC QKYNSAPLT FGGGTKVEIKR 3'

TABLE-US-00112 TABLE 45 2.7.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI LTQSPSSLSASVRDRVTI 1-20 SEQ ID NO: 228 TC CDR1 RASQDISNYLA 21-31 SEQ ID NO: 229 FR2 WYQQKPGKVPNLLIY 32-46 SEQ ID NO: 230 CDR2 AASTLQ 47-52 SEQ ID NO: 231 FR3 GVPSRFSGSGSGTDFTLTI 53-84 SEQ ID NO: 232 SSLQPEDVATYYC CDR3 QKYNSAPLT 85-93 SEQ ID NO: 233 FR4 FGGGTKVEIKR 94-104 SEQ ID NO: 234 *AA Residues of SEQ ID NO: 227

Antibody-2.8.1

TABLE-US-00113

[0304] Heavy chain variable region Nucleotide sequence (SEQ ID NO: 235) 5' CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGACACC CACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCA CTGGTGGAATGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTG GACTGGCTTACACTCATTTATTGGAATGATGATAAGCACTACAGCCCATC TCTGAAGAGCAGGCTTACCATCACCAAGGACACCTCCAAAAACCAGGTGG TCCTTAGAATGACCAACATGGACCCTGTGGACACAGCCACTTATTACTGT GCACACCTGCATTACGATATTTTGACTGGTTTTAACTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCAGCC 3' Amino acid sequence (SEQ ID NO: 236) 5' QITLKESGPTLVTPTQTLTLTCTFS GFSLSTGGMGVG WI RQPPGKALDWLT LIYWNDDKHYSPSLKS RLTITKDTSKNQVVLRMTNM DPVDTATYYCAH LHYDILTGFNFDY WGQGTLVTVSSA 3'

TABLE-US-00114 TABLE 46 2.8.1 Heavy chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI QITLKESGPTLVTPTQTL 1-25 SEQ ID NO: 237 TLTCTFS CDR1 GFSLSTGGMGVG 26-37 SEQ ID NO: 238 FR2 WIRQPPGKALDWLT 38-51 SEQ ID NO: 239 CDR2 LIYWNDDKHYSPSLKS 52-67 SEQ ID NO: 240 FR3 RLTITKDTSKNQVVLRMTN 68-99 SEQ ID NO: 241 MDPVDTATYYCAH CDR3 LHYDILTGFNFDY 100-112 SEQ ID NO: 242 FR4 WGQGTLVTVSSA 113-124 SEQ ID NO: 243 *AA Residues of SEQ ID NO: 236

TABLE-US-00115 Light chain variable region Nucleotide sequence (SEQ ID NO: 244) 5'GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGA GAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGA TGATGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTC CACAGCTCCTGATCTATACGCTTTCCTATCGGGCCTCTGGAGTCCCAGAC AGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAG GGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACGTATAGAGT TTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA 3' Amino acid sequence (SEQ ID NO: 245) 5'DIVMTQTPLSLPVTPGEPASISC RSSQSLLDSDDGNTYLD WYLQKP GQSPQLLIY TLSYRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQRIEFPLT FGGGTKVEIKR 3'

TABLE-US-00116 TABLE 47 2.8.1 Light chain V region domains. AA REGION SEQUENCE RESIDUES* SEQ ID FRI DIVMTQTPLSLPVTPGE 1-23 SEQ ID NO: 246 PASISC CDR1 RSSQSLLDSDDGNTYLD 24-40 SEQ ID NO: 247 FR2 WYLQKPGQSPQLLIY 41-55 SEQ ID NO: 248 CDR2 TLSYRAS 56-62 SEQ ID NO: 249 FR3 GVPDRFSGSGSGTDFTLKI 63-94 SEQ ID NO: 250 SRVEAEDVGVYYC CDR3 MQRIEFPLT 95-103 SEQ ID NO: 251 FR4 FGGGTKVEIKR 103-114 SEQ ID NO: 252 *AA Residues of SEQ ID NO: 245

Example 16

Use of Anti-GPNMB Antibodies as a Diagnostic Agent

Detection of GPNMB Antigen in a Sample:

[0305] The following is a protocol for an Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of GPNMB antigen in a sample. In the assay, wells of a microtiter plate, such as a 96-well microtiter plate or a 384-well microtiter plate, are adsorbed for several hours with a first fully human monoclonal antibody directed against GPNMB. The immobilized antibody serves as a capture antibody for any of the GPNMB that may be present in a test sample. The wells are rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

[0306] Subsequently the wells are treated with a test sample suspected of containing the GPNMB antigen, or with a solution containing a standard amount of GPNMB antigen. Such a sample may be, for example, a serum sample from a subject suspected of having levels of circulating GPNMB considered to be diagnostic of a pathology.

[0307] After rinsing away the test sample or standard, the wells are treated with a second fully human monoclonal anti-GPNMB antibody that is labeled by conjugation with biotin. The labeled anti-GPNMB antibody serves as a detecting antibody. After rinsing away excess second antibody, the wells are treated with avidin-conjugated horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of the antigen in the test samples is determined by comparison with a standard curve developed from the standard samples.

[0308] This ELISA assay provides a highly specific and very sensitive assay for the detection of the GPNMB antigen in a test sample.

Determination of GPNMB Antigen Concentration in Patients:

[0309] A sandwich ELISA can also be used to quantify GPNMB levels in human serum. The 2 fully human monoclonal anti-GPNMB antibodies used in the sandwich ELISA, recognize different epitopes on the GPNMB molecule. The ELISA is performed as follows: 50 .mu.l of capture anti-GPNMB antibody in coating buffer (0.1 M NaHCO.sub.3, pH 9.6) at a concentration of 2 .mu.g/mL is coated on ELISA plates (Fisher). After incubation at 4.degree. C. overnight, the plates are treated with 200 .mu.l of blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in PBS) for 1 hr at 25.degree. C. The plates are washed (3.times.) using 0.05% Tween 20 in PBS (washing buffer, WB). Normal or patient sera (Clinomics, Bioreclaimation) are diluted in blocking buffer containing 50% human serum. The plates are incubated with serum samples overnight at 4.degree. C., washed with WB, and then incubated with 100 .mu.l/well of biotinylated detection anti-GPNMB antibody for 1 hr at 25.degree. C. After washing, the plates are incubated with HRP-Streptavidin for 15 min, washed as before, and then treated with 100 .mu.l/well of o-phenylenediamine in H.sub.2O.sub.2 (Sigma developing solution) for color generation. The reaction is stopped with 50 .mu.l/well of H.sub.2SO.sub.4 (2M) and analyzed using an ELISA plate reader at 492 nm Concentration of GPNMB antigen in serum samples is calculated by comparison to dilutions of purified GPNMB antigen using a four parameter curve fitting program.

Staging of Cancer in a Patient:

[0310] It will be appreciated that based on the results set forth and discussed in the above diagnostic examples, it is possible to stage a cancer in a subject based on expression levels of the GPNMB antigen. For a given type of cancer (e.g., melanoma), samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the cancer. The concentration of the GPNMB antigen present in the blood samples is determined using a method that specifically determines the amount of the antigen that is present. Such a method includes an ELISA method, such as the method described in the previous diagnostic examples. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated.

[0311] In order to stage the progression of the cancer in a subject under study, or to characterize the response of the subject to a course of therapy, a sample of blood is taken from the subject and the concentration of the GPNMB antigen present in the sample is determined. The concentration so obtained is used to identify in which range of concentrations the value falls. The range so identified correlates with a stage of progression or a stage of therapy identified in the various populations of diagnosed subjects, thereby providing a stage in the subject under study.

Example 17

Diagnosing Cancer with Antibodies Against GPNMB

[0312] A subject suspected of having an ovarian cancer tumor is identified and a tissue sample from the suspected tumor is removed for testing. The removed tissue is then contacted with anti-GPNMB antibodies having a colorimetric label. A determination is made of whether the anti-GPNMB antibodies bind specifically to the removed tissue. Binding is indicative of cancerous tissue while the absence of binding is indicative of non-cancerous tissue. The patient's condition is diagnosed accordingly to facilitate subsequent testing, counseling, and/or treatment.

Example 18

Treating Cancer with Antibodies Against GPNMB

[0313] Targeting GPNMB on tumor cells is useful to treat a subject at risk for or afflicted with cancer. Such a subject would benefit from treatment with an anti-GPNMB antibody of the present invention. Typically, antibodies are administered in an outpatient setting by weekly administration at about 0.1-1.0 mg/kg dose by slow intravenous (IV) infusion. The appropriate therapeutically effective dose of an antibody is selected by a treating clinician and would range approximately from 1 .mu.g/kg to 20 mg/kg, from 1 .mu.g/kg to 10 mg/kg, from 1 .mu.g/kg to 1 mg/kg, from 10 .mu.g/kg to 1 mg/kg, from 10 .mu.g/kg to 100 .mu.g/kg, from 100 .mu.g/kg to 1 mg/kg, and from 500 .mu.g/kg to 5 mg/kg.

[0314] The antibodies are also used to prevent and/or to reduce severity and/or symptoms of disease associated with GPNMB-related disorders.

[0315] To test the clinical efficacy of antibodies in humans, individuals with cancer, particularly, but not limited to ovarian, lung or colon carcinoma are identified and randomized into treatment groups. Treatment groups include a group not receiving antibody treatment and groups treated with different doses of anti-GPNMB antibody. Individuals are followed prospectively and individuals receiving antibody treatment exhibit an improvement in their condition.

Example 19

The Specificity of the Anti-Tumor Effects of CR011-vcMMAE (CR011-ONC-1)

[0316] The study was performed to determine the anti-tumor effects of the constituent components of the antibody-drug conjugate and its formulation and to relate these effects to the anti-tumor effects of the intact immunoconjugate.

Results:

[0317] Mice were implanted by trocar with fragments of SK-ME-2 melanoma and, after the tumors became established, treatment with CR011-vcMMAE and various components was tested to demonstrate the specificity of anti-tumor effects of this agent. Control groups, dosed with either the phosphate-buffered saline (vehicle) or the excipients of the immunoconjugate preparation (3% DMSO, sucrose, phosphate medium) steadily increased in tumor size to a maximum of 2,000 mg, at which time they were removed from the study. No apparent or statistically significant anti-tumor effects were observed. However, CR011-vcMMAE treatment (at 5 mg/kg/treatment, q4 d.times.4) produced measurable inhibition after the first 2 doses. Tumor growth inhibition continued until no discernible tumor was detected in all 6 of the test animals (FIG. 4). In preliminary studies, tumor regression was complete and was not followed by regrowth of the tumor despite lengthy observation periods (up to 200 days).

Conclusions:

[0318] The regressions produced by the immunoconjugate were not due to the individual components of the immunoconjugate nor to components of the formulation of that immunoconjugate. This is demonstrated by the lack of tumor growth inhibition after treatment with CR011 antibody alone (group 3) or free monomethylauristatin E (group 4), where the doses applied were identical to that contained in the intact immunoconjugate. Furthermore, the lack of anti-tumor effects noted with free MMAE suggests that anti-tumor effects from MMAE as a result of slow release from the antibody-drug conjugate may not explain the anti-tumor effects of the immunoconjugate. Release of MMAE from antibody-MMAE conjugates has been shown to be a very slow process in vivo (T.sub.1/2.beta.=6.0 days in the case of the anti-CD30 antibody-Auristatin E immunoconjugate (Sanderson et al., Clin. Cancer Res. 11: 843-852 (2005)) and would provide for plasma or serum concentrations that would be considerably lower than the "bolus" doses used in this study, which were ineffective at slowing the growth of the human melanoma xenografts.

Example 20

CR011-vcMMAE Inhibits the Growth of Human SK-MEL-5 Melanoma Xenografts Leading to Complete Regression of Established Melanoma Tumors in Athymic Mice (CR011-ONC-3)

[0319] This study was performed to assess the potency and therapeutic efficacy of the antibody-drug conjugate, CR011-vcMMAE, against a second model of established human melanoma, the SK-MEL-5 xenograft.

Results:

[0320] Though unrelated in origin, the SK-MEL-5 expresses GPNMB on the surface of the cell membrane and is killed by CR011-vcMMAE in vitro. In this study, the anti-tumor effects of the CR011 immunoconjugate were examined, along with the vehicles PBS and saline, and the reference agents vinblastine and paclitaxel. In a manner similar to the SK-MEL-2 tumor, vinblastine produced a noticeable, but not significant tumor growth inhibition (P.ltoreq.0.21) when compared to saline and PBS control groups (FIG. 5). Soon after the commencement of treatment with paclitaxel, however, significant tumor growth inhibition was observed (P.ltoreq.0.039) at day 3 after treatment began, and this anti-tumor effect continued, producing 100% growth inhibition (stasis). The responses of SK-MEL-5-bearing test animals to vinblastine and paclitaxel were short-lived. After cessation of treatment at the maximally tolerated doses, tumors resumed rapid, progressive growth. One long-term, tumor-free survivor occurred in the paclitaxel group and one spontaneous regression occurred in the group treated with saline.

[0321] Substantial tumor growth inhibition, as well as tumor growth delay and complete regressions occurred in SK-MEL-5 tumor-bearing animals after treatment with CR011-vcMMAE, and these effects were dose-related. At 10 mg/kg/treatment, significant anti-tumor effects were noted as early as 7 days (the equivalent of 2 treatments) after treatments began, when compared to saline (P.ltoreq.0.0096), and as early as 10 days after treatment began when compared to PBS-treated controls (P=0.039). In a dose-related manner, CR011-vcMMAE produced tumor growth delay leading to complete regressions of established SK-MEL-5 melanoma xenografts (see tabular insert to FIG. 5 for proportions of animals with complete regressions). Complete regressions occurred at CR011-vcMMAE doses of 2.5 mg/kg/treatment, but not at 1.25 mg/kg/treatment.

[0322] As in previous studies, no indication of toxicity by the immunoconjugate occurred in treated animals as evidenced by mortality of effects on body weight or weight gain.

Conclusions:

[0323] CR011-vcMMAE exerts substantial, dose-dependent anti-tumor effects against established xenografts of the SK-MEL-5 human melanoma. After just one or two treatments significant tumor growth inhibition is noted and which leads to long-term tumor-free survivors. Complete regressions occurred at doses of .gtoreq.2.5 mg/kg i.v., q4 d.times.4.

Example 21

Pharmacokinetics of CR011-vcMMAE (CR011-PK-1A)

[0324] The purpose of this study was to determine the stability of CR011-vcMMAE in vivo after intravenous injection, the anticipated route of clinical administration.

Materials & Methods.

[0325] The CR011 antibody component of CR011-vcMMAE was measured by a sandwich style enzyme-linked immunosorbent assay (ELISA) where serum was added to the wells of microtiter plates coated with the cognate antigen (GPNMB, CG56972-03) for the CR011 antibody, and the amount of human antibody were detected with an anti-globulin conjugated to the signal generator (horseradish peroxidase).

Results:

[0326] Pharmacokinetics. The persistence of compound availability for antibody component of CR011-vcMMAE was examined in a pharmacokinetic study in athymic mice (study CR011-PK-1, FIG. 6). The serum concentration-time profile for the antibody-drug conjugate was determined in athymic mice after intravenous administration of CR011-vcMMAE and the results are presented in FIG. 6. Athymic mice receiving 1 or 10 mg/kg intravenously showed dose-proportional serum concentrations over the entire span of sampling times (42 days). The concentration-time pattern was bi-phasic. The initial phase (.alpha.), however, was minor as it contributed <2% of the total AUC. Nevertheless, the compound disappeared very slowly from the peripheral blood (T.sub.1/2.beta.=10.3 days) with serum concentrations of 1 .mu.g/mL and 10 .mu.g/mL remaining in the blood for 6 weeks after dosing.

[0327] Estimates for the pharmacokinetic parameters for CR011-vcMMAE are presented in Table 48. One parameter is noteworthy. The volume of distribution at steady state (Vss) is very low, approaching the theoretical minimum; this suggests that the com-pound does not distribute outside the extravascular space. The distribution pattern, as well as the (3-elimination phase for CR011-vcMMAE are in good agreement with values obtained for antibodies in general (see Reviews by Mahmood and Green, Clin. Pharmacokinet 44: 331-347 (2005); or Lobo et al. J. Pharm. Sci. 93: 2645-2668 (2004)) and agree with values obtained for an antibody-Auristatin E immunoconjugate with comparable drug loading (Hamblett et al., Clin. Cancer Res. 10: 7063-7070 (2004)).

TABLE-US-00117 TABLE 48 PK Parameters for CR011-vcMMAE after Intravenous Administration. Parameter Units 1 mg/kg 10 mg/kg A .mu.g/mL 8.97 74.6 B .mu.g/mL 9.82 113 Alpha 1/h 0.179 0.0812 Beta 1/h 0.00269 0.00281 AUC h * .mu.g/mL 3712 41210 Alpha-Half Life h 3.88 8.531 Beta-Half Life h 258 247 Volume mL/kg 53.2 53.2 Cmax .mu.g/mL 18.8 188 Cl mL/h/kg 0.269 0.243 MRT h 368 348 Vss mL/kg 99.0 84.5

[0328] Abbreviations: A: Pre-exponential constant for alpha phase; Alpha: Exponential rate constant for alpha phase; AUC Total area under the curve from 0 to infinity; B: Pre-exponential constant for beta phase; Beta: Exponential rate constant for beta phase; Cl: Total or systemic clearance; C.sub.max: Maximum observed concentration; MRT: Mean residence time; Volume: Volume of central compartment; Vss: Steady-state volume of distribution.

[0329] Estimates for pharmacokinetic parameters are presented in Table 48. One parameter is noteworthy. The volume of distribution steady state (Vss) is, approaching the theoretical minimum. These data suggest that the compound did not distribute outside the extravascular space. Taken together, these data are in good agreement with data on other immunoconjugates bearing the -vcMMAE cytotoxic moiety (see Hamblett et al., Clin. Cancer Res. 10: 7063-7070 (2004)).

Conclusions:

[0330] The CR011-vcMMAE antibody-drug conjugate has a serum-concentration profile which favors continuous exposure sufficient for disruption and eradication of melanoma xenografts. The immunoconjugate after i.v. administration has a sufficiently long half-life to ensure exposure of tumor cells for extended periods (T.sub.1/2.beta.=10.3 days), and may not require frequent dosing. The durability of CR011-vcMMAE in vivo (e.g., athymic mice) is comparable to other Auristatin E immunoconjugates.

Example 22

The Schedule Dependency of the Anti-Tumor Effects of CR011-vcMMAE (CR011-ONC-1)

[0331] The purpose of this study was to determine the extent to which the curative anti-tumor effects of the CR011 antibody-drug conjugate are dependent on the dosing regimen and, if possible, to determine the optimum dosing interval for this xenograft model.

Materials and Methods:

[0332] The protocol for this study is presented in Table 49. To test the hypothesis that curative anti-tumor effects are influenced by the dosing schedule, the anti-tumor effects of CR011-vcMMAE were measured at 5 different dosing intervals (i.e., 0, 1, 4, 8, and 16 days between treatments) and for each dosing interval 3 dosage levels were employed (i.e., cumulative doses of 2, 8, and 32 mg/kg); for each group, n=6 athymic mice.

[0333] Nota bene: Please note that, although all 5 sets of groups in this experiment (e.g., groups 5, 6, and 7 represent one set and received 32, 8, and 2 mg/kg cumulative dose, respectively) received the same cumulative doses, the first set receiving the "bolus dose" is different from the other 4 sets. The C.sub.max for each group in the "bolus" set was likely four-fold higher that the C.sub.max for the other 4 sets (see section on pharmacokinetics for dose-linearity after i.v. administration), since 4 sets of groups received 4 treatments, whereas the first set received only one "bolus" treatment (see column 7, Table 49 below).

TABLE-US-00118 TABLE 49 Protocol for the Dosing Interval Study (CR011-PHM-2). No. Cum. Dose Dosing Treat- Dose (mg/ Regi- Interval ments (mg/ Group Treatment ROA kg men (days) (n) kg) 1 Phosphate Buffered Saline Bolus 0 1 N.A. 2 CR011-AE i.v. 32 Bolus 0 1 32 3 CR011-AE i.v. 8 Bolus 0 1 8 4 CR011-AE i.v. 2 Bolus 0 1 2 5 CR011-AE i.v. 8 qd x4 1 4 32 6 CR011-AE i.v. 2 qd x4 1 4 8 7 CR011-AE i.v. 0.5 qd x4 1 4 2 8 CR011-AE i.v. 8 q4d x4 4 4 32 9 CR011-AE i.v. 2 q4d x4 4 4 8 10 CR011-AE i.v. 0.5 q4d x4 4 4 2 11 CR011-AE i.v. 8 q8d x4 8 4 32 12 CR011-AE i.v. 2 q8d x4 8 4 8 13 CR011-AE i.v. 0.5 q8d x4 8 4 2 14 CR011-AE i.v. 8 q16d x4 16 4 32 15 CR011-AE i.v. 2 q16d x4 16 4 8 16 CR011-AE i.v. 0.5 q16d x4 16 4 2 17 Excipients i.v. N.A. q16d x4 16 4 N.A.

Results:

[0334] For this study, the frequency of complete regressions with long-term tumor-free survivors was determined after 5 different dosing intervals were examined empirically (i.e., 0, 1, 4, 8, and 16 days between treatments). The aggregate responses for each set of groups, where a set is defined as 3 groups of graduated dosage levels but one dosage interval (groups 5, 6, and 7 represent 1 set, all of which were treated with a dosing interval of 1 day) are shown in FIG. 7. The aggregate responses for test animals responding to CR011-vcMMAE appear to suggest that bolus dosing and intervals of 1 day and 4 days provide a very slight advantage to the proportion of cures, compared to longer intervals, such as 8 days and 16 days between doses. However, this effect was not significant (P<0.2904). The data therefore suggest that the anti-tumor effects of CR011-vcMMAE in the SK-MEL-2 model are not schedule-dependent. This conclusion is strengthened by the fact that test animals in the bolus set (groups 2, 3, and 4), which were exposed to plasma concentrations approximately four-fold higher than any of the other groups, did not show any greater percentages of cured subjects.

[0335] The original design of this study was expanded to include an examination of the effects of various dosage levels. For each set, one group of animals received a cumulative dose of 8 mg/kg, which, from previous studies employing a dosing interval of 4 days, provided consistent therapeutic effects leading to long-term tumor-free animals. In addition, cumulative doses of 2 mg/kg and 32 mg/kg were employed.

[0336] The effects of dosage levels, in conjunction with various dosing intervals, are presented in FIG. 8. Athymic mice receiving a cumulative dose of 32 mg/kg showed complete regressions in 100% of each group, regardless of dosing interval; that is, a cumulative dose of 32 mg/kg is schedule-independent and represents a dose which is well above that sufficient for complete regressions in 100% of the test animals (5 groups of 6 animals/group=30 test animals). Animals receiving 8 mg/kg cumulative dose did not demonstrate schedule dependency and showed nearly the same proportions of complete regressions (i.e., 28/30=93%); Test animals receiving 2 mg/kg (cumulative dose), which was recognized in preliminary studies to be below the threshold for cures (using a standardized regimen of q4 d.times.4) appeared to be schedule dependent, though this was not significant, and produced a much lower proportion of complete regressors (i.e., 13%).

Conclusions:

[0337] The data from the dosing interval study suggests that the responses of SK-MEL-2 melanoma xenografts are not dependent on the schedule of administration of CR011-vcMMAE. While no advantage could be shown for bolus dosing or regimens with low dosing intervals, there is the suggestion that, below a certain threshold cumulative dose, there may be some advantage to combining multiple treatments into a single bolus dose.

Example 23

GPNMB Transcript Expression in Human Melanoma

[0338] GPNMB was recently shown to be expressed in glioblastoma and to mediate the in vitro and in vivo invasiveness of glioblastoma-derived tumor cells (see, e.g., Loging et al., Genome Res. 10:1393-1402 (2000); and Rich et al., J. Biol. Chem. 278:15951-15975 (2003)). To confirm and extend these findings to additional cancer types, we examined the expression of GPNMB transcripts in human cancer cell lines and tissues.

Material and Methods:

[0339] Total RNA was isolated using the RNeasy kit with a DNase digestion step (Qiagen Inc., Valencia Calif.). RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) as follows. RT: 50.degree. C. for 45 min and 95.degree. C. for 15 min for 1 cycle. PCR: 1 min at 95.degree. C., 1 min at 50.degree. C. and 2 min at 72.degree. C. for 30 cycles with final extension for 10 min at 72.degree. C. Products were separated on a 2% agarose/0.33% low melting point agarose gel and visualized by ethidium bromide staining. The integrity of each RNA sample was verified via RT-PCR with primers designed to amplify GAPDH. Specific primers (5'-3') used were:

TABLE-US-00119 GPNMB: (SEQ ID NO: 327) Forward-GAATTCAGAGTTAAACCTTGAG (SEQ ID NO: 328) Reverse-CAGGAATCTGATCTGTTACCAC MART-1: (SEQ ID NO: 329) Forward-CTGACCCTACAAGATGCCAAGAG (SEQ ID NO: 330) Reverse-ATCATGCATTGCAACATTTATTGATGGAG Tyrosinase: (SEQ ID NO: 331) Forward-TTGGCAGATTGTCTGTAGCC (SEQ ID NO: 332) Reverse-AGGCATTGTGCATGCTGCTT pMEL-17: (SEQ ID NO: 333) Forward-TATTGAAAGTGCCGAGATCC (SEQ ID NO: 334) Reverse-TGCAAGGACCACAGCCATC

[0340] RTQ-PCR analysis was performed with an ABI Prism 7700 Sequence Detection System using TaqMan reagents (PE Applied Biosystems, Foster City, Calif.). Equal quantities of normalized RNA's were used as a template in PCR reactions for 40 cycles with GPNMB-specific primers to obtain threshold cycle (C.sub.T) values. The following primers (5'-3') were used:

TABLE-US-00120 (SEQ ID NO: 335) Forward-TCAATGGAACCTTCAGCCTTA (SEQ ID NO: 336) Reverse-GAAGGGGTGGGTTTTGAAG (SEQ ID NO: 337) Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA

Result:

[0341] Our transcript expression analysis indicated that GPNMB was strongly expressed in a high percentage of human metastatic melanoma samples. Using RTQ-PCR, GPNMB was found to be highly expressed (CT<27.0) in 5/7 melanoma cell lines and 5/5 melanoma clinical specimens examined (Table 50). In contrast, GPNMB was not expressed in a renal carcinoma cell line, TK-10, that was used as a negative control in our experiments.

TABLE-US-00121 TABLE 50 GPNMB transcript expression in human melanoma cell lines and clinical specimens Sample Details Expression* Cell lines UACC-62 Met. Melanoma 21.2 M14 Met. Melanoma, amelanotic 22.2 SK-Mel-5 Met. Melanoma. axillary node 22.9 SK-Mel-28 Met. Melanoma, skin 24.1 WM-266-4 Met. Melanoma, skin 24.5 A-375 Met. Melanoma, skin 29.0 LOXIMVI Met. Melanoma, amelanotic 30.9 TK-10 Renal cell carcinoma 40.0 Clinical specimens #1 Met. Melanoma 26.6 #2 Melanoma 26.4 #3 Melanoma 26.9 #4 Met. Melanoma 24.1 #5 Met. Melanoma 25.3 *Threshold cycle (C.sub.T) values from RTQ-PCR analysis. Met: Metastatic.

[0342] To extend these results, we investigated the expression of GPNMB in a panel of 17 melanoma cell lines via semi-quantitative RT-PCR (Table 51). The results show that GPNMB transcript is highly expressed in 15/17 melanoma cell lines, weakly expressed in 1/17 melanoma cell line (A-375), and not detectable in 1/17 melanoma cell line (LOXIMVI) nor in the control TK-10.

TABLE-US-00122 TABLE 51 RT-PCR analysis Expression* Cell line Annotation GPNMB MART-1 Tyrosinase pMel-17 M14 Met. Melanoma, amelanotic +++ +++ +++ +++ SK-Mel-5 Met. Melanoma. axillary node +++ +++ +++ +++ SK-Mel-28 Met. Melanoma, skin +++ +++ +++ +++ WM-266-4 Met. Melanoma, skin +++ +++ +++ +++ SK-Mel-2 Met. Melanoma, skin +++ +++ +++ +++ UACC-257 Met. Melanoma +++ +++ +++ +++ A2058 Met. Melanoma, lymph node +++ +++ +++ +++ G361 Met. Melanoma, skin +++ +++ +++ +++ HT-144 Met. Melanoma, skin +++ +++ +++ +++ MEWO Met. Melanoma, lymph node +++ +++ +++ +++ SK-Mel-3 Met. Melanoma. Lymph node +++ +++ +++ +++ MALME-3M Met. Melanoma +++ +++ +++ +++ UACC-62 Met. Melanoma +++ +++ +++ - SK-Mel-24 Met. Melanoma, lymph node +++ - +++ - RPMI-7951 Met. Melanoma, lymph node +++ - + - A-375 Met. Melanoma, skin + - - - LOXIMVI Met. Melanoma, amelanotic - - - - TK-10 Renal cell carcinoma - - - - *RT-PCR analysis: Strongly (+++), weakly (+) or not detectable (-). Met: Metastatic.

[0343] Furthermore, comparing the expression of GPNMB transcript to known melanoma/melanocyte-associated gene transcripts (MART-1, tyrosinase and pMEL-17) in the melanoma cell lines (Table 51) demonstrated strong expression of MART-1, tyrosinase and pMEL-17 in 13/17, 14/17 and 12/17 melanoma cell lines, respectively. Notably, 12/17 samples co-expressed high levels of GPNMB and all three melanoma/melanocyte-associated genes. Both LOXIMVI and TK-10 cell lines, which had undetectable GPNMB expression, also lacked expression of the three melanoma/melanocyte-associated genes examined.

Example 24

Growth-Inhibitory Activity of CR011-vcMMAE is Dependent on GPNMB Expression

Material and Methods:

[0344] Flow cytometry: Quantitative analysis of GPNMB expression on the cell surface of cell lines was determined by flow cytometry. Approximately 1.times.10.sup.6 cells were harvested, washed and incubated with a saturating amount (10 .mu.g/mL) of either CR011 or isotype-matched control antibody in staining buffer containing PBS (pH 7.4), 4% FBS and 0.1% NaN3 for 30 min on ice, followed by washing and staining with R-Phycoerythrin (PE)-conjugated goat-anti-human antibody (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa.) at 1:100 for 30 min on ice. Cells were fixed in 1% paraformaldehyde/PBS and examined on a Becton Dickinson FACSCalibur flow cytometer. Data analysis was performed with Becton Dickinson Cell Quest software version 3.3 and the geometric mean fluorescence intensity ratio (GMR) was determined for each cell type.

[0345] Internalization of cell surface bound antibodies was assessed by a modified flow cytometry procedure. In brief, cell suspensions were labeled with 10 .mu.g/mL unconjugated or MMAE-conjugated CR011 for 30 min on ice. After washing cells, incubation was shifted to 37.degree. C. for 1 hr to allow internalization of bound antibodies. Cells that remained on ice (total surface bound) or that were incubated at 37.degree. C. (internalized) were stained with PE-conjugated goat-anti-human antibody at 1:100 for 30 min to detect CR011 retained on the cell surface. Labeled cells were analyzed by flow cytometry as described above. The percentage of antibody internalized was determined using the GMRs and the following formula:

Percent internalized=Total surface bound (4.degree. C.)-Total surface bound (37.degree. C.)/Total surface bound (4.degree. C.).times.100

[0346] Immunoprecipitation and immunoblot analysis: Cells were harvested and lysed on ice for 30 min in lysis buffer containing 1% NP-40, 0.15 M NaCl, 0.02 M Tris-HCl, 10% glycerol, 0.01 M EDTA and complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, Ind.). Supernatants were collected and the protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, Ill.). For immunoprecipitation, 2 .mu.g of primary antibody was added into 0.5-1 mg of total cell lysates and incubated at 4.degree. C. for 3 hrs, followed by incubation with protein-A-agarose (Amersham Biosciences, Upsala, Sweden) on ice for 2 hrs. The agarose beads were washed in ice-cold TBST (PBS with 0.1% Tween-20) Immunoprecipitates were recovered from supernatants after boiling in Laemmli sample buffer and centrifugation.

[0347] For immunoblot analysis, total cell lysates (50 .mu.g) or immunoprecipitates were resolved under reducing condition on 4-20% Tris-glycine gels (Invitrogen) and electrophoretically transferred to 0.45-.mu.m PVDF membranes (Invitrogen). Membranes were blocked with 3% BSA (Sigma, St. Louis, Mo.) in TBST for 3 hrs and probed with rabbit anti-GPNMB polyclonal antibody (1:1000) for 3 hrs. Peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch Labs) was added and incubated for 30 min. The membranes were washed in TBST and subjected to enhanced chemiluminescence (Amersham) following the manufacturer's protocol.

[0348] Clonogenic Assays: The growth-inhibitory activity of CR011-vcMMAE was determined by clonogenic assay. Cells were plated in 96-well plates and allowed to recover overnight. Unconjugated CR011, free MMAE, CR011-vcMMAE or isotype-matched vcMMAE conjugated antibody at various concentrations was added to sub-confluent cell cultures and incubated for 4 days at 37.degree. C. The cells were then transferred into 6-well plates and allowed to form colonies. Colonies were stained with Giemsa stain (Sigma) and counted. The surviving cell fractions were calculated based upon the ratio of the treated sample and the untreated control. The results were expressed as a percentage of control using GraphPad Prism Version 4 software. The IC50 was defined as the concentration resulting in a 50% reduction of colony formation compared to untreated control cultures.

Results:

[0349] To demonstrate that CR011-vcMMAE growth-inhibitory activity is dependent on GPNMB expression, full-length GPNMB protein was ectopically expressed in HEK293 cells. Immunoblot (FIG. 9A) and FACS (FIG. 9B) analyses confirmed that GPNMB was expressed in GPNMB/plasmid transfected cells. Treatment of cells with CR011-vcMMAE, followed by clonogenic assay, demonstrated that GPNMB-expressing HEK293 cells were more sensitive to CR011-vcMMAE-mediated growth-inhibition than were control cells devoid of GPNMB expression (FIG. 9C).

[0350] To further verify our findings, GPNMB-expressing SK-Mel-2 cells were transfected with siRNA to specifically inhibit endogenous GPNMB expression. Immunoblot and FACS analyses performed 2 and 4 days after transfection demonstrated that total GPNMB (FIG. 10A) and surface GPNMB (FIG. 10B) protein levels were significantly reduced in SK-Mel-2 cells after the transfection when compared to the control transfectants. The amount of GPNMB expression was reduced for at least 7 days after transfection. Treatment of these cells with CR011-vcMMAE demonstrated that SK-Mel-2 cells were less sensitive to the growth-inhibitory activity of CR011-vcMMAE following siRNA-mediated GPNMB knockdown (FIG. 10C). Taken together, these data indicate that the growth-inhibitory activity of CR011-vcMMAE required cell surface GPNMB expression.

Example 25

Cell Cycle Arrest and Induction of Apoptosis by CR011-vcMMAE

[0351] To evaluate CR011-vcMMAE's mechanism of growth inhibition, cell cycle analysis was performed.

Material and Methods:

[0352] The cell cycle effects of CR011-vcMMAE were evaluated after treating cells in complete growth medium for 24 or 48 hr. Briefly, cells were pulsed at the indicated times with 30 .mu.M of bromodeoxyuridine (BrdU, Sigma) for 30 min, harvested, fixed and permeabilized in methanol. Nascent DNA synthesis was detected by anti-bromodeoxyuridine-FITC (BD Biosciences, San Jose, Calif.) staining. Total DNA content was detected using propidium iodide (PI, Sigma). For apoptosis analysis, cells were treated as above and labeled with Annexin V-FITC followed by propidium iodide exclusion using the Annexin V-FITC Apoptosis Detection kit I (BD PharMingen, San Diego, Calif.) according to the manufacturer's protocols. Flow cytometry (as described in the previous Example) was used to assay both cell cycle and apoptosis studies.

Results:

[0353] GPNMB-positive SK-Mel-2 cells or negative TK-10 control cells were treated with CR011-vcMMAE for various lengths of time, followed by bromodeoxyuridine for 30 minutes to detect nascent DNA synthesis and finally, propidium iodide to detect total DNA content. DNA synthesis and cell cycle progression were determined by flow cytometry (Table 52).

TABLE-US-00123 TABLE 52 Cell cycle analysis of CR011-vcMMAE treated cells Treatment (ng/mL) % G.sub.1 % S-phase % G.sub.2/M % Sub-G.sub.1 SK-Mel-2 24 hour Untreated 55.2 30.0 9.9 0.5 CR011 (1000) 63.6 25.2 6.4 0.5 IgG.sub.2-vcMMAE (1000) 65.9 21.8 5.8 0.8 CR011-vcMMAE (100) 56.0 26.9 12.4 0.2 CR011-vcMMAE (1000) 43.7 20.0 28.5 1.1 TK-10 24 hour Untreated 39.7 43.7 7.0 0.5 CR011 (1000) 42.0 39.8 6.3 0.3 IgG.sub.2-vcMMAE (1000) 42.8 40.2 5.9 0.3 CR011-vcMMAE (100) 51.1 35.1 4.5 0.7 CR011-vcMMAE (1000) 52.6 34.2 3.9 0.8 Cell cycle analysis was carried out by flow cytometry and the percentages of cells in each phase of cell cycle were determined by CellQuest Software (Becton Dickinson).

[0354] Exposure of GPNMB-positive cells to 1000 ng/mL CR011-vcMMAE, but not to isotype control IgG2-vcMMAE for 24 hrs, resulted in a decreased percentage (10%) of cells in G1 and S-phase and an increased percentage (18.6%) of cells in G2/M when compared to untreated cells. In contrast, CR011-vcMMAE did not affect the cycling of GPNMB-negative cells. At 48 hr after the treatment, CR011-vcMMAE further reduced the percentage (11%) of cells in G1 and S-phase and increased the percentage (24%) of cells in G2/M.

[0355] The increase in the sub-G1 population following CR011-vcMMAE treatment suggested the onset of apoptosis. To investigate this possibility, analysis of apoptosis using Annexin-V surface binding and loss of propidium iodide (PI) exclusion was performed. Our results demonstrated that 1000 ng/mL of CR011-vcMMAE induced apoptosis specifically in GPNMB-expressing cells as indicated by an 11% increase in mono-stained (Annexin-V+/PI-) cells following 48 hr of CR011-vcMMAE treatment (Table 53).

TABLE-US-00124 TABLE 53 Induction of apoptosis in human melanoma cells by CR011-vcMMAE % % AnnV.sup.+/PI.sup.- AnnV.sup.-/PI.sup.+ AnnV.sup.+/PI.sup.+ % AnnV.sup.-/PI.sup.- % Treatment (ng/mL) UL UR LL LR SK-Mel-2 48 hour Untreated 1.23 1.23 94.37 3.16 CR011 (1000) 0.36 0.45 94.45 4.74 IgG.sub.2-vcMMAE (1000) 0.17 0.51 95.93 3.39 CR011-vcMMAE (100) 0.30 0.40 89.93 9.37 CR011-vcMMAE (1000) 2.08 2.02 82.08 13.83 TK-10 48 hour Untreated 0.54 0.66 96.92 1.87 CR011 (1000) 0.83 0.34 98.27 0.55 IgG.sub.2-vcMMAE (1000) 0.62 0.95 97.09 1.33 CR011-vcMMAE (100) 0.71 0.57 97.72 1.00 CR011-vcMMAE (1000) 0.86 0.83 97.75 0.56 Apoptosis analysis was carried out by flow cytometry and the percentages of cells in quadrants UL (upper left), UR (upper right), LL (lower left) and LR (lower right) were determined by CellQuest Software (Becton Dickinson). AnnV: Annexin V-FITC and PI: Propidium iodide.

[0356] In addition, an increase in dual-stained (Annexin-V+/PI+) cells following CR011-vcMMAE treatment indicated that the CR011 immunoconjugate enhanced cell death. Together, these results suggest that CR011-vcMMAE selectively induced G2/M cell cycle arrest followed by apoptotic cell death.

Example 26

CR011: A Naked Fully Human IgG1 for Use in Melanoma Therapy Exploiting the Mechanism of Antibody-Dependent Cellular Cytotoxicity (ADCC)

[0357] Fully Human Monoclonal Antibodies (mAb)-IgG2 to CG56972/GPNMB, an Antigen predominantly found on the surface of melanoma and brain tumor cells, were generated. The naked CR011 IgG2 mAb (mAb 1.15) had no effect on CG56972 expressing cells either in vitro or in vivo. Thus we examined whether isotype switching from an IgG2 to an IgG1 might enable the mAb to kill human melanoma cells through ADCC effector functions.

[0358] Briefly, to switch CR011 from an IgG2 to IgG1 antibody, double stranded DNA encoding constant region of IgG1 (allotype Gm(f)) was synthesized, and IgG2 constant region was replaced with IgG1 constant region using overlapped PCR approach. The sequences are described below:

TABLE-US-00125 CR011 mAb 1.15.1 mature heavy chain (IgG2): (SEQ ID NO: 394) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGLEWI GYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYCARG YNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CR011 mAb 1.15.1 mature heavy chain (IgG1): (SEQ ID NO: 395) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGLEWI GYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYCARG YNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0359] We first analyzed the binding properties of the IgG1 and IgG2 fully human monoclonal antibodies on SK-MEL-2 melanoma cells that have been shown to express CG56972 on the cell surface and bind CR011 IgG2. As shown in FIGS. 11B-D, both the IgG1 and IgG2 mAbs caused comparable FACS shifts on SK-MEL-2 cells compared to isotype control mAbs (FIG. 11A) indicating that both isotypes bind to CG56972/GPNMB with comparable saturation densities and affinities.

[0360] We next examined whether the CR011 IgG1 mAb could induce ADCC in SK-MEL-2 cells in culture in the presence of human PBMC. Human PBMC were isolated from whole blood using a Ficoll-Plaque. Briefly, in a 50 mL tube, 15 mL of PBS was added to 20 mL of whole blood which was underlayed with 10 mL Ficoll-Plaque and the tube was centrifuged at 2000 RPM. Mononuclear cells were collected from the interface and washed 3 times with PBS. The ADCC assay was carried out in a 96 well plate using a fluorescence assay for cytolysis from Perkin-Elmer (DELFIA EuTDA Cytotoxic assay). The procedure is based on loading target cells with a fluorescence enhancing ligand (BATDA, bis (acetoxymethyl) terpyridine-dicarboxylate). The hydrophic ligand penetrates the membrane quickly. Within the cell the esterbonds are hydrolyzed to form a hydrophilic ligand (TDA, terpyridine-dicarboxylic acid) which can no longer pass through the membrane. After cytolysis the ligand is released and introduced to the Europium solution. The europium and the ligand form a highly fluorescent and stable chelate (EuTDA). Fluorescence intensity are recorded using excitation and emission wavelengths as .lamda.ex=340 nm and .lamda.em=613 nm, respectively.

[0361] Antibody-dependent cell-mediated cytotoxicity on SK-MEL-2 cells was assayed in the presence of PBMC and CR011 monoclonal antibody using effector: target ratios of 10, 30, 60 and 100 and various concentrations of IgG1 or IgG2 mAb against CG56972/GPNMB (2, 5, 10 .mu.g/200 .mu.l). Our data showed that between 30 to 100 fold PBMC, IgG1 mAb caused cytolysis of SK-MEL-2 cells in a dose dependent manner (FIG. 12A) whereas IgG2 mAb did not show any cytolysis (FIG. 12B). Therefore, we conclude that CR011 IgG1 mAb to CG56972/GPNMB can kill CG56972/GPNMB expressing melanoma cells in vitro and potentially human melanoma in vivo through ADCC effector functions. CR011 IgG1 mAb can also be useful in combination with immune effector cytokines that could provide some clinical benefit in metastatic melanoma such as high dose IL-2, interferon-gamma or TNF-alpha. CR011 can also be used to treat melanoma in combination with vaccine immunotherapy, immunomodulators such as MDX-010, radiation therapy and/or chemotherapy.

Example 27

Treatment of Astrocytoma, Glioblastoma, Medulloblastoma and Other Tumors of the CNS

[0362] Astrocytoma/glioblastoma is a highly drug-refractory neoplasm representing significant unmet medical needs. We identified CG56972 as a human gene (also known as GPNMB) that is highly expressed in these human cancer tissues and cancer cell lines. CG56972 is a type I transmembrane protein potentially involved in vesicular trafficking with a very restricted expression pattern in human brain. We generated fully human monoclonal antibodies against the CG56972 extracellular domain (amino acids 23-480). Our lead monoclonal antibody, designated CR011-vcMMAE was biochemically characterized and tested for therapeutic activity against cell lines derived from human brain tumors of astrocytoma, glioblastoma, medulloblastoma or neuroectodermal origin.

[0363] Transcript expression analysis demonstrated highly elevated CG56972 mRNA in brain tumors derived from astrocytoma, glioblastomas, medulloblastoma and tumors of neuroectodermal origin with restricted low expression in normal brain. CR011 bound by FACS analysis surface CG56972 on brain cancer cell lines. CR011 mAbs western blotted the predicted 100 and 120 kDa gene products. Clonogenic assays demonstrated that CR011-vcMMAE mAbs inhibited the growth of brain cancer cell lines.

Material and Methods:

[0364] Cell lines and culture conditions: All human cell lines, SK-MEL-2, XF-498, SNB-78, U-118-MG, SF-539, H79MG, D392-MG, D534-MG, SK-N-SH, U-251, SF-295, D450-MG, U87MG, SF-268, T98G, and SW-1783 were obtained from the American Type Culture Collection (Manassas, Va.) or were purchased from the NCI (Bethesda, Md.). Cells were maintained in DMEM or RPMI (Invitrogen, Carlsbad, Calif.) containing 10% FBS (Gemini Bio-Products, Woodland, Calif.) and penicillin-streptomycin.

[0365] Real-Time Quantitative PCR (RTQ-PCR): Total RNA was isolated using the RNeasy kit with a DNase digestion step (Qiagen Inc., Valencia). RNA samples were derived from normal human tissues obtained commercially (Clontech, Palo Alto, Calif.; Invitrogen, Carlsbad, Calif.) or cell lines grown according to specifications. RNAs were harvested and PCR was performed as previously described (Shimkets R A et. al. Nat Biotechnol., 1999. 17-8: 798-803) using TaqMan.RTM. reagents (PE Applied Biosystems, Foster City, Calif.). RNAs were normalized utilizing human .beta.-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) TaqMan.RTM. probes according to the manufacturer's instructions. Equal quantities of normalized RNA were used as templates in PCR reactions with CG56972-specific reagents to obtain threshold cycle (CT) values. For graphic representation, CT numbers were converted to relative expression, relative to the sample exhibiting the highest level of expression. RTQ-PCR analysis was performed with an ABI Prism 7700 Sequence Detection System using TaqMan reagents (PE Applied Biosystems, Foster City, Calif.). The following primers (5'-3') were used:

TABLE-US-00126 (SEQ ID NO: 338) Forward-TCAATGGAACCTTCAGCCTTA (SEQ ID NO: 339) Reverse-GAAGGGGTGGGTTTTGAAG (SEQ ID NO: 340) Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA

[0366] CuraChip.TM.: Tissues were lysed in Trizol. Biotin-labeled cDNA was made by using 15 mg of total RNA with poly(T) primers. Gene expression was evaluated by hybridization to the proprietary CuraChip microarray (CuraGen, New Haven, Conn.) of 11,000 oligonucleotide probes. Slides were hybridized for 15 h at 30.degree. C. with constant rotation, washed for 30 min at room temperature (RT), incubated in streptavidin solution (4.degree. C., 30 min), washed three times for 15 min at RT, incubated in Cy3-conjugated detection buffer (4.degree. C., 30 min), and washed three times for 15 min at RT. Slides were scanned (GMS 418 Scanner, Genetic Microsystems, Woburn, Mass.) and analyzed by using IMAGENE software (BioDiscovery, Marina Del Rey, Calif.). Data was subjected to 90th percentile normalization, and expression of the CG56972 gene was analyzed in comparison to that of the housekeeping gene GAPDH. The oligonucleotide sequence used to detect CG56972 is 5'-TGATCAGTAAGGATTTCACCTCTGTTTGTA (SEQ ID NO: 341). The oligonucleotide sequence used to detect GAPDH is 5'-ACCTTGTCATGTACCATCAATAAAGTACCC (SEQ ID NO: 342), corresponding to by 1243-1272 of the GAPDH transcript (accession no. NM_002046).

[0367] Flow Cytometry: Quantitative analysis of CG56972 expression on the surface of cell lines was determined by flow cytometry (FACS). Approximately 1.times.10.sup.6 cells were harvested, washed and incubated with a saturating amount (10 .mu.g/mL) of either CR011 or isotype-matched control antibody in staining buffer containing PBS (pH 7.4), 4% FBS and 0.1% NaN3 for 30 min on ice, followed by washing and staining with R-Phycoerythrin (PE)-conjugated goat-anti-human antibody (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa.) at 1:100 for 30 min on ice. Cells were fixed in 1% paraformaldehyde/PBS and examined on a Becton Dickinson FACSCalibur flow cytometer. Data analysis was performed with Becton Dickinson Cell Quest software version 3.3 and the geometric mean fluorescence intensity ratio (GMR) was determined for each cell type.

[0368] Immunoblot analysis: SK-MEL-2, XF-498, SNB-78, U-118-MG, SF-539, H79MG, D392-MG, D534-MG, SK-N-SH, U-251, SF-295, D450-MG, U87MG, SF-268, T98G, and SW-1783 cells were harvested and lysed on ice for 30 min in lysis buffer containing 1% NP-40, 0.15 M NaCl, 0.02 M Tris-HCl, 10% glycerol, 0.01 M EDTA and complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, Ind.). Supernatants were collected and the protein concentration was determined with the BCA Protein Assay Kit (Pierce, Rockford, Ill.). For immunoblot analysis, 40 ul of total cell lysate from one well of confluent cells harvested from a 6 well Falcon tissue culture dish were boiled in Laemmli sample buffer, centrifuged and resolved under reducing condition on 4-20% Tris-glycine gels (Invitrogen). Gels were electrophoretically transferred to 0.45-.mu.m PVDF membranes (Invitrogen). Membranes were blocked with 3% BSA (Sigma, St. Louis, Mo.) in TBST for 3 hrs and probed with goat anti-GPNMB polyclonal IgG (R & D Systems; 1 .mu.g/mL, total 10 .mu.g)) for 3 hrs. Peroxidase-conjugated anti-goat secondary antibody (Jackson ImmunoResearch Labs) was added and incubated for 30 min. The membranes were washed in TBST and subjected to enhanced chemiluminescence (Amersham) following the manufacturer's protocol.

[0369] Clonogenic assays: The growth-inhibitory activity of CR011-vcMMAE was determined by clonogenic assay. Cells were plated in 96-well plates and allowed to recover overnight. CR011-vcMMAE or isotype-matched monoclonal antibody at various concentrations was added to sub-confluent cell cultures and incubated for 4 days at 37.degree. C. The cells were then transferred into 6-well plates and allowed to form colonies. Colonies were stained with Giemsa stain (Sigma) and counted. The surviving cell fractions were calculated based upon the ratio of the treated sample and the untreated control. The results were expressed as a percentage of control using GraphPad Prism Version 4 software. The IC50 was defined as the concentration resulting in a 50% reduction of colony formation compared to untreated control cultures.

Results:

1. CG56972 Transcript Expression in Human Astrocytoma, Glioblastoma, Medulloblastoma and Tumors of Neuroectodermal Origin.

[0370] We examined the expression of CG56972 transcripts in human cancer cell lines and tissues (FIGS. 13A and B). Our transcript expression analysis indicated that CG56972 was strongly expressed in all (15/15) human brain cancer cell lines tested (FIG. 13A) Using RTQ-PCR, CG56972 was found to be expressed in cells of mixed glioblastoma/astrocytoma origin, glioblastoma/gliomas, astrocytomas and metastatic neuroblastomas. The majority of brain or CNS tumor cell lines showed high level expression with CTs<27. Of note, CG56972 was found to be highly expressed (CT<27.0) in XF-498, U-118-MG, SNB-78, and SF-539 cells. As shown in FIG. 13B, CG56972 was also expressed at high levels in 4/5 glioma human biopsies and 1/4 medulloblastoma human biopsies. Using microarray analysis from an in house chip containing a large panel of human genes (FIG. 13C), CG56972 was found to be highly expressed in 5/9 brain cancers of astrocytoma or glioblastoma origin as well as 4/9 oligodendrogliomas. Our analysis of these tumor expression profiles showed that CG56972 message was detected to a much lesser degree in normal brain tissues. These data are also consistent with our immunohistochemical data that demonstrated the lack of CR011 staining in normal human brain including neurons and glial cells. Taken together, these data demonstrate that the CG56972 transcript is expressed at highly elevated quantities in brain cancer and oligodendroglioma cell lines and specimens isolated from human tumors.

2. Generation of Fully Human CR011 Monoclonal Antibodies to CG56972/GPNMB

[0371] The CG56972 protein is predicted to be a type I transmembrane glycoprotein. The highly elevated expression of CG56972 transcripts and the potential cell surface localization of this protein in human cancer samples encouraged us to generate monoclonal antibodies (mAbs) as a potential cancer therapeutic. Therefore, we cloned the human CG56972 extracellular domain (ECD; aa 23-480). Sequencing of the cloned cDNA revealed the presence of an in-frame 36-nt insertion, likely due to alternative splicing at the exon 6/7 boundary, which added an additional 12-aa (ATTLKSYDSNTP) (SEQ ID NO: 343) after residue 339 of the published GPNMB protein sequence. We verified the authenticity of 36-nt insertion via RT-PCR. The cDNA was next expressed in human HEK293 cells. The resultant protein was harvested, purified from the conditioned media and used as an immunogen to generate fully human mAbs against CG56972-ECD. Following immunization of XenoMouse.RTM., mAbs that specifically recognized the CG56972-ECD protein via ELISA were generated. Our lead mAb, designated 1.15 or CR011 against purified CG56972-ECD, exhibiting a Kd of 52 nM against purified CG56972-ECD protein, was selected for in depth characterization and will be the focus of the remainder of this example.

3. CR011 mAb 1.15 Detection of CG56972 Protein Expression in Human Brain Cancers

[0372] We further used CR011 monoclonal antibodies to examine the surface expression of CG56972 protein on a variety of brain cancer cell lines by flow cytometry (FIGS. 14A-F & Table 54). FACS analysis demonstrated that the brain cancer cell lines XF-498, U-118-MG, SNB-78 and SF-539; all positive for CG56972 transcript expression exhibited surface staining with CR011 monoclonal antibodies of at least 1.5-fold above isotype control mAb background (FIGS. 14B, 14C, 14D, and 14E, respectively). The cell line SF-268, that was the most weakly positive (CT>32) for the CG56972 transcript expression (FIG. 13C & Table 54) showed minimal surface staining as expected of around 1.5-fold above control mAb background. Our SK-MEL-2 melanoma cell line that is our positive control for CG56972 expression showed strong cell surface staining.

[0373] To investigate CG56972 protein expression in our panel of brain cancer cell lines, total cell lysates were harvest, resolved by SDS-PAGE, transferred to membrane filters and subjected to immunoblot analysis with a CG56972 polyclonal antibody. As shown in FIG. 15, the CG56972 polyclonal antibody detected two protein species that are differential glycosylation products of CG56972 of approximately 100 and 120 kDa from various brain cancer cell lines that have been shown to express CG56972 transcripts (FIG. 13A). CG56972 protein was highly expressed in XF-498, SNB-78 and H79-MG and SF-539 cells. Both p100 and p120 species were detected to a lesser extent in U-118-MG, U251, D534-MG and D450-MG. Little or no CG56972 protein was detected in weakly expressing CG56972 transcript cell line, SF-268. An isotype-matched control IgG2 antibody did not immunoprecipitate CG56972 from any of the cell lines examined. All of these data are consistent with the cell surface expression of the CG56972 protein of the predicted molecule weights in brain cancer.

4. In Vitro Growth-Inhibition of Astrocytoma/Glioblastoma Cell Lines with CR011-vcMMAE.

[0374] CG56972 possesses a very restricted human tissue expression pattern. In preliminary studies, CR011 did not inhibit the growth of CG56972-expressing cancer cell lines when used directly (data not shown). Since CG56972 is a cell surface molecule on brain cancers and melanoma, and since CR011 was internalized following incubation with CG56972-expressing cells, we evaluated whether CR011 would inhibit the growth of cancer cells when combined with a protein synthesis inhibitor (saporin)-conjugated secondary antibody. Our results indicated that CR011 could specifically inhibit the growth of CG56972-expressing cancer cells (data not shown). Thus, we conjugated CR011 directly to the cytotoxic drug monomethyl aurostatin E (MMAE) through a highly stable, but intracellular protease cleavable valine-citrulline (vc) linker. The resulting antibody-drug conjugate was named CR011-vcMMAE.

[0375] To investigate whether CR011-vcMMAE specifically inhibited the growth of brain cancer cells, clonogenic assays were performed to assess cell viability after CR011-vcMMAE treatment. As shown in FIGS. 16A-16F and Table 54, our results indicated that CG56972-expressing cells were sensitive to growth-inhibition induced by CR011-vvMMAE, but not cells that poorly expressed this antigen (see SF-268 and LOXIMVI) at concentrations of vcMMAE less than 3 .mu.g/mL. Strikingly, CR011-vcMMAE possessed IC50s of approximately 215, 450, 1250, and 1050 ng/mL on XF-498, SNB-78, U-118-MG and SF-539 cells (FIGS. 16A, 16B, 16C, and 16D, respectively, and Table 54). In these experiments, IC50s correlated with cell surface density as measured by FACS analysis. In contrast, conjugated control human IgG2 antibody-vcMMAE failed to inhibit the growth of any of the cell lines examined at concentrations up to 3 .mu.g/mL (Table 54) with IC50s exceeding 1.5 or 4.5 .mu.g/mL (Table 54).

TABLE-US-00127 TABLE 54 Summary of RTQ PCR, FACS and in vitro growth inhibition of human cancer cell lines with CR011-mAbs CR011-AE IC50 IgG2-AE IC50 Cell Line Description CT Values CR011 Fold Shift (ng/ml) (ng/ml) SK-MEL-2 melanoma ND 13.1, 21.4, 17.8 303 >1500 XF-498 glioblastoma +++ 10, 9.5 216 >1500 SNB-78 astrocytoma +++ 8.6 449 >1500 U-118-MG glioblastoma/astrocytoma +++ 7.4 1254 >1500 SF-539 glioblastoma + 5.4 1030 >4500 H79MG glioblastoma/astrocytoma ND 4.7, 3.9 ND ND D392-MG glioblastoma ND 3.1 ND ND D534-MG glioblastoma ND 2.3 ND ND SK-N-SH neuroblastoma + 2 ND ND U-251 glioblastoma +++ 1.9 ND ND SF-295 glioblastoma ++ 1.8 ND ND D450-MG glioblastoma ND 1.6 ND ND U87MG glioblastoma/astrocytoma ++ 1.5 ND ND SF-268 glioblastoma/astrocytoma + 1.5 >1500 >4500 T98G glioblastoma + 1.3 ND ND SW 1783 astrocytoma + 1.1 ND ND .sup.aCR011 vcMMAE (mAb 1.15): CT values were determined by RTQ PCR as described in Materials and Methods. Geometric Mean ratios (GMR) were determined by flow cytometric analysis. Antibody-Drug Cytotoxicity (ADC) or cell killing was determined by clonogenic assay as described. .sup.bIC.sub.50 value is the mean and SD of a representative clonogenic assay with each experiment performed in triplicate wells. ND: Not done.

Conclusion:

[0376] These data indicate that CR011-vcMMAE can be a highly potent and selective agent for the treatment of astrocytoma/glioblastoma and their metastasis as well as brain tumors of medulloblastoma and neuroectodermal origin. CR011-vcMMAE can also be useful for the treatment of melanoma metastasis to brain and other brain neoplasms such as neoplastic meningitis.

Example 28

Engineered Antibodies Derived from CR011

[0377] The CR011 bi-scFv's (see FIG. 17) of this work were designed to bind to a CD3 epitope of the T cell receptor on cytotoxic human T lymphocytes and, at the same time, to target diseased cells expressing GPNMB, with the net result of facilitating the lysis or destruction of the diseased cells.

[0378] The V.sub.L and V.sub.H domains of mAb CR011, clone 1.15 were used in the construction of 3 CR011 based engineered antibodies:

(1) CR011 single chain antibody (CR011 scFv) (2) CR011.times.anti-CD3 bispecific single chain antibody (bi-scFv), Linker set L4-L2-L4 (3) CR011.times.anti-CD3 bispecific single chain antibody (bi-scFv), Linker set L4-L4-L4

[0379] The components of the CR011 scFv protein were: Signal Peptide-V.sub.L (CR011)-Linker 4-V.sub.H (CR011)-Flag Tag. The components of the CR011.times.anti-CD3 bi-scFv (Linker set L4-L2-L4) protein were: Signal Peptide-V.sub.L (CR011)-Linker 4-V.sub.H (CR011)-Linker 2-V.sub.H (anti-CD3)-Linker 4-V.sub.L (anti-CD3)-Flag Tag. The components of the CR011.times.anti-CD3 bi-scFv (Linker set L4-L4-L4) protein were: Signal Peptide-V.sub.L (CR011)-Linker 4-V.sub.H (CR011)-Linker 4-V.sub.H (anti-CD3)-Linker 4-V.sub.L (anti-CD3)-Flag Tag.

[0380] The various DNA components outlined above were used to generate the three CR011 engineered antibody products. The DNA components were synthesized by Blue Heron and cloned into commercially available plasmid vectors by methods familiar to those skilled in the art. These plasmids were then used in PCRs to combine the components, indicated in the 3 examples above, to generate engineered antibody inserts for expression vectors. In the host expression system examples practicing this invention described below, we have used CHOK1 mammalian cells for the CR011 expression vectors, but expression is not limited to these cells; it will be recognized by those skilled in the art that the CR011 engineered antibodies of this invention can be expressed using other vectors, systems and cells, including but not limited to: pET vectors, inducible and constitutive promoters, and hosts may include E. coli, Bacillus species, yeast, including Pichia pastoris or insect cells. Other expression hosts can also include various plant species and transgenic animals such as goats.

[0381] SP (Signal Peptide): We incorporated a signal peptide in our constructs in order to express products that will be secreted. The signal peptide which was utilized for expression from CHO cells was derived from an immunoglobulin light chain leader peptide (Erik et al., 1986), or from the CR002 antibody (CuraGen).

[0382] Order of the bi-scFv components: The order of the antibody variable domains was fixed in both bi-scFv constructs as follows: V.sub.L1-L-V.sub.H1-L-V.sub.H2-L-V.sub.L2. Each of the 4 V domains was linked by a linker segment, L. V.sub.L1 and V.sub.H1 represent the immunoglobulin light and heavy chain variable domains respectively of CR011, and V.sub.H2 and V.sub.L2 represent the immunoglobulin heavy and light chain variable domains respectively of an anti-CD3 antibody that was used for both bi-scFv constructs. Other orders of the V domains can also be used for the 2 scFv components, as recognized by those skilled in the art, and the products evaluated for biological activity.

[0383] Tag: We used the 8 amino acid Flag tag at the C-terminus of the CR011 engineered antibodies to facilitate detection and purification of the products (Hickman et al., 2000).

[0384] Anti-CD3 scFv: The sequences of the V.sub.L and V.sub.H components of the anti-CD3 antibody used to generate the bi-scFv constructs may be found in the NCBI database under accession number CAE85148 (Lutterbuese et al.)

[0385] Linkers used in constructs: The sequence of L2, a short 5 amino acid linker that links the 2 monomer scFv components together in CR011.times.anti-CD3 bi-scFv (L4-L2-L4 linker set) is G.sub.4S (Mack et al., 1995). L4 is a 25 amino acid linker based on the 205C linker (Denzin et al., 1991): LSADDAKKDAAKKDDAKKDDAKKDL (SEQ ID NO: 344) and is used in both of the CR011 bi-scFv species to link the CR011 V.sub.L and V.sub.H and the anti-CD3 V.sub.H and V.sub.L. In the case of the CR011.times.anti-CD3 bi-scFv (L4-L4-L4 linker set), L4 is also used to link the 2 monomer scFv components together. For the CR011 scFv, the L4 linker was used to link the two variable domains together.

1. DNA Plasmid Constructs for Expression of CR011 scFv and CR011.times.Anti-CD3 Bi-scFv Species

[0386] CR011 scFv Flag tag: The PCR amplification product for generating the expression construct for CR011 scFv was generated from a synthetic DNA template (Blue Heron) using the F1/R1 primers followed by nested PCR with the F1 nested/R1 primer pair (Table 55) and Pfu Turbo DNA polymerase (Stratagene, cat#600322), as per the manufacturer's directions. A Sal I/EcoR I PCR fragment coding for the CR011 scFv cassette was cloned into the corresponding restriction sites of the pCTN vector (CuraGen Corporation, mammalian expression vector) using the Fast-Link DNA Ligation kit (Epicentre, cat# LK11025).

TABLE-US-00128 TABLE 55 Name Sequence F1 Primer 5'- TCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGTGAAATAGTGATGACGCA GTC (SEQ ID NO: 345) R1 Primer 5'- CCGGAATTCTTACTATTTGTCATCATCGTCCTTATAATCGCTAGCTGAGGAGACGGT (SEQ ID NO: 346) F1 Nested 5'- Primer ACGCGTCGACCCACCATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGG CTC (SEQ ID NO: 347) F2 Primer 5'- TCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGTGAAATAGTGATGACGCA GTC (SEQ ID NO: 348) R2 Primer 5'- CCGGAATTCTTACTATTTGTCATCATCGTCCTTATAATCGCTAGCTTTCAGCTCCAG (SEQ ID NO: 349) F2 Nested 5'- Primer ACGCGTCGACCCACCATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGG CTC (SEQ ID NO: 350) F3 Primer 5'- ACTCTGGCTCCCAGATACCACCGGAGAAATAGTGATGACGCAGTCTCCAGCCACC (SEQ ID NO: 351) R3 Primer 5'-CCGCTCGAGCTATTTGTCATCATCGTCCTTATAATCTTTCAGCTCCAGCTT (SEQ ID NO: 352) F3 Nested 5'- Primer TCTTCGCGACCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGC TCCCAGATACCACCGGA (SEQ ID NO: 353)

[0387] CR011.times.anti-CD3 bi-scFv (L4-L2-L4) linker set Flag tag: The PCR amplification product for the CR011.times.anti-CD3 bi-scFv having the (L4-L2-L4) linker set, was generated from a synthetic DNA template (Blue Heron) using the F2/R2 primers followed by nested PCR with the F2 nested/R2 primer pair (see Table 55 for sequences of oligonucleotides) and Pfu Turbo DNA polymerase (Stratagene, cat#600322), as per the manufacturer's directions. The Sal I/EcoR I PCR fragment having the coding sequence for the CR011.times.anti-CD3 (L4-L2-L4) bi-scFv was cloned into the corresponding sites of the pCTN vector using Fast-Link DNA Ligation kit (Epicentre, cat# LK11025).

[0388] CR011.times.anti-CD3 bi-scFv (L4-L4-L4) linker set Flag tag: The PCR amplification product for the CR011.times.anti-CD3 bi-scFv having the (L4-L4-L4) linker set, was generated from a synthetic DNA template (Blue Heron) using the F3/R3 primers followed by nested PCR with the F3 nested/R3 primer pair (Table 55) and Pfu Turbo DNA polymerase (Stratagene, cat#600322), as per the manufacturer's directions. The Nru I/Xho I PCR fragment having the coding sequence for the CR011.times.anti-CD3 (L4-L4-L4 linker set) bi-scFv was cloned into the corresponding sites of the pEE14.4FL2 expression vector (Lonza Biologics plc, 228 Bath Road, Slough, Berkshire SL1 4Dx, UK) using the Fast-Link DNA Ligation kit (Epicentre, cat# LK11025).

[0389] The DNA sequences of the above 3 expression construct inserts were verified by sequencing both strands of the relevant DNA products.

2. Protein Production of the CR011 Engineered Antibodies in CHOK1 Cells

[0390] Adherent Chinese Hamster Ovary (CHOK1) cells (ATCC catalog# CCL-61) were grown in DMEM media (Invitrogen, cat#10564-011) supplemented with 10% fetal bovine serum (Gemini, cat#100106), GS supplement (JRH Biosciences, cat#58672-100M) and 50 mg/L gentamicin (Invitrogen, cat#15750078).

[0391] CHOK1 cells were transfected with FuGENE 6 reagent (Roche, cat #1815075) according to the manufacturer's directions. Expression and secretion was verified by Western blotting performed ca. 48 hours after the transfections. Selection of stable secreted CR011 scFv and CR011.times.anti-CD3 bi-scFv (L4-L2-L4 linker set) lines were performed in selection media A (Table 56), while selection of a stable secreted CR011.times.anti-CD3 bi-scFv (L4-L4-L4 linker set) line was performed in selection media B (Table 57).

TABLE-US-00129 TABLE 56 CHOK1 (Adherent) Selection Media A Vendor Item No. Description DMEM-glutamine free JRH 51435-1000M Glutamine-Free Media for GS Biosciences System(TM) (DMEM/High Modified) 10% dialyzed FBS (heat JRH 12117-500M Fetal Bovine Serum, Dialyzed inactivated 56.degree. C. for 30 Biosciences (500 mL) minutes) 1X GS Supplement JRH 58672-100M GS Supplement (50X) Biosciences 50 mg/L gentamicin Invitrogen 15750078 Gentamicin Reagent Solution (50 mg/mL), liquid 1 mg/mL G418 Invitrogen 10131027 Geneticin (G418)

TABLE-US-00130 TABLE 57 CHOK1 (Adherent) Selection Media B Vendor Item No. Description DMEM-glutamine free JRH 51435-1000M Glutamine-Free Media for GS Biosciences System(TM) (DMEM/High Modified) 10% dialyzed FBS (heat JRH 12117-500M Fetal Bovine Serum, Dialyzed inactivated 56.degree. C. for 30 Biosciences (500 mL) minutes) 1X GS Supplement JRH 58672-100M GS Supplement (50X) Biosciences 50 mg/L gentamicin Invitrogen 15750078 Gentamicin Reagent Solution (50 mg/mL), liquid 25 .mu.M MSX Sigma M 5379 L-Methionine sulfoximine (MSX)

[0392] In each case, 8 out of 96 CR011 scFv and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv CHOK1 clones that were secreting products were expanded and archived. The best stable clones secreting products in each case were adapted to suspension culture in shake flasks with selection media C (Table 58) at 37.degree. C. and 5% CO.sub.2. Protein production for CR011 scFv and CR011.times.CD3 (L4-L2-L4 linker set) bi-scFv was carried out in 4 L of selection media D (Table 59) at 30.degree. C. and 5% CO.sub.2.

TABLE-US-00131 TABLE 58 CHOK1 Large Scale (Suspension) Selection Media C Vendor Item number Description Ex-Cell 302 JRH Biosciences 14324-1000M Ex-Cell 302 CHO Serum- free medium without L- glutamine (1000 mL) 5% FBS JRH Biosciences 12117-500M Fetal Bovine Serum, Dialyzed (500 mL) GS Supplement JRH Biosciences 58672-100M GS Supplement (50X) HT Supplement Invitrogen 11067030 HT Supplement (100X) 1 mg/mL G418 Invitrogen 10131027 Geneticin (G418)

TABLE-US-00132 TABLE 59 CHOK1 Large Scale (Suspension) Selection Media D Vendor Item number Description Ex-Cell 302 + Ex-Cell JRH 14324-1000M Ex-Cell 302 CHO Serum-free CD CHO (1:1) Biosciences medium without L-glutamine (1000 mL) JRH 14360-1000M CD CHO Medium (1000 mL) Biosciences 5% FBS JRH 12117-500M Fetal Bovine Serum, Dialyzed Biosciences (500 mL) GS Supplement JRH 58672-100M GS Supplement (50X) Biosciences HT Supplement Invitrogen 11067030 HT Supplement (100X) 1 mg/mL G418 Invitrogen 10131027 Geneticin (G418)

[0393] Only one out of two hundred CR011.times.anti-CD3 (L4-L4-L4 linker set) bi-scFv CHOK1 clones was found to produce a secreted product; this clone was expanded and archived. Protein production for the CR011.times.anti-CD3 (L4-L4-L4 linker set) clone was carried out using a cell factory apparatus (Nunc, cat#164327), in selection media B (Table 57), 1 mM sodium butyrate (Sigma, cat# B5887) at 37.degree. C. and 10% CO.sub.2.

3. Protein Purification of the CR011 Engineered Antibodies

[0394] Protein purification for the CR011 scFv Flag and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv Flag was accomplished in three chromatography steps, including affinity, ion exchange and size exclusion chromatographies. For the purification of CR011.times.anti-CD3 (L4-L4-L4 linker set) bi-scFv Flag protein, affinity and size exclusion chromatographies were used.

[0395] Affinity chromatography was performed using anti-FLAG M2 affinity gel (Sigma, cat# A2220-25 mL) as per the manufacturer's instructions on a BioCAD 700E instrument (Applied Biosystems). Ion exchange chromatography was performed on a MonoQ 5/50 GL column (Amersham, cat#17-5166-01) using 20 mM Tris-HCl pH7.5 as equilibration buffer and a gradient elution with 0-1 M NaCl. Size exclusion chromatography was performed using a Superdex 75/10/300 GL column (Amersham, cat#17-5174-01) following the manufacturer's protocols on BioCAD 700E (Applied Biosystems) liquid chromatography instrument.

[0396] The approximate yields from 1 L conditioned CHOK1 media were:

[0397] (1) CR011 scFv: 1.0 mg

[0398] (2) CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv: 0.5 mg

[0399] (3) CR011.times.anti-CD3 (L4-L4-L4 linker set) bi-scFv: 1.5 mg

[0400] The N-terminal amino acid sequence of the purified proteins was determined by Edman degradation, using methods known to those skilled in the art. The sequence of the first five amino acids was: E I V M T in each case (the mature N-terminus of the CR011 V.sub.L protein), indicating that accurate processing by signal peptidase had occurred to give a soluble, secreted product of the predicted sequence and size.

[0401] The DNA and amino acid sequences of the 3 CR011 engineered products are given below.

SEQ ID for CR011 scFv-(V.sub.L-L4-V.sub.H) Flag. The Signal peptide of Human kappa light chain was used as described in Kabat et al. 45 CLL-CL). There was a FLAG tag included at the C-terminus. The Kozak sequence CCACC was included in the 5' PCR primer.

TABLE-US-00133 (SEQ ID NO: 354) ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGTGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGAC AACAACTTAGTCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTG GCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGTAGTCTGCAGTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGTG GACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACTTTCCGCGGACGATG CGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAGAAAGACGATGCTAAA AAGGACCTGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC TTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCA GTTTTAATTACTACTGGAGCTGGATCCGCCACCACCCAGGGAAGGGCCTG GAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTCCAACCCGTC CCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCT CCCTGACGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT GCGAGAGGGTATAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCAGCTAGCGATTATAAGGACGATGATGACAAATAGT AA (SEQ ID NO: 355) MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVD NNLVWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPPWTFGQGTKVEIKLSADDAKKDAAKKDDAKKDDAK KDLQVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGL EWIGYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYC ARGYNWNYFDYWGQGTLVTVSSASDYKDDDDK

SEQ ID for CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv--The Signal peptide of Human kappa light chain was used as described in Kabat et al. 45 CLL-CL). There was a FLAG tag included at the C-terminus

TABLE-US-00134 (SEQ ID NO: 356) ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGTGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGAC AACAACTTAGTCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTG GCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGTAGTCTGCAGTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGTG GACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACTTTCCGCGGACGATG CGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAGAAAGACGATGCTAAA AAGGACCTGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC TTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCA GTTTTAATTACTACTGGAGCTGGATCCGCCACCACCCAGGGAAGGGCCTG GAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTCCAACCCGTC CCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCT CCCTGACGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT GCGAGAGGGTATAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCAGGAGGTGGTGGATCCGATATCAAACTGCAGCAGT CAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAG ACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAG GCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTT ATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGAC AAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGA CTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTG ACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCACTTTCCGCGGAC GATGCGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAGAAAGACGATGC TAAAAAGGACCTGGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTG CATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTA AGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATG GATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTG GCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCT GAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCAC GTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGCTAGCGATTATAAGGACG ATGATGACAAATAGTAA (SEQ ID NO: 357) MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVD NNLVWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPPWTFGQGTKVEIKLSADDAKKDAAKKDDAKKDDAK KDLQVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGL EWIGYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYC ARGYNWNYFDYWGQGTLVTVSSGGGGSDIKLQQSGAELARPGASVKMSCK TSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTD KSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSLSAD DAKKDAAKKDDAKKDDAKKDLDIQLTQSPAIMSASPGEKVTMTCRASSSV SYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEA EDAATYYCQQWSSNPLTFGAGTKLELKASDYKDDDDK

SEQ ID for CR011.times.anti-CD3 (L4-L4-L4 linker set) bi-scFv--The Signal peptide of CR002 was used. There was a FLAG tag included at the C-terminus.

TABLE-US-00135 (SEQ ID NO: 358) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGAGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGAC AACAACTTAGTCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTG GCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGTAGTCTGCAGTCT GAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCGTG GACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACTTTCCGCGGACGATG CGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAGAAAGACGATGCTAAA AAGGACCTGCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCC TTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCA GTTTTAATTACTACTGGAGCTGGATCCGCCACCACCCAGGGAAGGGCCTG GAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTCCAACCCGTC CCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCT CCCTGACGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT GCGAGAGGGTATAACTGGAACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCATTATCAGCGGATGACGCCAAGAAAGACGCAGCCA AAAAGGACGATGCAAAGAAGGATGACGCAAAGAAAGATTTAGATATCAAA CTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGAT GTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCT AGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATT GACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGA CATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCAT TACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCACT TTCCGCGGACGATGCGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAGA AAGACGATGCTAAAAAGGACCTGGACATTCAGCTGACCCAGTCTCCAGCA ATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAG TTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCC CCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTAT CGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAG CATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTA ACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGATTATAAG GACGATGATGACAAATAGCTCGAGCGG (SEQ ID NO: 359) METPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVD NNLVWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWPPWTFGQGTKVEIKLSADDAKKDAAKKDDAKKDDAK KDLQVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGL EWIGYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYC ARGYNWNYFDYWGQGTLVTVSSLSADDAKKDAAKKDDAKKDDAKKDLDIK LQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINP SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDH YCLDYWGQGTTLTVSSLSADDAKKDAAKKDDAKKDDAKKDLDIQLTQSPA IMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPY RFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKDYK DDDDK

4. Testing of the 3 CR011 Engineered Antibodies by ELISA, Flow Cytometry and Determination of Cytotoxicity:

[0402] ELISA: The binding of the CR011 engineered antibodies to purified recombinant GPNMB (2 .mu.g/mL) was measured using plates coated overnight at 4.degree. C. Plates were then blocked and washed. Various dilutions of the CR011 engineered antibodies were added into the wells. Plates were incubated for 1 h and washed. HRP-conjugated anti-FLAG M2 mAb (Sigma, St. Louis, Mo.) was added into the wells for 1 h, washed and the reaction developed with the TMB substrate reagent as described by the manufacturer (Pharmingen, San Jose, Calif.).

[0403] Binding of the CR011 scFv and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv product to GPNMB was first confirmed using ELISA, as shown in FIG. 18A. Plates were coated with human GPNMB protein tagged with His and V5. Coated plates were incubated with either supernatants containing CR011.times.anti-CD3 bi-scFv or purified CR011 scFv monomer. Binding of the recombinant mAbs (both monomer and dimer) was detected using anti-FLAG-HRP conjugated mAb M2 (Sigma). As can be seen in FIG. 18B, both anti-GPNMB antibody species described bind to the recombinant GPNMB protein, indicating that the specificity and binding activity of the engineered anti-GPNMB antibody, using the methods described in this example, was preserved.

[0404] Flow cytometry: The binding of the CR011 engineered antibodies to native proteins was analyzed by FACS. Briefly, human T cells and SK-Mel-5 cells were incubated with either the CR011 scFv or CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv (5 .mu.g/sample/100 .mu.l) with subsequent staining with mouse anti-FLAG mAb (Sigma) and PE-conjugated goat anti-mouse Ig F(ab').sub.2. (Jackson ImmunoResearch, West Grove, Pa.) Ten thousand events were collected and analyzed on a FACSCalibur instrument (Becton Dickinson, Mountain View, Calif.).

[0405] To confirm binding of the CR011 scFv and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv products to native GPNMB protein expressed on the cell surface, we used SK-Mel-5 cells which naturally express GPNMB. To verify binding of the bi-scFv to human CD3 molecules, we used purified human T cells. As a positive control we used native PE conjugated anti-CD3-PE and CR011 mAb. Binding of the CR011 scFv was detected using anti-FLAG mAb M2 with subsequent staining with PE conjugated anti mouse IgG, while for detection of CR011 mAb binding we used anti-human IgG-PE. Control anti-CD3 mAb bound to T cells, and control anti-GPNMB mAb bound to SK-Mel-5 tumor cells. We found that only the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv stained T cells; the CR011 scFv monomer did not bind CD3 positive T cells, as expected (see FIG. 19C (bottom panel)). Binding to SK-Mel-5 cells by either the CR011 scFv monomer or CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv was present at a low level (FIG. 19D (bottom panel)).

[0406] Cytotoxicity: The ability of CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv to redirect human T lymphocytes to kill relevant human tumor cells was measured by flow cytometry. Tumor cells were labeled with PKH2 green fluorescent linker kit (Sigma) and washed. Purified T cells were cultured O/N with PKH2-labeled tumor cells in the presence or absence of purified bi-scFv. Death of GPNMB positive tumor cells was measured by propidium iodine (PI) incorporation.

[0407] To evaluate the ability of the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv product to increase T cell mediated killing of GPNMB positive cells, we performed a cytotoxicity test. Purified T cells were cultured O/N with PKH2-labeled SK-Mel-S (GPNMB positive) tumor cells in the presence of various doses of purified CR011 scFv and CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv products.

Conclusion:

[0408] The CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv significantly increased killing of SK-Mel-S tumor cells by T lymphocytes (FIG. 20). In contrast, the addition of monospecific anti-GPNMB scFv did not increase killing of SK-Mel-S tumors. In addition, no cytotoxicity was observed when the tumor cells were cultured with the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv without T lymphocytes (FIG. 20). These data indicate that the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv provided sufficient bridging between T cells and SK-Mel-5 cells to induce cell death and that both components of this engineered CR011 bi-specific antibody were biologically active. Therefore the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv engineered antibody of the present invention may be used as a therapeutic to treat diseases, such as melanoma and other cancers where there are upregulated levels of GPNMB and T cells present.

[0409] Other methods of cytotoxicity analysis, including fluorescence and chromium release assays can be used to demonstrate the usefulness of the CR011.times.anti-CD3 (L4-L2-L4 linker set) bi-scFv in treating tumors. Other linkers may also be used to link the two scFv monomer components together, as in the CR011.times.anti-CD3 (linker set L4-L4-L4) molecule described supra.

Example 29

Optimized Production Process of CR011-vcMMAE

[0410] CR011AE is an antibody-drug conjugate composed of the anti-GPNMB (CG56972) fully human antibody CR011 conjugated with the toxin Auristatin E through a protease-cleavable linker. The toxin-to-antibody ratio is approx. 4.0 but may vary between 3.5 and 4.2. While the CR011 antibody is IgG2, it is therefore possible to append up to 12 toxin molecules per antibody molecule using the free thiols as a reactive site.

[0411] The structure of Maleimidocoaproyl-Valine-Citrullin-Monomethyl-Auristatin E (vcMMAE) is shown in FIG. 21.

[0412] Conjugation: A process of generating the drug-substance consisting of CR011 mAb with VCMMAE attached. An overview of the conjugation process is summarized in FIG. 22.

[0413] Briefly, the conjugation process for CR011 fully human antibody consists of the following 4 steps. 1) Buffer exchange and sucrose removal by diafiltration, 2) Disulfides reduction, 3) Conjugation to vcMMAE and finally, 4) Purification of conjugated CR011-vcMMAE by diafiltration. There are several assays throughout the process, i.e. in-process assays, which include Ellman's assay and determination of protein concentration. At the end of the process, the drug substance, i.e. the conjugate, is analyzed for drug-to-antibody ratio, free drug content and protein concentration.

[0414] Diafiltration of the bulk antibody: The bulk antibody originally formulated in phosphate pH 7-10% sucrose was buffer exchanged into the conjugation buffer (borate pH 9.0-NaCl) by diafiltration over 10 diavolumes. At the end of diafiltration, CR011 was diluted to .about.5.5 mg/mL and filtered through a set of two filters consisting of 1.2 and 0.22 .mu.m. The buffer exchange is required because sucrose interferes with reduction. In addition, high pH improves CR011 solubility.

[0415] CR011 reduction--General considerations: CR011 is produced as an IgG2 isotype product and contains 6 disulfide bridges in the hinge region. These disulfides can be reduced under mild conditions to give rise to 12 cysteine residues. Therefore, it is possible to maximally attach 12 vcMMAE drug molecules per antibody. For the process, however, the bulk antibody is only partially reduced because the aim is to generate conjugates with an average of 4 vcMMAE molecules. The reason for this is two-fold. First, it broadens the therapeutic window by decreasing potential systemic toxicity associated with MMAE. Second, it is difficult and sometimes impossible to produce fully-loaded conjugates with low aggregation because of greatly reduced solubility imparted by the hydrophobic drug.

[0416] Process: Tris-(carboxyethyl)-phosphine or TCEP was added at the 4:1 molar ratio (TCEP:mAb) to CR011 at a concentration of .about.5.5 mg/mL in the jacketed reactor equipped with an agitator set to 90 RPM. The reaction was allowed to proceed for 3 hours at 37.degree. C. in the presence of 1 mM EDTA. At the end, Ellman's assay was used to determine the amount of free thiols. Typically, it was 4.2 thiols per antibody. The reactor was then chilled to 4.degree. C.

[0417] CR011 conjugation--General considerations: TCEP was not fully consumed during the reduction. The left over TCEP was capable of reacting with vcMMAE. However, this spurious side reaction was slower compared to the conjugation reaction and can be mitigated by adding an excess of vcMMAE. The advantage of TCEP compared to DTT is that it does not require removal of the left-over reducing agent.

[0418] Process: vcMMAE was dissolved in DMSO and added at 20% molar excess to the reduced CR011 mAb. The reaction was allowed to proceed for 1 hour. The final concentration of DMSO is 4% (v/v). DMSO played a dual purpose in the process. It is required for solubilizing the drug and also it helps to solubilize the conjugate. At the end of conjugation, N-acetylcysteine was added to quench any unreacted drug.

[0419] CR011-vcMMAE purification: The temperature in the reactor was brought to room temperature. A 40% sucrose stock solution was used to adjust the final sucrose concentration to 10% (w/v) followed by a pH adjustment using 300 mM histidine HCl pH 5.0 buffer to a final pH of 6.0. The conjugate was then purified by diafiltration into 20 mM histidine pH 6.0-10% sucrose (w/v) buffer and using 10 diavolumes. At the end of diafiltration, the conjugate was concentrated to .about.7 mg/mL and filtered through a set of three filters consisting of 1.2, 0.45 and finally, 0.22 .mu.m.

[0420] CR011-vcMMAE formulation: The conjugate was formulated by adding Tween-20 to a final concentration of 0.02% and by diluting to 6 mg/mL (.+-.10%) using formulation buffer (20 mM histidine pH 6.0, 10% sucrose, 0.02% Tween-20). The conjugate was then stored at 4.degree. C. until pooling if more than one lot is being manufactured (a.k.a. staging time). After pooling, the final concentration was adjusted to 5.0 mg/mL (.+-.5%) and the drug substance was stored frozen.

1. Pre-Conjugation UF/DF: Removal of Sucrose

[0421] Experiments were performed to monitor the rate of removal of sucrose during UF/DF by Ellman's assay; and estimate the diavolumes needed to achieve the highest SH-per-Ab ratio.

[0422] It was found that it is desirable to conduct at least 6 diavolumes in order to remove sucrose to a level that does not impede CR011 reduction. To ensure robustness, at least 10 diavolumes should be utilized during the process.

2. The Effect of Percent DMSO on Aggregation in the Conjugation Reaction

[0423] Experiments were performed to determine the effect of DMSO in the conjugation reaction on: (1) aggregation; and (2) drug:Ab molar ratio (i.e. completeness of conjugation).

[0424] It was found that the percent aggregate in reaction with 12% DMSO was lower than in 15% DMSO, 4.4 and 3.0%, respectively. Formulation pH 9.0 buffer vs. pH 7.0 buffer did not have any effect on aggregation or yield, provided 10% sucrose was included in formulation. The percent aggregate in the 10%, 8%, 6%, and 4% (v/v) DMSO reactions were 2.7, 1.7, 1.0 and 0.5%, respectively. This suggests that CR011 and CR011 AE were very susceptible to aggregation when a higher percentage of DMSO is present.

[0425] All four conjugation reactions resulted in a final molar ratio of 4.0 drugs/Ab, suggesting that all four reactions went to completion. Safety margins for DMSO percentage in the conjugation reaction are 4-6%. This predicted to yield an aggregation level of 1% or less.

5. Investigation of Side Reaction During Conjugation of CR011 to vcMMAE

[0426] Experiments were performed to: (1) investigate the extent and the kinetics of the side reaction in which maleimide-drug is converted into an unreactive side product, which results in an incomplete conjugation and low drug-loading; (2) determine factors that influence the side reaction; and (3) determine whether the old vcMMAE lot (SGD1006-0-04) differed in reactivity compared to the new lot (SGD1006-0-06).

[0427] Reactions (100 .mu.l) containing vcMMAE at 30 .mu.M final concentration, were incubated in borate pH 9.0 buffer either in the absence or in the presence of 2-fold molar excess of TCEP (with respect to vcMMAE). The reactions were quenched at 0, 2, 7 or 15 min with excess NAcCys. The control consisted of vcMMAE in phosphate pH 7.0 buffer quenched at the 15 min time point. The chromatograms are shown on FIGS. 23A, 23E, and 23I.

[0428] In pH 7 phosphate buffer 15 min and in pH 9.0 borate buffer 0 min after addition of the drug a single Cys-quenched product with (rt=9.0 min) is formed (Compare A and B). In borate buffer pH 9.0 an unreactive side product is formed (rt=9.2 min) in a time-dependent fashion (B, C, D and E). In borate buffer and in the presence of TCEP (such as CR011 conjugation conditions), formation of the unreactive product is catalyzed resulting in >90% conversion of maleimide into succinimide after only 2 min of incubation (F through I). Both the old vcMMAE lot (SGD-1006-0-06) and the new lot (SGD-1006-0-04) exhibited similar reactivity towards high pH and TCEP, as well as similar kinetics.

[0429] FIG. 24 shows the LC-MS identification of the unreactive product as succinimidyl-VCMMAE (rt=9.2 min, m/z=1318). FIG. 25 shows the relative kinetics of formation of the succinimide in the presence or absence of TCEP.

Conclusions

[0430] The side product is a result of conjugation performed at pH 9 instead of pH 7.4 (PBS). Formation of the side product is greatly enhanced in the presence of TCEP. The major stable side product has been identified by LC-MS as succinimidyl-vcMMAE. Minor and less stable side-products remain to be identified. Both vcMMAE lots behaved similarly.

6. Overcoming the Side Reaction During Conjugation of CR011 to vcMMAE

[0431] Experiments were performed to investigate whether the side reaction can be overcome by providing a larger excess of the drug for conjugation.

[0432] Several ways to suppress the side reaction were proposed: (1) Conducting conjugation at lower pH, e.g. 8.5 instead of 9.0 (high risk due to reduced solubility of CR011); (2) Removal of the excess of TCEP by UF/DF (not practical); and (3) Elevation of the excess of VCMMAE added upfront (practical).

[0433] 100 mg of CR011 that was previously buffer exchanged into 50 mM borate-50 mM NaCl, was reduced with TCEP to generate 4.35 free thiols per Ab. The reaction was divided into two halves. For the first 50 mg half, a 10% excess of VCMMAE was added based on the 1 drug per thiol ratio. For the second half, a 20% excess was used. The conjugates were purified by UF/DF into 10 mM Histidine pH 6/10% sucrose solution. The results are summarized in Table 60.

TABLE-US-00136 TABLE 60 Preparation of CR011-VCMMAE conjugates using various excess of vcMMAE based on the 1 drug per thiol ratio. Drug-to-Ab ratios were determined by RP HPLC. VCMMAE Excess, % 10 20 SH per Ab 4.35 4.35 Drug-to-Ab ratio (in reaction) 3.9 4.1 Drug-to-Ab ratio (in final product) 3.7 4.0

Conclusions

[0434] Using 10% versus 20% excess of vcMMAE was compared in a 100 mg conjugation. The higher excess of vcMMAE afforded a drug-to-Ab ratio closer to the expected value, and therefore, has been determined to be optimal.

EQUIVALENTS

[0435] The foregoing description and Examples detail certain preferred embodiments of the antibodies and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the methods of making and using the antibodies described herein may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments described herein.

Sequence CWU 1

1

3951347DNAHomo sapiens 1aggtgcagct gcaggagtcg ggcccaggac tggtgaagcc ttcggagacc ctgtccctca 60cctgcactgt ctctggtgac tccatcagta attactactg gagctggatc cggcagcccc 120cagggaaggg actggagtgg attgggtatt tctattacag tgggagcacc aactacaacc 180cctccctcaa gagtcgagtc accatatcag tagacacgtc caagaaccag ttctccctga 240aactgagctc tgtgaccgct gcggacacgg ccgtgtatta ctgtgcgaga gataggggct 300gggctgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcc 3472116PRTHomo sapiens 2Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Gly Trp Ala Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 325PRTHomo sapiens 3Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 410PRTHomo sapiens 4Gly Asp Ser Ile Ser Asn Tyr Tyr Trp Ser 1 5 10 514PRTHomo sapiens 5Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 616PRTHomo sapiens 6Tyr Phe Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 732PRTHomo sapiens 7Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 87PRTHomo sapiens 8Asp Arg Gly Trp Ala Asp Tyr 1 5 912PRTHomo sapiens 9Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 10324DNAHomo sapiens 10gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagggccacc 60ctctcctgca gaaccagtca gagtattagc agcagctatt tagcctggta ccagcagaaa 120cctggccagg ttcccaggct cctcatctat ggtgcttcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttattgtcag cagtatggta gctcgatcac cttcggccaa 300gggacacgac tggagattaa acga 32411108PRTHomo sapiens 11Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Ile Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 1223PRTHomo sapiens 12Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 1312PRTHomo sapiens 13Arg Thr Ser Gln Ser Ile Ser Ser Ser Tyr Leu Ala 1 5 10 1415PRTHomo sapiens 14Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg Leu Leu Ile Tyr 1 5 10 15 157PRTHomo sapiens 15Gly Ala Ser Ser Arg Ala Thr 1 5 1632PRTHomo sapiens 16Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 178PRTHomo sapiens 17Gln Gln Tyr Gly Ser Ser Ile Thr 1 5 1811PRTHomo sapiens 18Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 1 5 10 19360DNAHomo sapiens 19caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agttttaatt actactggag ctggatccgc 120caccacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180tccaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240tccctgacgc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagaggg 300tataactgga actactttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc 36020120PRTHomo sapiens 20Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Phe 20 25 30 Asn Tyr Tyr Trp Ser Trp Ile Arg His His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Ser Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115 120 2130PRTHomo sapiens 21Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 227PRTHomo sapiens 22Ser Phe Asn Tyr Tyr Trp Ser 1 5 2314PRTHomo sapiens 23Trp Ile Arg His His Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 2416PRTHomo sapiens 24Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Ser Asn Pro Ser Leu Lys Ser 1 5 10 15 2532PRTHomo sapiens 25Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Thr 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 269PRTHomo sapiens 26Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr 1 5 2712PRTHomo sapiens 27Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 28327DNAHomo sapiens 28gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttgac aacaacttag tctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagtag tctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 300caagggacca aggtggaaat caaacga 32729109PRTHomo sapiens 29Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asp Asn Asn 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 3023PRTHomo sapiens 30Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 3111PRTHomo sapiens 31Arg Ala Ser Gln Ser Val Asp Asn Asn Leu Val 1 5 10 3215PRTHomo sapiens 32Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 337PRTHomo sapiens 33Gly Ala Ser Thr Arg Ala Thr 1 5 3432PRTHomo sapiens 34Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 3510PRTHomo sapiens 35Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr 1 5 10 3611PRTHomo sapiens 36Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 37369DNAHomo sapiens 37atcaccttga aggagtctgg tcctacgctg gtgaaaccca cacagaccct cacgctgacc 60tgcaccttct ctgggttctc actcagcgct ggtggagtgg gtgtgggctg gatccgtcag 120cccccaggaa aggccctgga gtggcttgca ctcatttatt ggaatgatga taagcgctac 180agcccatctc tgaggagcag gctcaccatc accaaggaca cctccaaaaa ccaggtggtc 240cttacaatta ccaacatgga ccctgtggac acagccacat attattgtgc acacagtcac 300tatgattacg attgggggag ttactttgac tactggggcc agggaaccct ggtcaccgtc 360tcctcagcc 36938123PRTHomo sapiens 38Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Ala Gly Gly 20 25 30 Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp 35 40 45 Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu 50 55 60 Arg Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His Ser His Tyr Asp Tyr Asp Trp Gly Ser Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 3924PRTHomo sapiens 39Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Phe Ser 20 4012PRTHomo sapiens 40Gly Phe Ser Leu Ser Ala Gly Gly Val Gly Val Gly 1 5 10 4114PRTHomo sapiens 41Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 1 5 10 4216PRTHomo sapiens 42Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu Arg Ser 1 5 10 15 4332PRTHomo sapiens 43Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 1 5 10 15 Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala His 20 25 30 4413PRTHomo sapiens 44Ser His Tyr Asp Tyr Asp Trp Gly Ser Tyr Phe Asp Tyr 1 5 10 4512PRTHomo sapiens 45Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 46342DNAHomo sapiens 46gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcttg gatagtgatg atggaaacac ctatttggac 120tggtacctgc agaagccagg acagtctcca cagctcctga tctatacgct ttcctatcgg 180gcctctggag tcccagacag gttcagtggc agtgggtcag gcactgattt cacactgaac 240atcagcaggg tggaggctga ggatgttgga gtttattact gcatgcaacg tatagagttt 300cctatcacct tcggccaagg gacacgactg gagattaaac ga 34247114PRTHomo sapiens 47Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Ile Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg 4823PRTHomo sapiens 48Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys 20 4917PRTHomo sapiens 49Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu 1 5 10 15 Asp 5015PRTHomo sapiens 50Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 517PRTHomo sapiens 51Thr Leu Ser Tyr Arg Ala Ser 1 5 5232PRTHomo sapiens 52Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Asn Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 539PRTHomo sapiens 53Met Gln Arg Ile Glu Phe Pro Ile Thr 1 5 5411PRTHomo sapiens 54Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 1 5 10 55360DNAHomo sapiens 55caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtgctaatt actactggac ctggatccgc 120cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180tgcaacccgt ccctcaagag tcgagttatc atatcagtag acacgtctaa gaaccagttc 240tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagaggg 300tataactgga actactttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc 36056120PRTHomo sapiens 56Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ala 20 25 30 Asn Tyr Tyr Trp Thr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Cys Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115 120 5724PRTHomo sapiens 57Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val 20 5812PRTHomo sapiens 58Gly Gly Ser Ile Ser Ser Ala Asn Tyr Tyr Trp Thr 1 5 10 5914PRTHomo sapiens 59Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 6016PRTHomo sapiens 60Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Cys Asn Pro Ser Leu Lys Ser 1 5 10 15 6132PRTHomo sapiens 61Arg Val Ile Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 629PRTHomo sapiens 62Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr 1 5 6312PRTHomo sapiens 63Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 64330DNAHomo sapiens 64gatatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca ggagagacct 120ggccaggctc ccagactcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataagt ggcctccgtg gacgttcggc 300caagggacca aggtggaaat cgaacgaact 33065109PRTHomo sapiens 65Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Glu Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Lys Trp Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg 100 105 6623PRTHomo sapiens 66Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 6711PRTHomo sapiens 67Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 6815PRTHomo sapiens 68Trp Tyr Gln Glu Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 697PRTHomo sapiens 69Gly Ala Ser Thr Arg Ala Thr 1 5 7032PRTHomo sapiens 70Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 7110PRTHomo sapiens 71Gln Gln Tyr Asn Lys Trp Pro Pro Trp Thr 1 5 10 7211PRTHomo sapiens 72Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg 1 5 10 73378DNAHomo sapiens 73cagctggtgg agtctggggg aggcgtggtc cagcctggga ggtccctgag actctcctgt 60gcagcctctg gattcgcctt cagtagctat ggcatgcact gggtccgcca ggctccaggc 120aaggggctgg agtgggtggc agttatatca tatgatggaa ataataaata ctatgcagac 180tccgtgaagg gccgattcac catctccaga gacaattcca agaacacgct gtatctgcaa 240atgaacagcc tgagagctga ggacacggct gtgtattact gtgcgagaga tctagtggtt 300cggggaatta gggggtacta ctactacttc ggtatggacg tctggggcca agggaccacg 360gtcaccgtct cctcagcc 37874126PRTHomo sapiens 74Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Gly Met 20 25 30 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val 35 40 45 Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Asp Leu Val Val Arg Gly Ile Arg Gly Tyr Tyr Tyr Tyr Phe Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 7523PRTHomo sapiens 75Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser 20 7610PRTHomo sapiens 76Gly Phe Ala Phe Ser Ser Tyr Gly Met His 1 5 10 7714PRTHomo sapiens 77Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 7817PRTHomo sapiens 78Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 7932PRTHomo sapiens 79Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 8018PRTHomo sapiens 80Asp Leu Val Val Arg Gly Ile Arg Gly Tyr Tyr Tyr Tyr Phe Gly Met 1 5 10 15 Asp Val 8112PRTHomo sapiens 81Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 1 5 10 82339DNAHomo sapiens 82gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaaggtct acaaactccg 300atcaccttcg gccaagggac acgactggag attaaacga 33983113PRTHomo sapiens 83Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 Arg 8423PRTHomo sapiens 84Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys 20 8516PRTHomo sapiens 85Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 8615PRTHomo sapiens 86Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 877PRTHomo sapiens 87Leu Gly Ser Asn Arg Ala Ser 1 5 8832PRTHomo sapiens 88Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 899PRTHomo sapiens 89Met Gln Gly Leu Gln Thr Pro Ile Thr 1 5 9011PRTHomo sapiens 90Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 1 5 10 91366DNAHomo sapiens 91caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt aactatggca ttcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtttg atggacgtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac gcggctgtgt attactgtgc gagagatccc 300tttgactatg gtgactcctt ctttgactac tggggccagg gcaccctggt caccgtctcc 360tcagcc 36692122PRTHomo sapiens 92Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Phe Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Phe Asp Tyr Gly Asp Ser Phe Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 9325PRTHomo sapiens 93Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 9410PRTHomo sapiens 94Gly Phe Thr Phe Ser Asn Tyr Gly Ile His 1 5 10 9514PRTHomo sapiens 95Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 9617PRTHomo sapiens 96Val Ile Trp Phe Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 9732PRTHomo sapiens 97Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr Tyr Cys Ala Arg 20 25 30 9812PRTHomo sapiens 98Asp Pro Phe Asp Tyr Gly Asp Ser Phe Phe Asp Tyr 1 5 10 9912PRTHomo sapiens 99Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 100315DNAHomo sapiens 100ctgactcagt ctccatcctc cctgtctgca tctgtaagag acagagtcac catcacttgc 60cgggcgagtc aggacattag caattattta gcctggtatc agcagaaacc agggaaagtt 120cctaatctcc tgatctatgc tgcatccact ttgcaatcag gggtcccatc tcggttcagt 180ggcagtggat ctgggacaga tttcactctc accatcagca gcctgcagcc tgaagatgtt 240gcaacttatt actgtcaaaa gtataacagt gccccgctca ctttcggcgg agggaccaag 300gtggagatca aacga 315101105PRTHomo sapiens 101Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Arg Asp Arg Val 1 5 10 15 Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Lys Val Pro Asn Leu Leu Ile Tyr Ala Ala 35 40 45 Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 50 55 60 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val 65 70 75 80 Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Leu Thr Phe Gly 85 90 95 Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 10220PRTHomo sapiens 102Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Arg Asp Arg Val 1 5 10 15 Thr Ile Thr Cys 20 10311PRTHomo sapiens 103Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala 1 5 10 10415PRTHomo sapiens 104Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Asn Leu Leu Ile Tyr 1 5 10 15 1056PRTHomo sapiens 105Ala Ala Ser Thr Leu Gln 1 5 10632PRTHomo sapiens 106Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 20 25 30 1079PRTHomo sapiens 107Gln Lys Tyr Asn Ser Ala Pro Leu Thr 1 5 10811PRTHomo sapiens 108Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 109366DNAHomo sapiens 109caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt gactactaca tgacctggat ccgccaggct 120ccagggaagg ggctggagtg ggtttcatac attagtatta gtggtagtat cacacactac 180gcagactcag tgaagggccg attcaccatg tccagggaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagacgga 300gcagcagctg gtacggatgc ttttgatatc tggggccacg ggacaaaggt caccgtctct 360tcagcc 366110122PRTHomo sapiens 110Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ile Ser Gly Ser Ile Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ala Ala Ala Gly Thr Asp Ala Phe Asp Ile Trp Gly 100 105 110 His Gly Thr Lys Val Thr Val Ser Ser Ala 115 120 11125PRTHomo sapiens 111Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 11210PRTHomo sapiens 112Gly Phe Thr Phe Ser Asp Tyr Tyr Met Thr 1 5 10 11314PRTHomo sapiens 113Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 11417PRTHomo sapiens 114Tyr Ile Ser Ile Ser Gly Ser Ile Thr His Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 11532PRTHomo sapiens 115Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 11612PRTHomo sapiens 116Asp Gly Ala Ala Ala Gly Thr Asp Ala Phe Asp Ile 1 5 10 11712PRTHomo sapiens 117Trp Gly His Gly Thr Lys Val Thr Val Ser Ser Ala 1 5 10 118321DNAHomo sapiens 118gagatagtga tgacgcagtc tccagccacc ctatctgtgt ctccagggga cagagccacc 60ctctcctgca gggccagtca gaatgttagc agcaacttgg cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctttggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctacagtct 240gaagattttg cagtttatta ctgtcagcag tatcattact ggcccacttt cggccctggg 300accaaagtgg atatcaaacg a 321119107PRTHomo sapiens 119Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Phe Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Tyr Trp Pro Thr 85 90 95 Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 12023PRTHomo sapiens 120Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Ala Thr Leu Ser Cys 20 12111PRTHomo sapiens 121Arg Ala Ser Gln Asn Val Ser Ser Asn Leu Ala 1 5 10 12215PRTHomo sapiens 122Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Phe 1 5 10 15 1237PRTHomo sapiens 123Gly Ala Ser Thr Arg Ala Thr 1 5 12432PRTHomo sapiens 124Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 1258PRTHomo sapiens 125Gln Gln Tyr His Tyr Trp Pro Thr 1 5 12611PRTHomo sapiens 126Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 1 5 10 127360DNAHomo sapiens 127cagctggtgc agtctggggc tgaggtgaag aagcctgggg cctcagtgaa ggtctcctgc 60aaggcttctg gatacacctt caccggcttc tatatgcact gggtgcgaca gacccctgga 120caagggcttg agtggatggg atggatcaac cctaacagtg gtggcacata ttatgtacag 180aagtttcagg gcagggtcac catgaccagg gacacgtcca tcagcacagt ctacatggag 240ctgagcaggt tgagatctga cgacacggcc gtatattact gtgcgagaga tgggtatagc 300agtggagagg actggttcga cccctggggc cagggaaccc tggtcaccgt ctcctcagcc 360128120PRTHomo sapiens 128Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Phe Tyr Met 20 25 30 His Trp Val Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Asn Pro Asn Ser Gly Gly Thr Tyr Tyr Val Gln Lys Phe Gln Gly 50 55 60 Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Val Tyr Met Glu 65 70 75 80 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Asp Gly Tyr Ser Ser Gly Glu Asp Trp Phe Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115 120 12923PRTHomo sapiens 129Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 1 5 10

15 Lys Val Ser Cys Lys Ala Ser 20 13010PRTHomo sapiens 130Gly Tyr Thr Phe Thr Gly Phe Tyr Met His 1 5 10 13114PRTHomo sapiens 131Trp Val Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 13217PRTHomo sapiens 132Trp Ile Asn Pro Asn Ser Gly Gly Thr Tyr Tyr Val Gln Lys Phe Gln 1 5 10 15 Gly 13332PRTHomo sapiens 133Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Val Tyr Met Glu 1 5 10 15 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 13412PRTHomo sapiens 134Asp Gly Tyr Ser Ser Gly Glu Asp Trp Phe Asp Pro 1 5 10 13512PRTHomo sapiens 135Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 136339DNAHomo sapiens 136gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg catagtggtg gaaagaccta tttgtattgg 120tacctgcaga ggccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acaccttccg 300ctcactttcg gcggagggac caaggtggag atcaaacga 339137113PRTHomo sapiens 137Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile His Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 13823PRTHomo sapiens 138Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 13916PRTHomo sapiens 139Lys Ser Ser Gln Ser Leu Leu His Ser Gly Gly Lys Thr Tyr Leu Tyr 1 5 10 15 14015PRTHomo sapiens 140Trp Tyr Leu Gln Arg Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr 1 5 10 15 1417PRTHomo sapiens 141Glu Val Ser Asn Arg Phe Ser 1 5 14232PRTHomo sapiens 142Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 1439PRTHomo sapiens 143Met Gln Ser Ile His Leu Pro Leu Thr 1 5 14411PRTHomo sapiens 144Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 145360DNAHomo sapiens 145caggtgcagc tggagcagtc ggggggaggc ctggtcaagc ctggggggtc cctgagattc 60tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcattc attagtagta gtagtagtta catatactac 180gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaggac 300tgggtgggag ctacctttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc 360146120PRTHomo sapiens 146Gln Val Gln Leu Glu Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Phe Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Trp Val Gly Ala Thr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115 120 14725PRTHomo sapiens 147Gln Val Gln Leu Glu Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Phe Ser Cys Ala Ala Ser 20 25 14810PRTHomo sapiens 148Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5 10 14914PRTHomo sapiens 149Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15017PRTHomo sapiens 150Phe Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 15132PRTHomo sapiens 151Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 15210PRTHomo sapiens 152Glu Asp Trp Val Gly Ala Thr Phe Asp Tyr 1 5 10 15312PRTHomo sapiens 153Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 154324DNAHomo sapiens 154gacattcagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagg aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct gatctatgct gcttccgctt tgaaattagg ggtcccatct 180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgatcac cttcggccaa 300gggacacgac tggacattaa acga 324155108PRTHomo sapiens 155Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ala Leu Lys Leu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg 100 105 15623PRTHomo sapiens 156Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 15711PRTHomo sapiens 157Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala 1 5 10 15815PRTHomo sapiens 158Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr 1 5 10 15 1597PRTHomo sapiens 159Ala Ala Ser Ala Leu Lys Leu 1 5 16032PRTHomo sapiens 160Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 20 25 30 1619PRTHomo sapiens 161Gln Lys Tyr Asn Ser Ala Pro Ile Thr 1 5 16211PRTHomo sapiens 162Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg 1 5 10 163396DNAHomo sapiens 163caggtgcagc tggagcagtc gggcccagga ctggtgaagc cttcacagaa cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtggtggtt atttctggag ctggatccgc 120cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gaacacctac 180tacaacccgt ccctcaagag tcgagttacc atatcagttg acacgtctaa gaaccagttc 240tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagac 300tattactatg atactagtgg tttttcctac cgttacgact ggtactacgg tatggacgtc 360tggggccaag ggaccacggt caccgtctcc tcagcc 396164132PRTHomo sapiens 164Gln Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Asn Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Phe Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Tyr Tyr Asp Thr Ser Gly Phe Ser Tyr Arg Tyr 100 105 110 Asp Trp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser Ala 130 16525PRTHomo sapiens 165Gln Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Asn Leu Ser Leu Thr Cys Thr Val Ser 20 25 16612PRTHomo sapiens 166Gly Gly Ser Ile Ser Ser Gly Gly Tyr Phe Trp Ser 1 5 10 16714PRTHomo sapiens 167Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 16816PRTHomo sapiens 168Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 16932PRTHomo sapiens 169Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 17021PRTHomo sapiens 170Asp Tyr Tyr Tyr Asp Thr Ser Gly Phe Ser Tyr Arg Tyr Asp Trp Tyr 1 5 10 15 Tyr Gly Met Asp Val 20 17112PRTHomo sapiens 171Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 1 5 10 172321DNAHomo sapiens 172gacatccagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaaatgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacaa cataatactt acccggcgtt cggccaaggg 300accaaggtgg aaatcaaacg a 321173107PRTHomo sapiens 173Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Ala 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 17423PRTHomo sapiens 174Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 17511PRTHomo sapiens 175Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10 17615PRTHomo sapiens 176Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr 1 5 10 15 1777PRTHomo sapiens 177Ala Ala Ser Ser Leu Gln Asn 1 5 17832PRTHomo sapiens 178Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 1798PRTHomo sapiens 179Leu Gln His Asn Thr Tyr Pro Ala 1 5 18011PRTHomo sapiens 180Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 181354DNAHomo sapiens 181cagctggtgc agtctggagc agaagtgaaa aagcccgggg agtctctgaa gatctcctgt 60cagggttctg gatacatctt taccaactac tggatcggct gggtgcgcca gatgcccggg 120aaaggcctgg agtggatggg ggtcatctat cctgatgact ctgataccag atacagcccg 180tccttccaag gccaggtcac catctcagcc gacaagtcca tcagcaccgc ctacctgcag 240tggagcagcc tgaaggcctc ggacaccgcc atatattact gtgcgagaca aaaatggcta 300caacacccct ttgactactg gggccaggga accctggtca ccgtctcctc agcc 354182118PRTHomo sapiens 182Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu 1 5 10 15 Lys Ile Ser Cys Gln Gly Ser Gly Tyr Ile Phe Thr Asn Tyr Trp Ile 20 25 30 Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Val 35 40 45 Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly 50 55 60 Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln 65 70 75 80 Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys Ala Arg 85 90 95 Gln Lys Trp Leu Gln His Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala 115 18323PRTHomo sapiens 183Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu 1 5 10 15 Lys Ile Ser Cys Gln Gly Ser 20 18410PRTHomo sapiens 184Gly Tyr Ile Phe Thr Asn Tyr Trp Ile Gly 1 5 10 18514PRTHomo sapiens 185Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly 1 5 10 18617PRTHomo sapiens 186Val Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly 18732PRTHomo sapiens 187Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln 1 5 10 15 Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys Ala Arg 20 25 30 18810PRTHomo sapiens 188Gln Lys Trp Leu Gln His Pro Phe Asp Tyr 1 5 10 18912PRTHomo sapiens 189Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 190327DNAHomo sapiens 190gaaattgtgt tgacgcagtc accaggcacc ctgtctttgt ctccagggga aagagtcacc 60ctctcatgca gggccagtca gagtgttagc agcagatact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagttta ttactgtcag cagtatggta gctcacctcg gacgttcggc 300caagggacca aggtggaaat caaacga 327191109PRTHomo sapiens 191Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Arg 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 19223PRTHomo sapiens 192Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys 20 19312PRTHomo sapiens 193Arg Ala Ser Gln Ser Val Ser Ser Arg Tyr Leu Ala 1 5 10 19415PRTHomo sapiens 194Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 1957PRTHomo sapiens 195Gly Ala Ser Ser Arg Ala Thr 1 5 19631PRTHomo sapiens 196Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr 20

25 30 1979PRTHomo sapiens 197Gln Gln Tyr Gly Ser Ser Pro Arg Thr 1 5 19811PRTHomo sapiens 198Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 199369DNAHomo sapiens 199caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt ttcaggacag ggtcaccatg accagggaca cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagatttc 300tttggttcgg ggagtctcct ctactttgac tactggggcc agggaaccct ggtcaccgtc 360tcctcagcc 369200123PRTHomo sapiens 200Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Phe Phe Gly Ser Gly Ser Leu Leu Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 20125PRTHomo sapiens 201Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 20210PRTHomo sapiens 202Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His 1 5 10 20314PRTHomo sapiens 203Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 20417PRTHomo sapiens 204Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Asp 20532PRTHomo sapiens 205Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 20613PRTHomo sapiens 206Asp Phe Phe Gly Ser Gly Ser Leu Leu Tyr Phe Asp Tyr 1 5 10 20712PRTHomo sapiens 207Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 208339DNAHomo sapiens 208gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg catagtggtg gaaagaccta tttgtattgg 120tacctgcaga ggccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acaccttccg 300ctcactttcg gcggagggac caaggtggag atcaaacga 339209113PRTHomo sapiens 209Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile His Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 21023PRTHomo sapiens 210Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 21116PRTHomo sapiens 211Lys Ser Ser Gln Ser Leu Leu His Ser Gly Gly Lys Thr Tyr Leu Tyr 1 5 10 15 21215PRTHomo sapiens 212Trp Tyr Leu Gln Arg Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr 1 5 10 15 2137PRTHomo sapiens 213Glu Val Ser Asn Arg Phe Ser 1 5 21432PRTHomo sapiens 214Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 2159PRTHomo sapiens 215Met Gln Ser Ile His Leu Pro Leu Thr 1 5 21611PRTHomo sapiens 216Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 217366DNAHomo sapiens 217caggtgcagc tggagcagtc ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcaat aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaaagatgag 300gaatactact atgtttcggg gcttgactac tggggccagg gaaccctggt caccgtctcc 360tcagcc 366218122PRTHomo sapiens 218Gln Val Gln Leu Glu Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Glu Glu Tyr Tyr Tyr Val Ser Gly Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 21925PRTHomo sapiens 219Gln Val Gln Leu Glu Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 22010PRTHomo sapiens 220Gly Phe Thr Phe Asn Asn Tyr Gly Met His 1 5 10 22114PRTHomo sapiens 221Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 22217PRTHomo sapiens 222Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 22332PRTHomo sapiens 223Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30 22412PRTHomo sapiens 224Asp Glu Glu Tyr Tyr Tyr Val Ser Gly Leu Asp Tyr 1 5 10 22512PRTHomo sapiens 225Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 226315DNAHomo sapiens 226ctgactcagt ctccatcctc cctgtctgca tctgtaagag acagagtcac catcacttgc 60cgggcgagtc aggacattag caattattta gcctggtatc agcagaaacc agggaaagtt 120cctaatctcc tgatctatgc tgcatccact ttgcaatcag gggtcccatc tcggttcagt 180ggcagtggat ctgggacaga tttcactctc accatcagca gcctgcagcc tgaagatgtt 240gcaacttatt actgtcaaaa gtataacagt gccccgctca ctttcggcgg agggaccaag 300gtggagatca aacga 315227105PRTHomo sapiens 227Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Arg Asp Arg Val 1 5 10 15 Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Lys Val Pro Asn Leu Leu Ile Tyr Ala Ala 35 40 45 Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 50 55 60 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val 65 70 75 80 Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Leu Thr Phe Gly 85 90 95 Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 22820PRTHomo sapiens 228Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Arg Asp Arg Val 1 5 10 15 Thr Ile Thr Cys 20 22911PRTHomo sapiens 229Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ala 1 5 10 23015PRTHomo sapiens 230Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Asn Leu Leu Ile Tyr 1 5 10 15 2316PRTHomo sapiens 231Ala Ala Ser Thr Leu Gln 1 5 23232PRTHomo sapiens 232Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 20 25 30 2339PRTHomo sapiens 233Gln Lys Tyr Asn Ser Ala Pro Leu Thr 1 5 23411PRTHomo sapiens 234Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 235372DNAHomo sapiens 235cagatcacct tgaaggagtc tggtcctacg ctggtgacac ccacacagac cctcacgctg 60acctgcacct tctctgggtt ctcactcagc actggtggaa tgggtgtggg ctggatccgt 120cagcccccag gaaaggccct ggactggctt acactcattt attggaatga tgataagcac 180tacagcccat ctctgaagag caggcttacc atcaccaagg acacctccaa aaaccaggtg 240gtccttagaa tgaccaacat ggaccctgtg gacacagcca cttattactg tgcacacctg 300cattacgata ttttgactgg ttttaacttt gactactggg gccagggaac cctggtcacc 360gtctcctcag cc 372236124PRTHomo sapiens 236Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Thr Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Gly 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Asp 35 40 45 Trp Leu Thr Leu Ile Tyr Trp Asn Asp Asp Lys His Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Arg Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Leu His Tyr Asp Ile Leu Thr Gly Phe Asn Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 23725PRTHomo sapiens 237Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Thr Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser 20 25 23812PRTHomo sapiens 238Gly Phe Ser Leu Ser Thr Gly Gly Met Gly Val Gly 1 5 10 23914PRTHomo sapiens 239Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Asp Trp Leu Thr 1 5 10 24016PRTHomo sapiens 240Leu Ile Tyr Trp Asn Asp Asp Lys His Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 24132PRTHomo sapiens 241Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Arg 1 5 10 15 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala His 20 25 30 24213PRTHomo sapiens 242Leu His Tyr Asp Ile Leu Thr Gly Phe Asn Phe Asp Tyr 1 5 10 24312PRTHomo sapiens 243Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 244342DNAHomo sapiens 244gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcttg gatagtgatg atggaaacac ctatttggac 120tggtacctgc agaagccagg gcagtctcca cagctcctga tctatacgct ttcctatcgg 180gcctctggag tcccagacag gttcagtggc agtgggtcag gcactgattt cacactgaaa 240atcagcaggg tggaggctga ggatgttgga gtttattact gcatgcaacg tatagagttt 300ccgctcactt tcggcggagg gaccaaggtg gagatcaaac ga 342245114PRTHomo sapiens 245Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg 24623PRTHomo sapiens 246Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys 20 24717PRTHomo sapiens 247Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu 1 5 10 15 Asp 24815PRTHomo sapiens 248Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 2497PRTHomo sapiens 249Thr Leu Ser Tyr Arg Ala Ser 1 5 25032PRTHomo sapiens 250Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 2519PRTHomo sapiens 251Met Gln Arg Ile Glu Phe Pro Leu Thr 1 5 25211PRTHomo sapiens 252Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 25394PRTHomo sapiensVARIANT(1)..(1)Xaa is E or Q 253Xaa Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Xaa Leu Ser Leu Thr 1 5 10 15 Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Xaa Xaa Tyr Xaa Trp Xaa 20 25 30 Trp Ile Arg Xaa His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile 35 40 45 Tyr Tyr Ser Gly Xaa Thr Tyr Xaa Asn Pro Ser Leu Lys Ser Arg Val 50 55 60 Xaa Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Xaa Leu Ser 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 25412PRTHomo sapiensVARIANT(7)..(7)Xaa is A, F, or G 254Gly Gly Ser Ile Ser Ser Xaa Xaa Tyr Xaa Trp Xaa 1 5 10 25516PRTHomo sapiensVARIANT(7)..(7)Xaa is S or N 255Tyr Ile Tyr Tyr Ser Gly Xaa Thr Tyr Xaa Asn Pro Ser Leu Lys Ser 1 5 10 15 25696PRTHomo sapiensVARIANT(30)..(30)Xaa is Y or F 256Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Xaa Tyr Met 20 25 30 His Trp Val Arg Gln Xaa Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Asn Pro Asn Ser Gly Gly Thr Xaa Tyr Xaa Gln Lys Phe Gln Xaa 50 55 60 Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Xaa Tyr Met Glu 65 70 75 80 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 25710PRTHomo sapiensVARIANT(7)..(7)Xaa is Y or F 257Gly Tyr Thr Phe Thr Gly Xaa Tyr Met His 1 5 10 25817PRTHomo sapiensVARIANT(10)..(10)Xaa is N or Y 258Trp Ile Asn Pro Asn Ser Gly Gly Thr Xaa Tyr Xaa Gln Lys Phe Gln 1 5 10 15 Xaa 2599PRTHomo sapiensVARIANT(1)..(1)Xaa is Y or D 259Xaa Xaa Xaa Gly Ser Gly Ser Xaa Xaa 1 5 26098PRTHomo sapiensVARIANT(12)..(12)Xaa is K or T 260Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Xaa Pro Thr Gln Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser

Xaa Xaa Gly 20 25 30 Xaa Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Xaa Trp 35 40 45 Leu Xaa Leu Ile Tyr Trp Asn Asp Asp Lys Xaa Tyr Ser Pro Ser Leu 50 55 60 Xaa Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu Xaa Xaa Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala His 26112PRTHomo sapiensVARIANT(6)..(6)Xaa is T or A 261Gly Phe Ser Leu Ser Xaa Xaa Gly Xaa Gly Val Gly 1 5 10 26216PRTHomo sapiensVARIANT(9)..(9)Xaa is R or H 262Leu Ile Tyr Trp Asn Asp Asp Lys Xaa Tyr Ser Pro Ser Leu Xaa Ser 1 5 10 15 2639PRTHomo sapiensVARIANT(1)..(1)Xaa is Y or H 263Xaa Tyr Asp Ile Leu Thr Gly Xaa Xaa 1 5 2647PRTHomo sapiensVARIANT(4)..(4)Xaa is V or D 264Tyr Asp Tyr Xaa Trp Gly Ser 1 5 26598PRTHomo sapiensVARIANT(5)..(5)Xaa is V or E 265Gln Val Gln Leu Xaa Xaa Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa Xaa Tyr 20 25 30 Gly Xaa His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Xaa Asp Gly Xaa Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Xaa Ala Val Tyr Tyr Cys 85 90 95 Ala Xaa 26610PRTHomo sapiensVARIANT(5)..(5)Xaa is S or N 266Gly Phe Thr Phe Xaa Xaa Tyr Gly Xaa His 1 5 10 26717PRTHomo sapiensVARIANT(4)..(4)Xaa is Y or F 267Val Ile Trp Xaa Asp Gly Xaa Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2687PRTHomo sapiensVARIANT(7)..(7)Xaa is S or L 268Tyr Tyr Tyr Gly Ser Gly Xaa 1 5 2695PRTHomo sapiensVARIANT(5)..(5)Xaa is Y or S 269Asp Tyr Gly Asp Xaa 1 5 27098PRTHomo sapiensVARIANT(31)..(31)Xaa is D or S 270Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Tyr 20 25 30 Xaa Met Xaa Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Xaa Ser Gly Ser Xaa Xaa Xaa Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Xaa Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 27110PRTHomo sapiensVARIANT(6)..(6)Xaa is D or S 271Gly Phe Thr Phe Ser Xaa Tyr Xaa Met Xaa 1 5 10 27217PRTHomo sapiensVARIANT(4)..(4)Xaa is S or I 272Tyr Ile Ser Xaa Ser Gly Ser Xaa Xaa Xaa Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 27310PRTHomo sapiensVARIANT(1)..(1)Xaa is G or D 273Xaa Xaa Ala Ala Ala Gly Ala Phe Asp Ile 1 5 10 27498PRTHomo sapiensVARIANT(1)..(1)Xaa is E or Q 274Xaa Val Gln Leu Xaa Xaa Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Xaa Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Xaa Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 27517PRTHomo sapiensVARIANT(1)..(1)Xaa is S or F 275Xaa Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2769PRTHomo sapiensVARIANT(1)..(1)Xaa is G or D 276Xaa Xaa Val Gly Ala Thr Phe Asp Tyr 1 5 27796PRTHomo sapiensVARIANT(26)..(26)Xaa is T or A 277Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Xaa Phe Ser Ser Tyr Gly Met 20 25 30 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val 35 40 45 Ile Ser Tyr Asp Gly Xaa Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 85 90 95 27810PRTHomo sapiensVARIANT(3)..(3)Xaa is T or A 278Gly Phe Xaa Phe Ser Ser Tyr Gly Met His 1 5 10 27917PRTHomo sapiensVARIANT(7)..(7)Xaa is S or N 279Val Ile Ser Tyr Asp Gly Xaa Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2809PRTHomo sapiensVARIANT(1)..(1)Xaa is I or D 280Xaa Xaa Xaa Val Arg Gly Xaa Xaa Xaa 1 5 28197PRTHomo sapiensVARIANT(27)..(27)Xaa is G or D 281Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Xaa Ser Ile Ser Xaa Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Xaa Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg 28210PRTHomo sapiensVARIANT(2)..(2)Xaa is G or D 282Gly Xaa Ser Ile Ser Xaa Tyr Tyr Trp Ser 1 5 10 28316PRTHomo sapiensVARIANT(2)..(2)Xaa is I or F 283Tyr Xaa Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 2846PRTHomo sapiensVARIANT(1)..(1)Xaa is S or D 284Xaa Xaa Gly Trp Asp Tyr 1 5 28596PRTHomo sapiensVARIANT(21)..(21)Xaa is K or Q 285Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu 1 5 10 15 Lys Ile Ser Cys Xaa Gly Ser Gly Tyr Xaa Phe Thr Xaa Tyr Trp Ile 20 25 30 Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Xaa 35 40 45 Ile Tyr Pro Xaa Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly 50 55 60 Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln 65 70 75 80 Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Xaa Tyr Tyr Cys Ala Arg 85 90 95 28610PRTHomo sapiensVARIANT(3)..(3)Xaa is S or I 286Gly Tyr Xaa Phe Thr Xaa Tyr Trp Ile Gly 1 5 10 28717PRTHomo sapiensVARIANT(1)..(1)Xaa is I or V 287Xaa Ile Tyr Pro Xaa Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly 2888PRTHomo sapiensVARIANT(1)..(1)Xaa is R or K 288Xaa Trp Leu Gln Xaa Phe Asp Tyr 1 5 289470PRTHomo sapiens 289Lys Arg Phe His Asp Val Leu Gly Asn Glu Arg Pro Ser Ala Tyr Met 1 5 10 15 Arg Glu His Asn Gln Leu Asn Gly Trp Ser Ser Asp Glu Asn Asp Trp 20 25 30 Asn Glu Lys Leu Tyr Pro Val Trp Lys Arg Gly Asp Met Arg Trp Lys 35 40 45 Asn Ser Trp Lys Gly Gly Arg Val Gln Ala Val Leu Thr Ser Asp Ser 50 55 60 Pro Ala Leu Val Gly Ser Asn Ile Thr Phe Ala Val Asn Leu Ile Phe 65 70 75 80 Pro Arg Cys Gln Lys Glu Asp Ala Asn Gly Asn Ile Val Tyr Glu Lys 85 90 95 Asn Cys Arg Asn Glu Ala Gly Leu Ser Ala Asp Pro Tyr Val Tyr Asn 100 105 110 Trp Thr Ala Trp Ser Glu Asp Ser Asp Gly Glu Asn Gly Thr Gly Gln 115 120 125 Ser His His Asn Val Phe Pro Asp Gly Lys Pro Phe Pro His His Pro 130 135 140 Gly Trp Arg Arg Trp Asn Phe Ile Tyr Val Phe His Thr Leu Gly Gln 145 150 155 160 Tyr Phe Gln Lys Leu Gly Arg Cys Ser Val Arg Val Ser Val Asn Thr 165 170 175 Ala Asn Val Thr Leu Gly Pro Gln Leu Met Glu Val Thr Val Tyr Arg 180 185 190 Arg His Gly Arg Ala Tyr Val Pro Ile Ala Gln Val Lys Asp Val Tyr 195 200 205 Val Val Thr Asp Gln Ile Pro Val Phe Val Thr Met Phe Gln Lys Asn 210 215 220 Asp Arg Asn Ser Ser Asp Glu Thr Phe Leu Lys Asp Leu Pro Ile Met 225 230 235 240 Phe Asp Val Leu Ile His Asp Pro Ser His Phe Leu Asn Tyr Ser Thr 245 250 255 Ile Asn Tyr Lys Trp Ser Phe Gly Asp Asn Thr Gly Leu Phe Val Ser 260 265 270 Thr Asn His Thr Val Asn His Thr Tyr Val Leu Asn Gly Thr Phe Ser 275 280 285 Leu Asn Leu Thr Val Lys Ala Ala Ala Pro Gly Pro Cys Pro Pro Pro 290 295 300 Pro Pro Pro Pro Arg Pro Ser Lys Pro Thr Pro Ser Leu Ala Thr Thr 305 310 315 320 Leu Lys Ser Tyr Asp Ser Asn Thr Pro Gly Pro Ala Gly Asp Asn Pro 325 330 335 Leu Glu Leu Ser Arg Ile Pro Asp Glu Asn Cys Gln Ile Asn Arg Tyr 340 345 350 Gly His Phe Gln Ala Thr Ile Thr Ile Val Glu Gly Ile Leu Glu Val 355 360 365 Asn Ile Ile Gln Met Thr Asp Val Leu Met Pro Val Pro Trp Pro Glu 370 375 380 Ser Ser Leu Ile Asp Phe Val Val Thr Cys Gln Gly Ser Ile Pro Thr 385 390 395 400 Glu Val Cys Thr Ile Ile Ser Asp Pro Thr Cys Glu Ile Thr Gln Asn 405 410 415 Thr Val Cys Ser Pro Val Asp Val Asp Glu Met Cys Leu Leu Thr Val 420 425 430 Arg Arg Thr Phe Asn Gly Ser Gly Thr Tyr Cys Val Asn Leu Thr Leu 435 440 445 Gly Asp Asp Thr Ser Leu Ala Leu Thr Ser Thr Leu Ile Ser Val Pro 450 455 460 Asp Arg Asp Pro Ala Ser 465 470 29026DNAArtificial Sequencechemically synthesized primer 290caccatggac tggcacctgg aggatc 2629126DNAArtificial Sequencechemically synthesized primer 291caccatggac tggacctgga gacatc 2629225DNAArtificial Sequencechemically synthesized primer 292caccatggac tggacctgga gggtc 2529325DNAArtificial Sequencechemically synthesized primer 293caccatggac tggatttgga ggatc 2529425DNAArtificial Sequencechemically synthesized primer 294caccatggac acactttgct cacac 2529526DNAArtificialchemically synthesized primer 295caccatggaa gttggggctg agctgg 2629626DNAArtificial Sequencechemically synthesized primer 296caccatggag ttgtggactg agctgg 2629726DNAArtificial Sequencechemically synthesized primer 297caccatggag tttgggctgt agctgg 2629825DNAArtificial Sequencechemically synthesized primer 298caccatggaa ctggggctcc gctgg 2529925DNAArtificial Sequencechemically synthesized primer 299caccatggag ttggggctgt gctgg 2530025DNAArtificial Sequencechemically synthesized primer 300caccatggag ttttggctga gctgg 2530125DNAArtificial Sequencechemically synthesized primer 301caccatgacg gagtttgggc tgagc 2530226DNAArtificial Sequencechemically synthesized primer 302caccatgaaa gcacctgtgg ttcttc 2630325DNAArtificial Sequencechemically synthesized primer 303caccatgaaa catctgtggt tcttc 2530425DNAArtificial Sequencechemically synthesized primer 304caccatgggg tcaaccgcca tcctc 2530528DNAArtificial Sequencechemically synthesized primer 305caccatgtct gtctccttcc tcatcttc 2830621DNAArtificial Sequencechemically synthesized primer 306atggggtccc aggttcacct c 2130722DNAArtificial Sequencechemically synthesized primer 307atgttgccat cacaactcat tg 2230898PRTHomo sapiens 308Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 309369DNAHomo sapiens 309caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt ttcaggacag ggtcaccatg accagggaca cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagatttc 300tttggttcgg ggagtctcct ctactttgac tactggggcc agggaaccct ggtcaccgtc 360tcctcagcc 369310123PRTHomo sapiens 310Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Phe Phe Gly Ser Gly Ser Leu Leu Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 31125PRTHomo sapiens 311Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 31210PRTHomo sapiens 312Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His 1 5 10 31314PRTHomo sapiens 313Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 31417PRTHomo sapiens 314Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Asp 31532PRTHomo sapiens 315Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Arg Leu Arg Ser Asp Asp Thr

Ala Val Tyr Tyr Cys Ala Arg 20 25 30 31613PRTHomo sapiens 316Asp Phe Phe Gly Ser Gly Ser Leu Leu Tyr Phe Asp Tyr 1 5 10 31712PRTHomo sapiens 317Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 1 5 10 318339DNAHomo sapiens 318gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg catagtggtg gaaagaccta tttgtattgg 120tacctgcaga ggccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acaccttccg 300ctcactttcg gcggagggac caaggtggag atcaaacga 339319113PRTHomo sapiens 319Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile His Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 32023PRTHomo sapiens 320Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 32116PRTHomo sapiens 321Lys Ser Ser Gln Ser Leu Leu His Ser Gly Gly Lys Thr Tyr Leu Tyr 1 5 10 15 32215PRTHomo sapiens 322Trp Tyr Leu Gln Arg Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr 1 5 10 15 3237PRTHomo sapiens 323Glu Val Ser Asn Arg Phe Ser 1 5 32432PRTHomo sapiens 324Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 3259PRTHomo sapiens 325Met Gln Ser Ile His Leu Pro Leu Thr 1 5 32611PRTHomo sapiens 326Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 32722DNAArtificial Sequencechemically synthesized primer 327gaattcagag ttaaaccttg ag 2232822DNAArtificial Sequencechemically synthesized primer 328caggaatctg atctgttacc ac 2232923DNAArtificial Sequencechemically synthesized primer 329ctgaccctac aagatgccaa gag 2333029DNAArtificial Sequencechemically synthesized primer 330atcatgcatt gcaacattta ttgatggag 2933120DNAArtificial Sequencechemically synthesized primer 331ttggcagatt gtctgtagcc 2033220DNAArtificial Sequencechemically synthesized primer 332aggcattgtg catgctgctt 2033320DNAArtificial Sequencechemically synthesized primer 333tattgaaagt gccgagatcc 2033419DNAArtificial Sequencechemically synthesized primer 334tgcaaggacc acagccatc 1933521DNAArtificial Sequencechemically synthesized primer 335tcaatggaac cttcagcctt a 2133619DNAArtificial Sequencechemically synthesized primer 336gaaggggtgg gttttgaag 1933725DNAArtificial Sequencechemically synthesized primer 337ctcactgtga aagctgcagc accag 2533821DNAArtificial Sequencechemically synthesized primer 338tcaatggaac cttcagcctt a 2133919DNAArtificial Sequencechemically synthesized primer 339gaaggggtgg gttttgaag 1934025DNAArtificial Sequencechemically synthesized primer 340ctcactgtga aagctgcagc accag 2534130DNAArtificial Sequencechemically synthesized 341tgatcagtaa ggatttcacc tctgtttgta 3034230DNAArtificial Sequencechemically synthesized 342accttgtcat gtaccatcaa taaagtaccc 3034312PRTHomo sapiens 343Ala Thr Thr Leu Lys Ser Tyr Asp Ser Asn Thr Pro 1 5 10 34425PRTArtificial Sequencechemically synthesized 344Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 1 5 10 15 Lys Lys Asp Asp Ala Lys Lys Asp Leu 20 25 34560DNAArtificial Sequencechemically synthesized primer 345tctcttcctc ctgctactct ggctcccaga taccaccggt gaaatagtga tgacgcagtc 6034657DNAArtificial Sequencechemically synthesized primer 346ccggaattct tactatttgt catcatcgtc cttataatcg ctagctgagg agacggt 5734760DNAArtificial Sequencechemically synthesized primer 347acgcgtcgac ccaccatgga agccccagcg cagcttctct tcctcctgct actctggctc 6034860DNAArtificial Sequencechemically synthesized primer 348tctcttcctc ctgctactct ggctcccaga taccaccggt gaaatagtga tgacgcagtc 6034957DNAArtificial Sequencechemically synthesized primer 349ccggaattct tactatttgt catcatcgtc cttataatcg ctagctttca gctccag 5735060DNAArtificial Sequencechemically synthesized primer 350acgcgtcgac ccaccatgga agccccagcg cagcttctct tcctcctgct actctggctc 6035155DNAArtificial Sequencechemically synthesized primer 351actctggctc ccagatacca ccggagaaat agtgatgacg cagtctccag ccacc 5535251DNAArtificial Sequencechemically synthesized primer 352ccgctcgagc tatttgtcat catcgtcctt ataatctttc agctccagct t 5135374DNAArtificial Sequencechemically synthesized primer 353tcttcgcgac caccatggaa accccagcgc agcttctctt cctcctgcta ctctggctcc 60cagataccac cgga 74354852DNAArtificial Sequencechemically synthesized primer 354atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 120ctctcctgca gggccagtca gagtgttgac aacaacttag tctggtacca gcagaaacct 180ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagtag tctgcagtct 300gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 360caagggacca aggtggaaat caaactttcc gcggacgatg cgaaaaagga tgctgcgaag 420aaagatgacg ctaagaaaga cgatgctaaa aaggacctgc aggtgcagct gcaggagtcg 480ggcccaggac tggtgaagcc ttcacagacc ctgtccctca cctgcactgt ctctggtggc 540tccatcagca gttttaatta ctactggagc tggatccgcc accacccagg gaagggcctg 600gagtggattg ggtacatcta ttacagtggg agcacctact ccaacccgtc cctcaagagt 660cgagttacca tatcagtaga cacgtctaag aaccagttct ccctgacgct gagctctgtg 720actgccgcgg acacggccgt gtattactgt gcgagagggt ataactggaa ctactttgac 780tactggggcc agggaaccct ggtcaccgtc tcctcagcta gcgattataa ggacgatgat 840gacaaatagt aa 852355282PRTHomo sapiens 355Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Asp Asn Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110 Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 130 135 140 Lys Lys Asp Asp Ala Lys Lys Asp Leu Gln Val Gln Leu Gln Glu Ser 145 150 155 160 Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr 165 170 175 Val Ser Gly Gly Ser Ile Ser Ser Phe Asn Tyr Tyr Trp Ser Trp Ile 180 185 190 Arg His His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr 195 200 205 Ser Gly Ser Thr Tyr Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile 210 215 220 Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Thr Leu Ser Ser Val 225 230 235 240 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asn Trp 245 250 255 Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270 Ala Ser Asp Tyr Lys Asp Asp Asp Asp Lys 275 280 3561617DNAArtificial Sequencechemically synthesized 356atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 120ctctcctgca gggccagtca gagtgttgac aacaacttag tctggtacca gcagaaacct 180ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagtag tctgcagtct 300gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 360caagggacca aggtggaaat caaactttcc gcggacgatg cgaaaaagga tgctgcgaag 420aaagatgacg ctaagaaaga cgatgctaaa aaggacctgc aggtgcagct gcaggagtcg 480ggcccaggac tggtgaagcc ttcacagacc ctgtccctca cctgcactgt ctctggtggc 540tccatcagca gttttaatta ctactggagc tggatccgcc accacccagg gaagggcctg 600gagtggattg ggtacatcta ttacagtggg agcacctact ccaacccgtc cctcaagagt 660cgagttacca tatcagtaga cacgtctaag aaccagttct ccctgacgct gagctctgtg 720actgccgcgg acacggccgt gtattactgt gcgagagggt ataactggaa ctactttgac 780tactggggcc agggaaccct ggtcaccgtc tcctcaggag gtggtggatc cgatatcaaa 840ctgcagcagt caggggctga actggcaaga cctggggcct cagtgaagat gtcctgcaag 900acttctggct acacctttac taggtacacg atgcactggg taaaacagag gcctggacag 960ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta caatcagaag 1020ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta catgcaactg 1080agcagcctga catctgagga ctctgcagtc tattactgtg caagatatta tgatgatcat 1140tactgccttg actactgggg ccaaggcacc actctcacag tctcctcact ttccgcggac 1200gatgcgaaaa aggatgctgc gaagaaagat gacgctaaga aagacgatgc taaaaaggac 1260ctggacattc agctgaccca gtctccagca atcatgtctg catctccagg ggagaaggtc 1320accatgacct gcagagccag ttcaagtgta agttacatga actggtacca gcagaagtca 1380ggcacctccc ccaaaagatg gatttatgac acatccaaag tggcttctgg agtcccttat 1440cgcttcagtg gcagtgggtc tgggacctca tactctctca caatcagcag catggaggct 1500gaagatgctg ccacttatta ctgccaacag tggagtagta acccgctcac gttcggtgct 1560gggaccaagc tggagctgaa agctagcgat tataaggacg atgatgacaa atagtaa 1617357537PRTArtificial Sequencechemically synthesized 357Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Asp Asn Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110 Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 130 135 140 Lys Lys Asp Asp Ala Lys Lys Asp Leu Gln Val Gln Leu Gln Glu Ser 145 150 155 160 Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr 165 170 175 Val Ser Gly Gly Ser Ile Ser Ser Phe Asn Tyr Tyr Trp Ser Trp Ile 180 185 190 Arg His His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr 195 200 205 Ser Gly Ser Thr Tyr Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile 210 215 220 Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Thr Leu Ser Ser Val 225 230 235 240 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asn Trp 245 250 255 Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270 Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu 275 280 285 Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr 290 295 300 Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln 305 310 315 320 Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn 325 330 335 Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser 340 345 350 Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 355 360 365 Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp 370 375 380 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Leu Ser Ala Asp 385 390 395 400 Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala Lys Lys Asp Asp 405 410 415 Ala Lys Lys Asp Leu Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met 420 425 430 Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser 435 440 445 Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro 450 455 460 Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr 465 470 475 480 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser 485 490 495 Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 500 505 510 Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala 515 520 525 Ser Asp Tyr Lys Asp Asp Asp Asp Lys 530 535 3581677DNAArtificial Sequencechemically synthesized 358atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 120ctctcctgca gggccagtca gagtgttgac aacaacttag tctggtacca gcagaaacct 180ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagtag tctgcagtct 300gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 360caagggacca aggtggaaat caaactttcc gcggacgatg cgaaaaagga tgctgcgaag 420aaagatgacg ctaagaaaga cgatgctaaa aaggacctgc aggtgcagct gcaggagtcg 480ggcccaggac tggtgaagcc ttcacagacc ctgtccctca cctgcactgt ctctggtggc 540tccatcagca gttttaatta ctactggagc tggatccgcc accacccagg gaagggcctg 600gagtggattg ggtacatcta ttacagtggg agcacctact ccaacccgtc cctcaagagt 660cgagttacca tatcagtaga cacgtctaag aaccagttct ccctgacgct gagctctgtg 720actgccgcgg acacggccgt gtattactgt gcgagagggt ataactggaa ctactttgac 780tactggggcc agggaaccct ggtcaccgtc tcctcattat cagcggatga cgccaagaaa 840gacgcagcca aaaaggacga tgcaaagaag gatgacgcaa agaaagattt agatatcaaa 900ctgcagcagt caggggctga actggcaaga cctggggcct cagtgaagat gtcctgcaag 960acttctggct acacctttac taggtacacg atgcactggg taaaacagag gcctggacag 1020ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta caatcagaag 1080ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta catgcaactg 1140agcagcctga catctgagga ctctgcagtc tattactgtg caagatatta tgatgatcat 1200tactgccttg actactgggg ccaaggcacc actctcacag tctcctcact ttccgcggac 1260gatgcgaaaa aggatgctgc gaagaaagat gacgctaaga aagacgatgc taaaaaggac

1320ctggacattc agctgaccca gtctccagca atcatgtctg catctccagg ggagaaggtc 1380accatgacct gcagagccag ttcaagtgta agttacatga actggtacca gcagaagtca 1440ggcacctccc ccaaaagatg gatttatgac acatccaaag tggcttctgg agtcccttat 1500cgcttcagtg gcagtgggtc tgggacctca tactctctca caatcagcag catggaggct 1560gaagatgctg ccacttatta ctgccaacag tggagtagta acccgctcac gttcggtgct 1620gggaccaagc tggagctgaa agattataag gacgatgatg acaaatagct cgagcgg 1677359555PRTArtificial Sequencechemically synthesized 359Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Asp Asn Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110 Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 130 135 140 Lys Lys Asp Asp Ala Lys Lys Asp Leu Gln Val Gln Leu Gln Glu Ser 145 150 155 160 Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr 165 170 175 Val Ser Gly Gly Ser Ile Ser Ser Phe Asn Tyr Tyr Trp Ser Trp Ile 180 185 190 Arg His His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr 195 200 205 Ser Gly Ser Thr Tyr Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile 210 215 220 Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Thr Leu Ser Ser Val 225 230 235 240 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asn Trp 245 250 255 Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270 Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 275 280 285 Lys Lys Asp Asp Ala Lys Lys Asp Leu Asp Ile Lys Leu Gln Gln Ser 290 295 300 Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 305 310 315 320 Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln 325 330 335 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 340 345 350 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr 355 360 365 Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 370 375 380 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His 385 390 395 400 Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 405 410 415 Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Asp Ala 420 425 430 Lys Lys Asp Asp Ala Lys Lys Asp Leu Asp Ile Gln Leu Thr Gln Ser 435 440 445 Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 450 455 460 Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser 465 470 475 480 Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser 485 490 495 Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 500 505 510 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 515 520 525 Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 530 535 540 Glu Leu Lys Asp Tyr Lys Asp Asp Asp Asp Lys 545 550 555 360100PRTHomo sapiens 360Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Arg 100 36198PRTHomo sapiens 361Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 36298PRTHomo sapiens 362Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 36398PRTHomo sapiens 363Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys 36498PRTHomo sapiens 364Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 36599PRTHomo sapiens 365Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg 36697PRTHomo sapiens 366Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg 36798PRTHomo sapiens 367Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg 368101PRTHomo sapiens 368Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Ile Glu Phe Pro 100 36996PRTHomo sapiens 369Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 37095PRTHomo sapiens 370Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro 85 90 95 371100PRTHomo sapiens 371Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro 100 37295PRTHomo sapiens 372Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro 85 90 95 373100PRTHomo sapiens 373Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile Gln Leu Pro 100 37495PRTHomo sapiens 374Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro 85 90 95 37517DNAHomo sapiens 375ggtataactg gaacgac 1737620DNAHomo sapiens 376ggtatagtgg gagctactac 2037731DNAHomo sapiens 377gtattactat ggttcgggga gttattataa c 3137837DNAHomo sapiens 378gtattatgat tacgtttggg ggagttatcg ttatacc 3737931DNAHomo sapiens 379gtattactat gatagtagtg gttattacta c 3138031DNAHomo sapiens 380gtattacgat attttgactg gttattataa c 3138116DNAHomo sapiens 381tgactacggt gactac 1638220DNAHomo sapiens 382gtagagatgg ctacaattac 2038321DNAHomo sapiens 383gggtatagca gcagctggta c 2138421DNAHomo sapiens 384gggtatagca gtggctggta c 2138515PRTHomo sapiens 385Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 15 38615PRTHomo sapiens 386Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 38716PRTHomo sapiens 387Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5

10 15 38820PRTHomo sapiens 388Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 1 5 10 15 Thr Val Ser Ser 20 38912PRTHomo sapiens 389Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 39012PRTHomo sapiens 390Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 39112PRTHomo sapiens 391Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 1 5 10 39212PRTHomo sapiens 392Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 39312PRTHomo sapiens 393Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 1 5 10 394445PRTHomo sapiens 394Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Phe 20 25 30 Asn Tyr Tyr Trp Ser Trp Ile Arg His His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Ser Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 395449PRTHomo sapiens 395Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Phe 20 25 30 Asn Tyr Tyr Trp Ser Trp Ile Arg His His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Ser Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Thr Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Asn Trp Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys

Claims

1. A method of treating cancer by administering to a patient in need thereof an isolated monoclonal human antibody that specifically binds GPNMB comprising a combination of a variable heavy chain and a variable light chain selected from the group consisting of: (a) the variable heavy chain amino acid sequence of: SEQ ID NO: 20 and the variable light chain amino acid sequence of: SEQ ID NO: 29; (b) the variable heavy chain amino acid sequence of SEQ ID NO:74 and the variable light chain amino acid sequence of SEQ ID NO: 83; (c) the variable heavy chain amino acid sequence of SEQ ID NO: 164 and the variable light chain amino acid sequence of SEQ ID NO: 173; (d) the variable heavy chain amino acid sequence of SEQ ID NO: 38 and the variable light chain amino acid sequence of SEQ ID NO: 47; and (e) the variable heavy chain amino acid sequence of SEQ ID NO: 310 and the variable light chain amino acid sequence of SEQ ID NO: 319.

2. A method of treating cancer by administering to a patient in need thereof an isolated monoclonal human antibody that specifically binds GPNMB comprising a heavy chain polypeptide comprising an amino acid sequence comprising three complementarity determining regions (CDRs) and a light chain polypeptide comprising an amino acid sequence comprising three CDRs, where the three heavy chain CDRs and the three light chain CDRS are selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 comprising the amino acid sequence 33, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a light chain CDR2 comprising the amino acid sequence 51, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53; (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 78, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a light chain CDR2 comprising the amino acid sequence 87, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 166, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 170, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 175, a light chain CDR2 comprising the amino acid sequence 177, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 179; and (e) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 312, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 316, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 321, a light chain CDR2 comprising the amino acid sequence 323, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 325.

3. The method of claim 2, wherein the antibody is selected from the group consisting of: Mab1.15.1, Mab1.2.2, Mab2.10.2, Mab2.22.1, and Mab2.6.1.

4. The method of claim 2, wherein said antibody is an IgG1 antibody.

5. A method of treating cancer by administering to a patient in need thereof an immunoconjugate comprising the antibody of claim 2 and a cytotoxic agent.

6. The method of claim 5, wherein said antibody comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 comprising the amino acid sequence 33, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

7. The method of claim 5, wherein the cytotoxic agent is auristatin E (dolastatin-10).

8. A method of treating cancer by administering to a patient in need thereof a pharmaceutical composition comprising the immunoconjugate of claim 5.

9. A method of treating cancer by administering to a patient in need thereof a single chain Fv antibody that specifically binds GPNMB, wherein the anti-GPNMB single chain Fv antibody comprises a VL domain of a monoclonal human anti-GPNMB antibody linked to a VH domain of said anti-GPNMB antibody, wherein the VL domain comprises an amino acid sequence comprising three complementarity determining regions (CDRs) and the VH domain comprises an amino acid sequence comprising three CDRs, where the three heavy chain CDRs and the three light chain CDRS are selected from the group consisting of: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 comprising the amino acid sequence 33, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a light chain CDR2 comprising the amino acid sequence 51, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 53; (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 78, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 80, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a light chain CDR2 comprising the amino acid sequence 87, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 166, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 170, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 175, a light chain CDR2 comprising the amino acid sequence 177, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 179; and (e) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 312, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 316, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 321, a light chain CDR2 comprising the amino acid sequence 323, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 325.

10. The method of claim 9 wherein the single chain Fv antibody further comprises a single chain Fv antibody that specifically binds CD3, wherein the anti-CD3 single chain Fv antibody comprises a VH domain of an anti-CD3 antibody linked to a VL domain of said anti-CD3 antibody.

11. A method of treating cancer by administering to a patient in need thereof an immunoconjugate comprising the single chain Fv antibody of claim 9 and a cytotoxic agent.

12. The method of claim 9 wherein the single chain Fv antibody comprises the amino acid sequence of SEQ ID NO: 355.

13. The method of claim 10 wherein the single chain Fv antibody comprises the amino acid sequence of SEQ ID NO: 357 or SEQ ID NO: 359.

14. The method of claim 8, wherein said immunoconjugate has a concentration of 5 mg/ml.

15. The method of claim 8 wherein the pharmaceutical composition further comprising sucrose, histidine, histidine hydrochloride monohydrate, and polysorbate 20.

16. The method of claim 8 wherein the pharmaceutical composition comprises 5 mg/ml of the immunoconjugate, 10% sucrose, 10 mM histidine, 10 mM histidine hydrochloride monohydrate, and 0.02% (w/v) polysorbate 20.

17. The method of claim 5, wherein the cytotoxic agent is auristatin E (dolastatin-10).

18. The method of claim 6, wherein said immunoconjugate has a concentration of 5 mg/ml.

19. The method of claim 6 wherein the immunoconjugate is administered in the form of a pharmaceutical composition comprising 5 mg/ml of the immunoconjugate, 10% sucrose, 10 mM histidine, 10 mM histidine hydrochloride monohydrate, and 0.02% (w/v) polysorbate 20.

20. A method of treating cancer by administering to a patient in need thereof an isolated monoclonal human antibody that specifically binds GPNMB wherein said antibody comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 comprising the amino acid sequence 33, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

21. A method of treating cancer by administering to a patient in need thereof an isolated monoclonal human antibody that specifically binds GPNMB comprising the variable heavy chain amino acid sequence of SEQ ID NO: 20, and the variable light chain amino acid sequence of SEQ ID NO: 29.

22. A method of treating cancer by administering to a patient in need thereof an isolated monoclonal human antibody that specifically binds GPNMB which is Mab 1.15.1.

23. The method of claim 20 wherein the antibody is administered in the form of an immunoconjugate comprising a cytotoxic agent.

24. The method of claim 21 wherein the antibody is administered in the form of an immunoconjugate comprising a cytotoxic agent.

25. The method of claim 22 wherein the antibody is administered in the form of an immunoconjugate comprising a cytotoxic agent.

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