Insulin-like growth factor (IGF-I) is a 7.5-kD polypeptide that circulates in plasma in high concentrations and is detectable in most tissues. IGF-I stimulates cell differentiation and cell proliferation, and is required by most mammalian cell types for sustained proliferation.

These cell types include, among others, human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, neural cells, myeloid cells, chondrocytes, osteoblasts and bone marrow stem cells.

The first step in the transduction pathway leading to IGF-I-stimulated cellular proliferation or differentiation is binding of IGF-I or IGF-II (or insulin at supraphysiological concentrations) to the IGF-I receptor. The IGF-I receptor (IGF-IR) is composed of two types of subunits: an alpha subunit (a 130-135 kD protein that is entirely extracellular and functions in ligand binding) and a beta subunit (a 95-kD transmembrane protein, with transmembrane and cytoplasmic domains). The IGF-IR is initially synthesized as a single chain proreceptor polypeptide that is processed by glycosylation, proteolytic cleavage, and covalent bonding to assemble into a mature 460-kD heterotetramer comprising two alpha-subunits and two beta- subunits. The beta subunit (s) possesses ligand-activated tyrosine kinase activity. This activity is implicated in the signaling pathways mediating ligand action which involve autophosphorylation of the beta-subunit and phosphorylation of IGF-IR substrates.

There is considerable evidence for a role for IGF-I and/or IGF-IR in the maintenance of tumor cells in vitro and in vivo. IGF-IR levels are elevated in tumors of lung (Kaiser et al., J. Cancer Res. Clin. Oncol. 119: 665-668,1993 ; Moody et al., Life Sciences 52: 1161-1173, 1993; Macauley et al., Cancer Res. , 50: 2511-2517,1990), breast (Pollak et al., Cancer Lett.

38: 223-230,1987 ; Foekens et al., Cancer Res. 49: 7002-7009,1989 ; Cullen et al., Cancer Res. 49: 7002-7009,1990 ; Arteaga et al., J. Clin. Invest. 84: 1418-1423,1989), prostate and colon (Remaole-Bennet et al., J. Clin. Endocrinol. Metab. 75: 609-616,1992 ; Guo et al., Gastroenterol. 102: 1101-1108,1992). In addition, IGF-I appears to be an autocrine stimulator of human gliomas (Sandberg-Nordqvist et al :, Cancer Res. 53: 2475-2478,1993), while IGF-I stimulated the growth of fibrosarcomas that overexpressed IGF-IR (Butler et al., Cancer Res. 58: 3021-27,1998). Further, individuals with"high normal"levels of IGF-I have an increased risk of common cancers compared to individuals with IGF-I levels in the"low normal"range (Rosen et al., Trends Endocrinol. Metab. 10: 136-41,1999). For a review of the role IGF-I/IGF-I receptor interaction plays in the growth of a variety of human tumors, see Macaulay, Br. J. Cancer, 65: 311-320,1992.

Calorie restriction is the most effective and reproducible intervention for increasing the life span in a variety of animal species, including mammals. It is also the most potent, broadly acting cancer-prevention regimen in experimental carcinogenesis models. A key biological mechanism underlying many of its beneficial effects is the insulin-like growth factor- 1 pathway (Hursting et al., Annu. Rev. Med. 54: 131-52,2003).

In view of the roles that IGF-I and IGF-IR have in such disorders as cancer and other proliferative disorders when IGF-I and/or IGF-IR are overexpressed, antibodies to IGF-IR have been produced that block binding of IGF-I or IGF-II to IGF-IR. Such antibodies are described, for example, in WO 02/05359, published July 11,2002. The text of these publications, including all sequences described, is hereby incorporated by reference. It is desirable to use such high-affinity human anti-IGF-IR antibodies to treat relevant diseases in humans.

Summary of the Invention The present invention relates to a method for the treatment or prevention of a disorder wherein said disorder is selected from the group consisting of multiple myeloma, liquid tumor, liver cancer, thymus disorder, T-cell mediated auto-immune disease, endocronological disorder, ischemia, and neurodegenerative disorder in a mammal comprising administering to said mammal an amount of a human anti-IGF-IR antibody that is effective in treating said disorder. In one embodiment, the method also comprises administering to said mammal said antibody in combination with an agent selected from the group consisting of a corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascular agent, and anti-proliferative agent.

The liquid tumor is preferably acute lymphocytic leukemia (ALL) or chronic milogenic leukemia (CML). The liver cancer is preferably hepatoma, hepatocellular carcinoma, cholangiocarcinoma, angiosarcomas, hemangiosarcomas, or hepatoblastoma. The thymus disorder is preferably thymoma or thyroiditis. The T-cell mediated autoimmune disease is preferably Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE), Grave's Disease, Hashimoto's Thyroiditis, Myasthenia Gravis, Auto-Immune Thyroiditis, or Bechet's Disease. The endocronological disorder is preferably Diabetes II, hyperthyroidism, hypothyroidism, thyroiditis, hyperadrenocorticism, and hypoadrenocorticism. The ischemia is preferably post-cardiac ischemia. The neurodegenerative disorder is preferably Alzheimer's Disease.

Where the antibody is administered in combination with an anti-proliferative agent, the agent is preferably selected from the group consisting of farnesyl protein transferase inhibitors, avE. 3 inhibitors, avß5 inhibitors, p53 inhibitors, and PDGFR inhibitors.

Where the antibody is administered in combination with an anti-vascular agent, the agent is preferably selected from the group consisting of bevacizumab or rhuMAb-VEGF.

Where the antibody is administered in combination with an anti-emetic agent, the agent is preferably selected from the group consisting of ondansetron hydrochloride, granisetron hydrochloride, metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, or tropisetron.

Where the antibody is administered in combination with a vaccine, the vaccine is preferably selected from GM-CSF DNA and cell-based vaccines, dendritic cell vaccines, recombinant viral vaccines, heat shock protein (HSP) vaccines, allogeneic or autologous tumor vaccines. In one embodiment, the vaccine is peptide, DNA, or cell based.

Where the antibody is administered in combination with an analgesic agent, the agent is preferably selected from the group consisting of ibuprofen, naproxen, choline magnesium trisalicylate, or oxycodone hydrochloride.

In a preferred embodiment, the mammal is a human.

In one embodiment, the antibody that binds to IGF-IR has the following properties: a binding affinity for human IGF-IR of Kd of 8 x 10-9 or less; inhibition of binding between human IGF-IR and IGF-I with an IC50 of less than 100 nM; and comprises a heavy chain amino acid sequence comprising human FR1, FR2, and FR3 amino acid sequences that correspond to those of the VH DP-35, VIV-4/4. 35, VH DP-47, or VH DP-71 gene, or conservative substitutions or somatic mutations therein, wherein the FR sequences are linked with CDR1, CDR2, and CDR3 sequences, and wherein the antibody also comprises CDR regions in its light chain from the A27, A30, or 012 gene.

Alternatively, the antibody competes for binding with an antibody having heavy and light chain amino acid sequences of an antibody selected from the group consisting of 2.12. 1, 2.13. 2,2. 14.3, 3.1. 1,4. 9.2, 4.17. 3, and 6.1. 1. For example, the antibody can bind to the epitope to which an antibody binds that has heavy and light chain amino acid sequences of an antibody selected from the group consisting of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3, and 6.1. 1.

In another embodiment, the invention is practiced using an antibody that comprises a heavy chain comprising the amino acid sequences of CDR-1, CDR-2, and CDR-3, and a light chain comprising the amino acid sequences of CDR-1, CDR-2, and CDR-3, of an antibody selected from the group consisting of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3, and 6.1. 1, or sequences having changes from said CDR sequences selected from the group consisting of conservative changes, wherein said conservative changes are selected from the group consisting of replacement of nonpolar residues by other nonpolar residues, replacement of polar charged residues by other polar uncharged residues, replacement of polar charged residues by other polar charged residues, and substitution of structurally similar residues; and non-conservative substitutions, wherein said non-conservative substitutions are selected from

the group consisting of substitution of polar charged residue for polar uncharged residues and substitution of nonpolar residues for polar residues, additions and deletions.

In a preferred embodiment, the antibody comprises a heavy chain comprising the amino acid sequences of CDR-1, CDR-2, and CDR-3, and a light chain comprising the amino acid sequences of CDR-1, CDR-2, and CDR-3, of an antibody selected from the group consisting of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3, or 6.1. 1. In another embodiment, the antibody comprises a heavy chain amino acid sequence derived from human gene DP-47 and a light chain amino acid derived from human gene A30.

The invention also relates to a pharmaceutical composition for treatment of a disorder in a mammal comprising an amount of a human anti-IGF-IR antibody that is effective in treating said disorder and a pharmaceutically acceptable carrier, wherein said disorder is selected from the group consisting of multiple myeloma, liquid tumor, liver cancer, thymus disorder, T-cell mediated autoimmune disease, endocronological disorder, ischemia, and neurodegenerative disorder. In one embodiment, the invention relates to a combination pharmaceutical composition that also comprises an amount of a corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascular agent, or an anti-proliferative agent that, in combination with said antibody, is effective in treating said disorder.

The invention also relates to use of an amount of a human anti-IGF-IR antibody in the preparation of a composition for the treatment of a disorder in a mammal that is effective in treating said disorder, wherein said disorder is selected from the group consisting of multiple myeloma, liquid tumor, liver cancer, thymus disorder, T-cell mediated autoimmune disease, endocronological disorder, ischemia, and neurodegenerative disorder.

Brief Description of the Drawings Figs. 1A-1C show alignments of the nucleotide sequences of the light chain variable regions from six human anti-IGF-IR antibodies to each other and to germline sequences. Fig.

1A shows the alignment of the nucleotide sequences of the variable region of the light chain (VL) of antibodies 2.12. 1 (SEQ ID NO: 1) 2.13. 2 (SEQ ID NO: 5), 2.14. 3 (SEQ ID NO: 9) and 4.9. 2 (SEQ ID NO: 13) to each other and to the germline VK A30 sequence (SEQ ID NO: 39).

Fig. 1B shows the alignment of the nucleotide sequence of VL of antibody 4.17. 3 (SEQ ID NO: 17) to the germline VK 012 sequence (SEQ ID NO: 41). Fig. 1C shows the alignment of the nucleotide sequence of VL of antibody 6.1. 1 (SEQ ID NO: 21) to the germline VK A27 sequence (SEQ ID NO: 37). The alignments also show the CDR regions of the VL from each antibody. The consensus sequences for Figs. 1A-1C are shown in SEQ ID NOS: 53-55, respectively.

Figs. 2A-2D show alignments of the nucleotide sequences of the heavy chain variable regions from six human anti-IGF-IR antibodies to each other and to germline sequences. Fig.

2A shows the alignment of the nucleotide sequence of the VH of antibody 2.12. 1 (SEQ ID

NO: 3) to the germline VH DP-35 sequence (SEQ ID NO: 29). Fig. 2B shows the alignment of the nucleotide sequence of the VH of antibody 2.14. 3 (SEQ ID NO: 11) to the germline VIV- 4/4.35 sequence (SEQ ID NO: 43). Figs. 2C-1 and 2C-2 show the alignments of the nucleotide sequences of the VH of antibodies 2.13. 2 (SEQ ID NO: 7), 4.9. 2 (SEQ ID NO: 15) and 6.1. 1 (SEQ ID NO: 23) to each other and to the germline VH DP-47 sequence (SEQ ID NO: 31). Fig. 2D shows the alignment of the nucleotide sequence of the VH of antibody 4.17. 3 (SEQ ID NO: 19) to the germline VH DP-71 sequence (SEQ ID NO: 35). The alignment also shows the CDR regions of the antibodies.. The consensus sequences for Figs. 2A-2D are shown in SEQ ID NOS: 56-59, respectively.

Fig. 3A shows the number of mutations in different regions of the heavy and light chains of 2.13. 2 and 2.12. 1 compared to the germline sequences. Figs. 3A-D show alignments of the amino acid sequences from the heavy and light chains of antibodies 2.13. 2 and 2.12. 1 with the germline sequences from which they are derived. Fig. 3B shows an alignment of the amino acid sequence of the heavy chain of antibody 2.13. 2 (SEQ ID NO: 45) with that of germline sequence DP-47 (3-23)/D6-19/JH6 (SEQ ID NO: 46). Fig. 3C shows an alignment of the amino acid sequence of the light chain of antibody 2.13. 2 (SEQ ID NO: 47) with that of germline sequence A30/Jk2 (SEQ ID NO: 48). Fig. 3D shows an alignment of the amino acid sequence of the heavy chain of antibody 2.12. 1 (SEQ ID NO: 49) with that of germline sequence DP-35 (3-11)/D3-3/JH6 (SEQ ID NO: 50). Fig. 3E shows an alignment of the amino acid sequence of the light chain of antibody 2.12. 1 (SEQ ID NO: 51) with that of germline sequence A30/Jk1 (SEQ ID NO: 52). For Figures 3B-E, the signal sequences are in italic, the CDRs are underlined, the constant domains are bold, the framework (FR) mutations are highlighted with a plus sign ("+") above the amino acid residue and CDR mutations are highlighted with an asterisk above the amino acid residue.

Fig. 4 shows that anti-IGF-IR antibodies 2.13. 2 and 4.9. 2 reduce IGF-IR phosphotyrosine signal in 3T3-IGF-IR tumors.

Fig. 5 shows that anti-IGF-IR antibody 2.13. 2 inhibits 3T3-IGF-IR tumor growth in vivo.

Detailed Description of the invention All patents, patent applications, and other references cited herein are hereby incorporated by reference in their entireties.

The antibody can also be used with other agents useful in treating abnormal IGF-IR activity, including, but not limited to different anti-IGF-IR antibodies such as those described in WO 02/053596, and other agents also capable of blocking IGF-IR.

Conjoint (combination) treatment described herein may be achieved by way of the simultanenous, sequential or separate dosing of the individual components of the treatment.

The antibody can be administered to treat or prevent initial disease, or to treat or prevent recurrence. It can be employed to treat early or advanced disease.

The term"treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term"treatment", as used herein, unless otherwise indicated, refers to the act of treating as"treating"is defined immediately above.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The following terms, unless otherwise indicated, shall be understood to have the following meanings: An"antibody"refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F (ab') 2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ), or Chothia & Lesk J. Mol. Biol 196: 901-917 (1987); Chothia et al. Nature 342: 878-883 (1989).

An"isolated antibody"is an antibody that (1) is not associated with naturally- associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Examples of isolated antibodies include an anti-IGF-IR antibody that has been affinity purified using IGF-IR is an isolated

antibody, an anti-IGF-IR antibody that has been synthesized by a hybridoma or other cell line in vitro, and a human anti-IGF-IR antibody derived from a transgenic mouse.

The term"chimeric antibody"refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In a preferred embodiment, one or more of the CDRs are derived from a human anti-IGF-IR antibody. In a more preferred embodiment, all of the CDRs are derived from a human anti-IGF-IR antibody.

In another preferred embodiment, the CDRs from more than one human anti-IGF-IR antibodies are mixed and matched in a chimeric antibody. Further, the framework regions may be derived from one of the same anti-IGF-IR antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.

The term"epitope"includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar sides chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

An antibody is said to specifically bind an antigen when the dissociation constant is <1 IlM, preferably < 100 nM and most preferably : 10 nM.

As applied to polypeptides, the term"substantial identity"means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution"is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e. g. , charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e. g. , Pearson, Methods Mol. Biol. 24: 307-31 (1994), herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine ; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamin ; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate- aspartate, and asparagine-glutamine.

Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino-and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253: 164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

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 mutations 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).

The term patient includes human and veterinary subjects.

Human antibodies avoid certain of the problems associated with antibodies that possess mouse or rat variable and/or constant regions. Therefore, in one embodiment, the invention provides humanized anti-IGF-IR antibodies. More preferred are fully human anti- human IGF-IR antibodies. Fully human anti-IGF-IR antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized monoclonal antibodies (Mabs) and thus to increase the efficacy and safety of the administered antibodies.

The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as inflammation and cancer, which may require repeated antibody administrations. In another embodiment, the invention provides an anti-IGF-IR antibody that does not bind complement.

In another aspect of the invention, the anti-IGF-IR antibodies bind to IGF-IR with high affinity. In one embodiment, the anti-IGF-IR antibody binds to IGF-IR with a Kd of 1 x 10-8 M or less. In a more preferred embodiment, the antibody binds to IGF-IR with a Kd or 1 x 10-9 M or less. In an even more preferred embodiment, the antibody binds to IGF-IR with a Kd or 5 x

10-'° M or less. In another preferred embodiment, the antibody binds to IGF-IR with a Kd or 1 x 10-'° M or less. In another preferred embodiment, the antibody binds to IGF-IR with substantially the same Kd as an antibody selected from 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2, 4.17. 3 or 6.1. 1. In another preferred embodiment, the antibody binds to IGF-IR with substantially the same Kd as an antibody that comprises one or more CDRs from an antibody selected from 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1.

The invention also employs an anti-IGF-IR antibody that binds the same antigen or epitope as a human anti-IGF-IR antibody. Further, the invention can employ an anti-IGF-IR antibody that cross-competes with a human anti-IGF-IR antibody. In a preferred embodiment, the human anti-IGF-IR antibody is 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1. In another preferred embodiment, the human anti-IGF-IR comprises one or more CDRs from an antibody selected from 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1 The invention can also be practiced using an anti-IGF-IR antibody that comprises variable sequences encoded by a human K gene. In a preferred embodiment, the variable sequences are encoded by either the VK A27, A30 or 012 gene family. In a preferred embodiment, the variable sequences are encoded by a human VK A30 gene family. In a more preferred embodiment, the light chain comprises no more than ten amino acid substitutions from the germline VK A27, A30 or 012, preferably no more than six amino acid substitutions, and more preferably no more than three amino acid substitutions. In a preferred embodiment, the amino acid substitutions are conservative substitutions.

In a preferred embodiment, the VL of the anti-IGF-IR antibody contains the same amino acid substitutions, relative to the germline amino acid sequence, as any one or more of the VL of antibodies 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1.

In another preferred embodiment, the light chain comprises an amino acid sequence that is the same as the amino acid sequence of the VL of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2, 4.17. 3 or 6.1. 1. In another highly preferred embodiment, the light chain comprises amino acid sequences that are the same as the CDR regions of the light chain of 2.12. 1,2. 13.2, 2.14. 3, 3.1. 1,4. 9.2, 4.17. 3 or 6.1. 1. In another preferred embodiment, the light chain comprises an amino acid sequence from at least one CDR region of the light chain of 2.12. 1,2. 13.2, 2.14. 3, 3.1. 1,4. 9.2, 4.17. 3 or 6. 1.1.

The present invention can also be carried out using an anti-IGF-IR antibody or portion thereof comprising a human heavy chain or a sequence derived from a human heavy chain.

In one embodiment, the heavy chain amino acid sequence is derived from a human VH DP- 35, DP-47, DP-70, DP-71 or VIV-4/4. 35 gene family. In a preferred embodiment, the heavy chain amino acid sequence is derived from a human VH DP-47 gene family. In a more preferred embodiment, the heavy chain comprises no more than eight amino acid changes

from germline VH DP-35, DP-47, DP-70, DP-71 or VIV-4/4. 35, more preferably no more than six amino acid changes, and even more preferably no more than three amino acid changes.

In a preferred embodiment, the VH of the anti-IGF-IR antibody contains the same amino acid substitutions, relative to the germline amino acid sequence, as any one or more of the VH of antibodies 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1. In another embodiment, the amino acid substitutions are made in the same position as those found in any one or more of the VH of antibodies 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.17. 3., 4.9. 2 or 6.1. 1, but conservative amino acid substitutions are made rather than using the same amino acid.

In another preferred embodiment, the heavy chain comprises an amino acid sequence that is the same as the amino acid sequence of the VH of 2.12. 1,2. 13.2, 2.14. 3, 3.1. 1,4. 9.2, 4.17. 3 or 6.1. 1. In another highly preferred embodiment, the heavy chain comprises amino acid sequences that are the same as the CDR regions of the heavy chain of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1. In another preferred embodiment, the heavy chain comprises an amino acid sequence from at least one CDR region of the heavy chain of 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17.3 or 6.1. 1. In another preferred embodiment, the heavy chain comprises amino acid sequences from CDRs from different heavy chains. In a more preferred embodiment, the CDRs from different heavy chains are obtained from 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2,4. 17. 3 or 6. 1.1.

In another embodiment, the invention employs an anti-IGF-IR antibody that inhibits the binding of IGF-I to IGF-IR or the binding of IGF-II to IGF-IR. In a preferred embodiment, the IGF-IR is human. In another preferred embodiment, the anti-IGF-IR antibody is a human antibody. In another embodiment, the antibody or portion thereof inhibits binding between IGF-IR and IGF-I with an IC50 of no more than 100 nM. In a preferred embodiment, the ICgo is no more than 10 nM. In a more preferred embodiment, the IC50 is no more than 5 nM. The IC, go can be measured by any method known in the art. Typically, an IC50 can be measured by ELISA or RIA. In a preferred embodiment, the IC5o is measured by RIA.

In another embodiment, the invention employs an anti-IGF-IR antibody that prevents activation of the IGF-IR in the presence of IGF-I. In another aspect of the invention, the antibody causes the downregulation of IGF-IR from a cell treated with the antibody. In a preferred embodiment, the antibody is selected 2.12. 1,2. 13.2, 2.14. 3,3. 1.1, 4.9. 2, or 6.1. 1, or comprises a heavy chain, light chain or antigen-binding region thereof.

Human antibodies can be produced by immunizing a non-human animal comprising of some or all of the human immunoglobulin locus with an IGF-IR antigen. In a preferred embodiment, the non-human animal is a XENOMOUSE, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e. g. , Green et al. Nature Genetics 7: 13-21 (1994) and United States Patents 5,916, 771,5, 939,598, 5,985, 615,5, 998,209, 6,075, 181,6, 091,001,

6,114, 598 and 6,130, 364. See also WO 91/10741, published July 25,1991, WO 94/02602, published February 3,1994, WO 96/34096 and WO 96/33735, both published October 31, 1996, WO 98/16654, published April 23,1998, WO 98/24893, published June 11,1998, WO 98/50433, published November 12,1998, WO 99/45031, published September 10,1999, WO 99/53049, published October 21,1999, WO 00 09560, published February 24,2000 and WO 00/037504, published June 29,2000. The XENOMOUSE TM produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human Mabs. A second generation XENOMOUSE TM contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and K light chain loci. See Mendez et al. Nature Genetics 15: 146-156 (1997), Green and Jakobovits J. Exp. Med. 188: 483-495 (1998), the disclosures of which are hereby incorporated by reference.

The IGF-IR antigen can be administered with a adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.

The nucleic acid molecule encoding the variable region of the light chain may be derived from the A30, A27 or 012 VK gene. In a preferred embodiment, the light chain is derived from the A30 VK gene. In an even more preferred embodiment, the nucleic acid molecule encoding the light chain contains no more than ten amino acid changes from the germline A30 VK gene, preferably no more than six amino acid changes, and even more preferably no more than three amino acid changes.

In one embodiment, the antibody contains no greater than ten amino acid changes in either the VH or VL regions of the mutated anti-IGF-IR antibody compared to the anti-IGF-IR antibody prior to mutation. In a more preferred embodiment, there are no more than five amino acid changes in either the VH or VL regions of the mutated anti-IGF-IR antibody, more preferably no more than three amino acid changes. In another embodiment, there are no more than fifteen amino acid changes in the constant domains, more preferably, no more than ten amino acid changes, even more preferably, no more than five amino acid changes.

SEQ ID NOS: 2,6, 10,14, 18 and 22 provide the amino acid sequences of the variable regions of six anti-IGF-IR K'light chains. SEQ ID NOS: 4,8, 12,16, 20 and 24 provide the amino acid sequences of the variable regions of six anti-IGF-IR heavy chains.

SEQ ID NO: 26 depicts the amino acid sequence and SEQ ID NO: 25 depicts the nucleic acid

sequence encoding the constant region of the light chain of the anti-IGF-IR antibodies 2.12. 1, 2.13. 2,2. 14.3, 3.1. 1,4. 9.2, 4.17. 3 and 6.1. 1. SEQ ID NO: 28 depicts the amino acid sequence and SEQ ID NO: 27 depicts the nucleic acid sequence encoding the constant region of the heavy chain of the anti-IGF-IR antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17. 3 and 6.1. 1. SEQ ID NOS: 30,32, 34,36 and 44 provide the amino acid sequences of the germline heavy chains DP-35, DP-47, DP-70, DP-71 and VIV-4, respectively. SEQ ID NO: 33 provides the nucleotide sequence of the germline heavy chain DP-70. SEQ ID NOS: 38, 40 and 42 provide the amino acid sequences of the three germline K light chains from which the six anti-IGF-IR K light chains are derived.

In another preferred embodiment, the invention relates to the use of anti-lGF-1R in the prevention of aging.

In another embodiment, the invention relates to pharmaceutical compositions for the treatment of a mammal that requires activation of IGF-IR, wherein the pharmaceutical composition comprises a therapeutical effective amount of an activating antibody of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising activating antibodies may be used to treat animals that lack sufficient IGF-I or IGF- li.

The anti-IGF-IR antibodies can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody and a pharmaceutically acceptable carrier. As used herein,"pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutical acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e. g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e. g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the

antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the anti-IGF-IR antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution 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 by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The antibodies can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intraperitoneal, subcutaneous, intramuscular, intravenous or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In one embodiment, the antibodies can be administered as a single dose or may be administered as multiple doses.

In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e. g. , Sustained and Controlled Release Drug Delivery Systems, J. R.

Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the antibody may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets,

buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an anti-IGF-IR antibody is coformulated with and/or coadministered with one or more additional therapeutic agents, such as anti-emetics, cancer vaccines, analgesics, anti-vascular agents, and anti-proliferative agents.

The pharmaceutical composition may include a"therapeutically effective amount"or a "prophylactically effective amount"of an antibody or antibody portion of the invention. A "therapeutically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A"prophylactically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e. g. , a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

Pharmaceutical composition comprising the antibody or comprising a combination therapy comprising the antibody and one or more additional therapeutic agents may be formulated for single or multiple doses. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects 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. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in

individuals. A particularly useful formulation is 5 mg/ml anti-IGF-IR antibody in a buffer of 20mM sodium citrate, pH 5.5, 140mM NaCI, and 0. 2mg/ml polysorbate 80.

An exemplary,, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.

It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. In one embodiment, the therapeutically or prophylactically effective amount of an antibody or antigen-binding portion thereof is administered along with one or more additional therapeutic agents.

The antibody employed in the method of the invention can be labeled. This can be done by incorporation of a detectable marker, e. g. , incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e. g. , streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following : radioisotopes or radionuclides (e. g., 3H, 14C, 15N, 35S, 90y, 99Tc,"'in, 1251, 1311), fluorescent labels (e. g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e. g., horseradish peroxidase, ß-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e. g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The antibodies employed in the present invention are preferably derived from cells that express human immunoglobulin genes. Use of transgenic mice is known in the art to produce such"human"antibodies. One such method is described in Mendez et al. Nature Genetics 15: 146-156 (1997), Green and Jakobovits J. Exp. Med. 188: 483-495 (1998), and U. S. Patent Application Serial 08/759,620 (filed December 3,1996). The use of such mice to obtain human antibodies is also described in U. S. Patent Applications 07/466,008 (filed January 12,1990), 07/610,515 (filed November 8,1990), 07/919,297 (filed July 24,1992), 07/922,649 (filed July 30,1992), filed 08/031,801 (filed March 15,1993), 08/112,848 (filed August 27,1993), 08/234,145 (filed April 28,1994), 08/376,279 (filed January 20,1995), 08/430,938 (filed April 27,1995), 08/464, 584 (filed June 5,1995), 08/464,582 (filed June 5,

1995), 08/463,191 (filed June 5,1995), 08/462,837 (filed June 5,1995), 08/486,853 (filed June 5,1995), 08/486,857 (filed June 5,1995), 08/486,859 (filed June 5,1995), 08/462,513 (filed June 5,1995), 08/724,752 (filed October 2,1996), and 08/759,620 (filed December 3, 1996). See also Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188: 483-495 (1998). See also European Patent EP 0 463 151 (grant published June 12,1996), International Patent Application WO 94/02602 (published February 3,1994), International Patent Application WO 96/34096 (published October 31,1996), and WO 98/24893 (published June 11,1998).

As noted above, the invention encompasses use of antibody fragments (included herein in the definition of"antibody"). Antibody fragments, such as Fv, F (ab') 2 and Fab may be prepared by cleavage of the intact protein, e. g. by protease or chemical cleavage.

Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F (ab') 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

In one approach, consensus sequences encoding the heavy and light chain J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.

C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

Expression vectors for use in obtaining the antibodies employed in the invention include plasmids, retroviruses, cosmids, YACs, EBV derived episomes, and the like. A convenient vector is normally one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons.

Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e. g. SV-40 early promoter, (Okayama et al. Mol. Cell. Bio. 3: 280 (1983) ), Rous sarcoma virus LTR (Gorman et al. P. N. A. S. 79: 6777 (1982) ), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41 : 885 (1985) ) ; native Ig promoters, etc.

Antibodies that are generated for use in the invention need not initially possess a particular desired isotype. Rather, the antibody as generated can possess any isotype and can be isotype switched thereafter using conventional techniques. These include direct recombinant techniques (see e. g. , U. S. Patent 4,816, 397), and cell-cell fusion techniques (see e. g. , U. S. Patent Application 08/730,639 (filed October 11,1996).

As noted above, the effector function of the antibodies of the invention may be changed by isotype switching to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeutic uses. Furthermore, dependence on complement for cell killing can be avoided through the use of bispecifics, immunotoxins, or radiolabels, for example.

Bispecific antibodies can be generated that comprise (i) two antibodies: one with a specificity for IGF-IR and the other for a second molecule (ii) a single antibody that has one chain specific for IGF-IR and a second chain specific for a second molecule, or (iii) a single chain antibody that has specificity for IGF-IR and the other molecule. Such bispecific antibodies can be generated using well known techniques, e. g. , Fanger et al. Immunol Methods 4: 72-81 (1994), Wright and Harris, supra, and Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992).

Antibodies for use in the invention also include"kappabodies" (III et al."Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions"Protein Eng 10: 949-57 (1997) ),"minibodies" (Martin et al."The affinity- selection of a minibody polypeptide inhibitor of human interleukin-6"EMBO J 13: 5303-9 (1994) ),"diabodies" (Holliger et al."'Diabodies' : small bivalent and bispecific antibody fragments"PNAS USA 90: 6444-6448 (1993) ), and"janusins" (Traunecker et al."Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells"EMBO J 10: 3655-3659 (1991) and Traunecker et al."Janusin : new molecular design for bispecific reagents"Int J Cancer Suppl 7 : 51-52 (1992) ) may also be prepared.

The antibodies employed can be modified to act as immunotoxins by conventional techniques. See e. g., Vitetta Immunol Today 14: 252 (1993). See also U. S. Patent 5,194, 594.

Radiolabeled antibodies can also be prepared using well-known techniques. See e. g., Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds. , Lippincott Raven (1996) ). See also U. S. Patents. 4,681, 581, 4, 735, 210, 5,101, 827,5, 102,990 (RE 35,500), 5,648, 471, and 5,697, 902.

Particular antibodies useful in practice of the invention include those described in WO 02/053596, which further describes antibodies 2.12. 1,2. 13.2., 2.14. 3,3. 1.1, 4.9. 2, and 4.17. 3.

As disclosed in that published application, hybridomas producing these antibodies were deposited in the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on December 12,2000 with the following deposit numbers: Hybridoma Deposit No.

2.12. 1 PTA-2792 2.13. 2 PTA-2788 2.14. 3 PTA-2790 3.1. 1 PTA-2791 4.9. 2 PTA-2789

4.17. 3 PTA-2793 These antibodies are either fully human IgG2 or IgG4 heavy chains with human kappa light chains. In particular the invention concerns use of antibodies having amino acid sequences of these antibodies.

Antibodies employed in the invention preferably possess very high affinities, typically possessing Kds of from about 10-9 through about 10-11 M, when measured by either solid phase or solution phase.

Antibodies used in the present invention can be expressed in cell lines other than hybridoma cell lines. Sequences encoding the cDNAs or genomic clones for the particular antibodies can be used for transformation of suitable mammalian or nonmammalian host cells. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U. S. Patents 4,399, 216,4, 912,040, 4,740, 461, and 4,959, 455. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, particle bombardment, encapsulation of the polynucleotide (s) in liposomes, peptide conjugates, dendrimers, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, NSOo, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e. g. , Hep G2). Non-mammalian cells can also be employed, including bacterial, yeast, insect, and plant cells. Site directed mutagenesis of the antibody CH2 domain to eliminate glycosylation may be preferred in order to prevent changes in either the immunogenicity, pharmacokinetic, and/or effector functions resulting from non-human glycosylation. The glutamin synthase system of expression is discussed in whole or part in connection with European Patents 216 846,256 055, and 323 997 and European Patent Application 89303964.4.

Antibodies for use in the invention can also be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. Transgenic antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e. g. , U. S. Patents 5,827, 690,5, 756,687, 5,750, 172, and 5,741, 957.

The antibody, with or without an additional agent, may be administered once, but more preferably is administered multiple times. The antibody may be administered from three times daily to once every six months. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months. The antibody may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, intratumor or topical route.

In certain embodiments, the antibody may be administered in an aerosol or inhaleable form. Dry aerosol in the form of finely divided solid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. The pharmaceutical formulations of the present invention may be administered in the form of an aerosol spray using for example, a nebulizer such as those described in U. S. Pat. Nos.

4,624, 251 issued Nov. 25,1986 ; 3,703, 173 issued Nov. 21,1972 ; 3,561, 444 issued Feb. 9, 1971 and 4,635, 627 issued Jan. 13,1971.

Hubbard, R. C. et al. (Proc. Natl. Acad. Sci. (USA) 86: 680-684,1989) disclose the administration of a relatively large protein alpha. sub. 1-antitrypsin (AAt) via the pulmonary epithelial surface for the treatment of alpha anti-trypsin deficiency. AAt, a 45,000 dalton molecular weight single-chain polypeptide that functions as an inhibitor of neutrophil elastase was administered to sheep in an aerosol form. Aerosolized AAt remained fully functional and intact in the tissues of the mammal and diffused across the alveolar epithelium, as evidenced by the presence of AAt in the lung, lymph and blood tissue.

The antibody may be administered at a site distant from the site of the tumor. The antibody may also be administered continuously via a minipump. The antibody may be administered once, at least twice or for at least the period of time until the condition is treated, palliated or cured. The antibody generally will be administered for as long as the tumor is present provided that the antibody causes the tumor or cancer to stop growing or to decrease in weight or volume. The antibody will generally be administered as part of a pharmaceutical composition as described supra. The dosage of antibody will generally be in the range of 0.1- 100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. The serum concentration of the antibody may be measured by any method known in the art. The antibody may also be administered prophylactically in order to prevent a cancer or tumor from occurring. This may be especially useful in patients that have a"high normal"level of IGF-I because these patients have been shown to have a higher risk of developing common cancers. See Rosen et al., supra.

Co-administration of the antibody with an additional therapeutic agent (combination therapy) encompasses administering a pharmaceutical composition comprising the anti-IGF-

IR antibody and the additional therapeutic agent and administering two or more separate pharmaceutical compositions, one comprising the anti-IGF-IR antibody and the other (s) comprising the additional therapeutic agent (s). Further, although co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the antibody and additional therapeutic agents are administered at different times. For instance, the antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody may be administered prior to or subsequent to treatment of the disorder with the additional therapeutic agent. Similarly, administration of the anti-IGF-IR antibody may be administered prior to or subsequent to other therapy, such as radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLE) : Effects of the Antibodies of the Invention on IGF-IR in vivo We induced tumors in athymic mice according to published methods (V. A. Pollack et al.,"Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774 : Dynamics of receptor inhibition in situ and antitumor effects in athymic mice,"J. Pharmacol. Exp. Ther. 291: 739-748 (1999). Briefly, we injected IGF-IR-transfected NIH-3T3 cells (5x103) suboutaneously into 3-4 week-old athymic (nulnu) mice with 0.2 ml of Matrigel preparation. We then injected mice with an antibody of the invention intraperitoneally after established (i. e. approximately 400 mm3) tumors formed.

After 24 hours, we extracted the tumors, homogenized them and determined the level of IGF-IR. To determine IGF-IR levels, we diluted the SC-713 antibody in Blocking buffer to a final concentration of 4 ug/ml and added 100 pi to each well of a Reacti-Bind Goat anti-rabbit (GAR) coated plate (Pierce). We incubated the plates at room temperature for 1 hour with shaking and then washed the plates five times with wash buffer. We then weighed tumor samples that had been prepared as described above and homogenized them in lysis buffer (1 ml/100 mg). We diluted 12. 5 pl of tumor extract with lysis buffer to a final volume of 100 pl and added this to each well of a 96-well plate. We incubated the plates at room temperature with shaking for 1-2 hours and then washed the plates five times with Wash buffer. We then added 100, ul of biotinylated anti-lGF-IR antibody in Blocking buffer to each well and incubated

at room temperature with shaking for 30 minutes. We then washed the plates five times with wash buffer. We developed the plates probed with anti-IGF-IR antibody by adding 100 pi of streptavidin-HRP diluted in Blocking buffer to each well, incubating at room temperature with shaking for 30 minutes. We developed the plates by adding 100 pl of the TMB microwell substrate per well and stopped color development with the addition 100 pl 0.9 M H2SO4. We then quantitated the signal by measuring the OD45onm. The signal was normalized to total protein.

We observed that intraperitoneal injection of an antibody of this invention, particularly 2.13. 2 and 4.9. 2, resulted in inhibition of IGF-IR activity as measured by a decrease of both IGF-IR phosphotyrosine (phosphorylated IGF-IR) and total IGF-IR protein (Figure 4).

Furthermore, this inhibition was responsive to the dose of antibody injected (Figure 4). These data demonstrate that the antibodies of the invention are able to target the IGF-IR in vivo in a manner analogous to what we observed in vitro.

EXAMPLE 11 : Growth Inhibition (TGI) of 3T3/lGF-IR Cell Tumors We tested whether anti-IGF-IR antibodies of the invention would function to inhibit tumor growth. We induced tumors as described above (Example I) and when established, palpable tumors formed (i. e. 250 mm3, within 6-9 days), we treated the mice with a single, 0.20 ml dose of antibody by intraperitoneal injection. We measured tumor size by Vernier calipers across two diameters every third day and calculated the volume using the formula (length x [width] 2)/2 using methods established by Geran, et al.,"Protocols for screening chemical agents and natural products against animal tumors and other biological systems," Cancer Chemother. Rep. 3: 1-104.

When we performed this analysis with an antibody of the invention, we found that a single treatment with antibody 2.13. 2 alone inhibited the growth of IGF-IR-transfected NIH- 3T3 cell-induced tumors (Figure 5).

Detailed Description Of The Drawings Figs. 1A-1C show alignments of the nucleotide sequences of the light chain variable regions from six human anti-IGF-IR antibodies to each other and to germline sequences. Fig.

1A shows the alignment of the nucleotide sequences of the variable region of the light chain (VL) of antibodies 2.12. 1 (SEQ ID NO: 1) 2.13. 2 (SEQ ID NO: 5), 2.14. 3 (SEQ ID NO: 9) and 4.9. 2 (SEQ ID NO: 13) to each other and to the germline VK A30 sequence (SEQ ID NO: 39).

Fig. 1B shows the alignment of the nucleotide sequence of VL of antibody 4.17. 3 (SEQ ID NO: 17) to the germline VK 012 sequence (SEQ ID NO: 41). Fig. 1C shows the alignment of the nucleotide sequence of VL of antibody 6.1. 1 (SEQ ID NO: 21) to the germline VK A27 sequence (SEQ ID NO: 37). The alignments also show the CDR regions of the VL from each antibody. The consensus sequences for Figs. 1A-1C are shown in SEQ ID NOS: 53-55, respectively.

Figs. 2A-2D show alignments of the nucleotide sequences of the heavy chain variable regions from six human anti-IGF-IR antibodies to each other and to germline sequences. Fig.

2A shows the alignment of the nucleotide sequence of the VH of antibody 2.12. 1 (SEQ ID NO: 3) to the germline VH DP-35 sequence (SEQ ID NO: 29). Fig. 2B shows the alignment of the nucleotide sequence of the VH of antibody 2.14. 3 (SEQ ID NO: 11) to the germline VIV- 4/4. 35 sequence (SEQ ID NO: 43). Figs. 2C-1 and 2C-2 show the alignments of the nucleotide sequences of the VH of antibodies 2.13. 2 (SEQ ID NO: 7), 4.9. 2 (SEQ ID NO: 15) and 6.1. 1 (SEQ ID NO: 23) to each other and to the germline VH DP-47 sequence (SEQ ID NO: 31). Fig. 2D shows the alignment of the nucleotide sequence of the VH of antibody 4.17. 3 (SEQ ID NO: 19) to the germline VH DP-71 sequence (SEQ ID NO: 35). The alignment also shows the CDR regions of the antibodies. The consensus sequences for Figs. 2A-2D are shown in SEQ ID NOS: 56-59, respectively.

Fig. 3A shows the number of mutations in different regions of the heavy and light chains of 2.13. 2 and 2.12. 1 compared to the germline sequences. Figs. 3A-D show alignments of the amino acid sequences from the heavy and light chains of antibodies 2.13. 2 and 2.12. 1 with the germline sequences from which they are derived. Fig. 3B shows an alignment of the amino acid sequence of the heavy chain of antibody 2.13. 2 (SEQ ID NO: 45) with that of germline sequence DP-47 (3-23) /D6-19/JH6 (SEQ ID NO: 46). Fig. 3C shows an alignment of the amino acid sequence of the light chain of antibody 2.13. 2 (SEQ ID NO: 47) with that of germline sequence A30/Jk2 (SEQ ID NO: 48). Fig. 3D shows an alignment of the amino acid sequence of the heavy chain of antibody 2.12. 1 (SEQ ID NO: 49) with that of germline sequence DP-35 (3-11)/D3-3/JH6 (SEQ ID NO: 50). Fig. 3E shows an alignment of the amino acid sequence of the light chain of antibody 2.12. 1 (SEQ ID NO: 51) with that of germline sequence A30/Jk1 (SEQ ID NO: 52). For Figures 3B-E, the signal sequences are in italic, the CDRs are underlined, the constant domains are bold, the framework (FR) mutations are highlighted with a plus sign ("+") above the amino acid residue and CDR mutations are highlighted with an asterisk above the amino acid residue.

Figure 4 shows that anti-IGF-IR antibodies 2.13. 2 and 4.9. 2 reduce IGF-IR phosphotyrosine signal in 3T3-IGF-IR tumors.

Figure 5 shows that anti-IGF-IR antibody 2.13. 2 inhibits 3T3-IGF-IR tumor growth in vivo.

SEQUENCE LISTING <110> Cohen, Bruce D.

Bedian, Vahe Obrocea, Mihail Gomez-Navarro, Jesus Cusmano, John D.

Wang, Huifen F.

Page, Kelly L.

Guyot, Deborah J.

<120> USES OF ANTI-INSULIN-LIKE GROWTH FACTOR I RECEPTOR ANTIBODIES <130> PC25232A <140> <141> <160> 60 <170> PatentIn Ver. 2.1 <210> 1 <211> 291 <212> DNA <213> Homo sapiens <400> 1 tgcatctgta ggagacagag tcaccttcac ttgccgggca agtcaggaca ttagacgtga 60 tttaggctgg tatcagcaga aaccagggaa agctcctaag cgcctgatct atgctgcatc 120 ccgtttacaa agtggggtcc catcaaggtt cagcggcagt ggatctggga cagaattcac 180 tctcacaatc agcagcctgc agcctgaaga ttttgcaact tattactgtc tacagcataa 240 taattatcct cggacgttcg gccaagggac cgaggtggaa atcatacgaa c 291 <210> 2 <211> 136 <212> PRT <213> Homo sapiens <400> 2 Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp 1 5 10 15 Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 20 25 30 Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser 35 40 45 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 50 55 60 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn 65 70 75 80 Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val Glu Ile Ile Arg

85 90 95 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 100 105 110 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125 Pro Arg Glu Ala Lys Val Gln Trp 130 135 <210> 3 <211> 352 <212> DNA <213> Homo sapiens <400> 3 gggaggcttg gtcaagcctg gaggtccctg agactctcct gtgcagcctc tggattcact 60 ttcagtgact actatatgag ctggatccgc caggctccag ggaaggggct ggaatgggtt 120 tcatacatta gtagtagtgg tagtaccaga gactacgcag actctgtgaa gggccgattc 180 accatctcca gggacaacgc caagaactca ctgtatctgc aaatgaacag cctgagagcc 240 gaggacacgg ccgtgtatta ctgtgtgaga gatggagtgg aaactacttt ttactactac 300 tactacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc ag 352 <210> 4 <211> 174 <212> PRT <213> Homo sapiens <400> 4 Gly Arg Leu Gly Gln Ala Trp Arg Ser Leu Arg Leu Ser Cys Ala Ala 1 5 10 15 Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala 20 25 30 Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser 35 40 45 Thr Arg Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 50 55 60 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 65 70 75 80 Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Gly Val Glu Thr Thr 85 90 95 Phe Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ser Cys Ala 165 170 <210> 5 <211> 322 <212> DNA <213> Homo sapiens <400> 5 gacatccaga tgacccagtt tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatcccgtt tgcacagagg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtttacaa cataatagtt acccgtgcag ttttggccag 300 gggaccaagc tggagatcaa ac 322 <210> 6 <211> 107 <212> PRT <213> Homo sapiens <400> 6 Asp Ile Gln Met Thr Gln Phe 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 Arg Leu His Arg 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 Cys 85 90 95 Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 375 <212> DNA <213> Homo sapiens <400> 7

aggtgcagct gttggagtct gggggaggct tggtacagcc tggggggtcc ctgagactct 60 cctgtacagc ctctggattc acctttagca gctatgccat gaactgggtc cgccaggctc 120 cagggaaggg gctggagtgg gtctcagcta ttagtggtag tggtggtacc acattctacg 180 cagactccgt gaagggccgg ttcaccatct ccagagacaa ttccaggacc acgctgtatc 240 tgcaaatgaa cagcctgaga gccgaggaca cggccgtata ttactgtgcg aaagatcttg 300 gctggtccga ctcttactac tactactacg gtatggacgt ctggggccaa gggaccacgg 360 tcaccgtctc ctcag 375 <210> 8 <211> 124 <212> PRT <213> Homo sapiens <400> 8 Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 302 <212> DNA <213> Homo sapiens <400> 9 tcctccctgt ctgcatctgt aggagacaga gtcaccttca cttgccgggc aagtcaggac 60 attagacgtg atttaggctg gtatcagcag aaaccaggga aagctcctaa gcgcctgatc 120 tatgctgcat cccgtttaca aagtggggtc ccatcaaggt tcagcggcag tggatctggg 180 acagaattca ctctcacaat cagcagcctg cagcctgaag attttgcaac ttattactgt 240 ctacagcata ataattatcc tcggacgttc ggccaaggga ccgaggtgga aatcatacga 300 ac 302 <210> 10 <211> 100 <212> PRT

<213> Homo sapiens <400> 10 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg 1 5 10 15 Ala Ser Gln Asp Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro 20 25 30 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser 35 40 45 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 50 55 60 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 65 70 75 80 Leu Gln His Asn Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val 85 90 95 Glu Ile Ile Arg 100 t <210> 11 <211> 338 <212> DNA <213> Homo sapiens <400> 11 gggcccagga ctggtgaagc cttcggagac cctgtccctc acctgcactg tctctggtgg 60 ctccatcagt aattactact ggagctggat ccggcagccc gccgggaagg gactggagtg 120 gattgggcgt atctatacca gtgggagccc caactacaac ccctccctca agagtcgagt 180 caccatgtca gtagacacgt ccaagaacca gttctccctg aagctgaact ctgtgaccgc 240 cgcggacacg gccgtgtatt actgtgcggt aacgattttt ggagtggtta ttatctttga 300 ctactggggc cagggaaccc tggtcaccgt ctcctcag 338 <210> 12 <211> 112 <212> PRT <213> Homo sapiens <400> 12 Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr 1 5 10 15 Val Ser Gly Gly Ser Ile Ser Asn Tyr Tyr Trp Ser Trp Ile Arg Gln 20 25 30 Pro Ala Gly Lys Gly Leu Glu Trp Ile Gly Arg Ile Tyr Thr Ser Gly 35 40 45 Ser Pro Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val 50 55 60

<210> 13 <211> 322 <212> DNA <213> Homo sapiens <210> 14 <211> 107 <212> PRT <213> Homo sapiens

<212> DNA <213> Homo sapiens <400> 15 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtat cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatctg 300 ggctacggtg acttttacta ctactactac ggtatggacg tctggggcca agggaccacg 360 gtcaccgtct cctcag 376 <210> 16 <211> 125 <212> PRT <213> Homo sapiens <400> 16 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ile Thr 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 Leu Gly Tyr Gly Asp Phe Tyr Tyr Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 17 <211> 279 <212> DNA <213> Homo sapiens <400> 17 caggagacag agtcaccatc acttgccggg caagtcagag cattagtacc tttttaaatt 60 ggtatcagca gaaaccaggg aaagccccta aactcctgat ccatgttgca tccagtttac 120 aaggtggggt cccatcaagg ttcagtggca gtggatctgg gacagatttc actctcacca 180 tcagcagtct gcaacctgaa gattttgcaa cttactactg tcaacagagt tacaatgccc 240 cactcacttt cggcggaggg accaaggtgg agatcaaac 279

<210> 18 <211> 92 <212> PRT <213> Homo sapiens <400> 18 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr 1 5 10 15 Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 20 25 30 Ile His Val Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser 35 40 45 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Ala Pro 65 70 75 80 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 85 90 <210> 19 <211> 341 <212> DNA <213> Homo sapiens <400> 19 cccaggactg gtgaagcctt cggagaccct gtccctcacc tgcactgtct ctggtggctc 60 catcagtagt tactactgga gttggatccg gcagccccca gggaagggac tggagtggat 120 tgggtatatc tattacagtg ggagcaccaa ctacaacccc tccctcaaga gtcgagtcac 180 catatcagta gacacgtcca agaaccagtt ctccctgaag ctgagttctg tgaccgctgc 240 ggacacggcc gtgtattact gtgccaggac gtatagcagt tcgttctact actacggtat 300 ggacgtctgg ggccaaggga ccacggtcac cgtctcctca g 341 <210> 20 <211> 113 <212> PRT <213> Homo sapiens <400> 20 Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val 1 5 10 15 Ser Gly Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro 20 25 30 Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser 35 40 45 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp 50 55 60

Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala 65 70 75 80 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Ser Ser Ser Phe Tyr 85 90 95 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser <210> 21 <211> 274 <212> DNA <213> Homo sapiens <400> 21 agagccaccc tctcctgtag ggccagtcag agtgttcgcg gcaggtactt agcctggtac 60 cagcagaaac ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact 120 ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc 180 agactggagc ctgaagattt tgcagtgttt tactgtcagc agtatggtag ttcacctcgn 240 acgttcggcc aagggaccaa ggtggaaatc aaac 274 <210> 22 <211> 91 <212> PRT <213> Homo sapiens <400> 22 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr 1 5 10 15 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 20 25 30 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 35 40 45 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 50 55 60 Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg 65 70 75 80 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 85 90 <210> 23 <211> 367 <212> DNA <213> Homo sapiens <400> 23

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggt attactggga gtggtggtag tacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcca 300 gggactacgg tgattatgag ttggttcgac ccctggggcc agggaaccct ggtcaccgtc 360 tcctcag 367 <210> 24 <211> 122 <212> PRT <213> Homo sapiens <400> 24 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr 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 Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 320 <212> DNA <213> Homo sapiens <400> 25 gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60 gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120 ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180 gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240 aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300 gcttcaacag gggagagtgt 320 <210> 26 <211> 106 <212> PRT

<213> Homo sapiens <400> 26 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 27 <211> 978 <212> DNA <213> Homo sapiens <400> 27 gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300 aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960 tccctgtctc cgggtaaa 978 <210> 28 <211> 326 <212> PRT <213> Homo sapiens <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90-95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn.

225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 29 <211> 296 <212> DNA <213> Homo sapiens <400> 29 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaga 296 <210> 30 <211> 98 <212> PRT <213> Homo sapiens <400> 30 Gln 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 <210> 31 <211> 296 <212> DNA <213> Homo sapiens <400> 31 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaga 296 <210> 32 <211> 98 <212> PRT <213> Homo sapiens <400>, 32 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 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 <210> 33 <211> 296 <212> DNA <213> Homo sapiens <400> 33 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60 acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 120 cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 180 aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 240 ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagaga 296 <210> 34 <211> 98 <212> PRT <213> Homo sapiens <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45 Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 35 <211> 293 <212> DNA <213> Homo sapiens <400> 35 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aga 293 <210> 36 <211> 97 <212> PRT <213> Homo sapiens <400> 36 Gln 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 <210> 37

<211> 290 <212> DNA <213> Homo sapiens <210> 38 <211> 96 <212> PRT <213> Homo sapiens <210> 39 <211> 288 <212> DNA <213> Homo sapiens <210> 40 <211> 96 <212> PRT <213> Homo sapiens

<210> 41 <211> 288 <212> DNA <213> Homo sapiens <210> 42 <211> 96 <212> PRT <213> Homo sapiens

<210> 43 <211> 293 <212> DNA <213> Homo sapiens <210> 44 <211> 97 <212> PRT <213> Homo sapiens <210> 45 <211> 470 <212> PRT <213> Homo sapiens

Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 46 <211> 470 <212> PRT <213> Homo sapiens <400> 46 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110 Tyr Tyr Cys Ala Lys Gly Tyr Ser Ser Gly Trp Tyr Tyr Tyr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 47 <211> 236 <212> PRT <213> Homo sapiens <400> 47 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Phe Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu His Arg Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser Tyr Pro Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 48 <211> 236 <212> PRT <213> Homo sapiens <400> 48 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile G1n Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 ( Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 49 <211> 470 <212> PRT <213> Homo sapiens <400> 49 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly 1 5 10 15 Val Gln Cys Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Arg Asp Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Val Arg Asp Gly Val Glu Thr Thr Phe Tyr Tyr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 50 <211> 473 <212> PRT <213> Homo sapiens <400> 50 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Val Leu Arg Phe Leu Glu Trp Leu Leu Tyr Tyr 115 120 125 Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 130 135 140 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 165 170 175 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180 185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 195 200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 210 215 220 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys 245 250 255 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 325 330 335

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln '450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 51 <211> 236 <212> PRT <213> Homo sapiens <400> 51 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg Ala Ser 35 40 45 Gln Asp Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val Glu Ile 115 120 125

Ile Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 52 <211> 236 <212> PRT <213> Homo sapiens <400> 52 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 53 <211> 326 <212> DNA <213> Artificial Sequence <400> 53 gacatccaga tgacccagty tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 wtcacttgcc gggcaagtca ggrcattaga mrtgatttag gctggtwtca gcagaaacca 120 gggaaagcyc ctaagcgcct gatctatgct gcatccmrwt trcammgwgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcmg cctgcagcct 240 gaagattttg caacttatta ctgtytacar cataatartt aycckybsns kttyggcsrr 300 gggaccrags tggaratcaw acgaac 326 <210> 54 <211> 322 <212> DNA <213> Artificial Sequence <400> 54 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgyaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagy asctwtttaa attggtatca gcagaaacca 120 gggaaagccc ctaarctcct gatcyatgyt gcatccagtt trcaargtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacartr ccccayychc tttcggcgga 300 gggaccaagg tggagatcaa ac 322 <210> 55 <211> 325 <212> DNA <213> Artificial Sequence <400> 55 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgya gggccagtca gagtgttmgc rgcagstact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgtw ttactgtcag cagtatggta gytcacctcs nacgttcggc 300

caagggacca aggtggaaat caaac 325 <210> 56 <211> 376 <212> DNA <213> Artificial Sequence <400> 56 caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt cacyttcagt gactactaya tgagctggat ccgccaggct 120 ccagggaagg ggctggartg ggtttcatac attagtagta gtggtagtac cakakactac 180 gcagactctg tgaagggccc attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgy gagagatgga 300 gtggaaacta ctttttacta ctactactac ggtatggacg tctggggcca agggaccacg 360 gtcaccgtct cctcag 376 <210> 57 <211> 358 <212> DNA <213> Artificial Sequence <400> 57 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt arttactact ggagctggat ccggcagccc 120 gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcmc caactacaac 180 ccctccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240 aagctgarct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcggt aacgattttt 300 ggagtggtta ttatctttga ctactggggc cagrganccc tggtcaccgt ctcctcag 358 <210> 58 <211> 418 <212> DNA <213> Artificial Sequence <400> 58 caggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtrcag cctctggatt cacctttagc agctatgcca tgarctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagst attastggka gtggtggtab yacatwctac 180 gcagactccg tgaagggccc gttcaccatc tccagagaca attccargam cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatctk 300 ggctrsksyg actyttacta ctactactac ggtatggacg tctggggcca agggacyacg 360 gtgattatga gttggttcga cccctggggc cagggaaccc tggtcaccgt ctcctcag 418 <210> 59 <211> 364 <212> DNA <213> Artificial Sequence <400> 59 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagytggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgact caccatatca gtagacacgt ccaagaacca gttctccctg 240

<210> 60 <211> 15 <212> PRT <213> Artificial Sequence