Treatment with anti-alpha2 integrin antibodies

TECHNICAL FIELD

The present invention relates to treatment of cancer. More specifically the invention relates to methods of treating cancer by administering antibodies directed to .alpha.2.beta.1 integrin.

BACKGROUND OF THE INVENTION

The integrin .alpha.2.beta.1 (Very late antigen 2; VLA-2) is expressed on a variety of cell types including platelets, vascular endothelial cells, epithelial cells, activated monocytes/macrophages, fibroblasts, leukocytes, lymphocytes, activated neutrophils and mast cells. (Hemler, Annu Rev Immunol 8:365:365-400 (1999); Wu and Santoro, Dev. Dyn. 206:169-171 (1994); Edelson et. al., Blood. 103(6):2214-20 (2004); Dickeson et al, Cell Adhesion and Communication. 5: 273-281 (1998)). The most typical ligands for .alpha.2.beta.1 include collagen and laminin, both of which are found in extracellular matrix. Typically the I-domain of the .alpha.2 integrin binds to collagen in a divalent-cation dependent manner whereas the same domain binds to laminin through both divalent-cation dependent and independent mechanisms. (Dickeson et al, Cell Adhesion and Communication. 5: 273-281 (1998)) The specificity of the .alpha.2.beta.1 integrin varies with cell type and serves as a collagen and/or laminin receptor for particular cell types, for example .alpha.2.beta.1 integrin is known as a collagen receptor for platelets and a laminin receptor for endothelial cells. (Dickeson et al, J. Biol. Chem. 272: 7661-7668 (1997)) Echovirus-1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepellin and multiple collectins and the C1q complement protein are also ligands for .alpha.2.beta.1 integrin. (Edelson et al., Blood 107(1): 143-50 (2006)) The .alpha.2.beta.1 integrin has been implicated in several biological and pathological processes including collagen-induced platelet aggregation, cell migration on collagen, cell-dependent reorganization of collagen fibers as well as collagen-dependent cellular responses that result in increases in cytokine expression and proliferation, (Gendron, J. Biol. Chem. 278:48633-48643 (2003); Andreasen et al., J. Immunol. 171:2804-2811 (2003); Rao et al., J. Immunol. 165(9):4935-40 (2000)), aspects of T-cell, mast cell, and neutrophil function (Chan et. al., J. Immunol. 147:398-404 (1991); Dustin and de Fougerolles, Curr Opin Immunol 13:286-290 (2001), Edelson et. al., Blood. 103(6):2214-20 (2004), Werr et al., Blood 95:1804-1809 (2000), aspects of delayed type hypersensitivity contact hypersensitivity and collagen-induced arthritis (de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000); Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002)), mammary gland ductal morphogenesis (Keely et. al., J. Cell Sci. 108:595-607 (1995); Zutter et al., Am. J. Pathol. 155(3):927-940 (1995)), epidermal wound healing (Pilcher et. al., J. Biol. Chem. 272:181457-54 (1997)), and processes associated with VEGF-induced angiogenesis (Senger et al., Am. J. Pathol. 160(1):195-204 (2002)).

Integrin/ligand interactions can facilitate leukocyte extravasation into inflamed tissues (Jackson et al., J. Med. Chem. 40:3359-3368 (1997); Gadek et al., Science 295(5557):1086-9 (2002), Sircar at al., Bioorg. Med. Chem. 10:2051-2066 (2002)), and play a role in downstream events following the initial extravasation of leukocytes from the circulation into tissues in response to inflammatory stimuli, including migration, recruitment and activation of pro-inflammatory cells at the site of inflammation (Eble J. A., Curr. Phar. Des. 11(7):867-880 (2005)). Some antibodies that block .alpha.2.beta.1 integrin were reported to show impact on delayed hypersensitivity responses and efficacy in a murine model of rheumatoid arthritis and a model of inflammatory bowel disease (Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002); de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000) and were reported to attenuate endothelial cell proliferation and migration in vitro (Senger et al., Am. J. Pathol. 160(1):195-204 (2002), suggesting that the blocking of .alpha.2.beta.1 integrin might prevent/inhibit abnormal or higher than normal angiogenesis, as observed in various cancers.

.alpha.2.beta.1 integrin is the only collagen-binding integrin expressed on platelets and has been implicated to play some role in platelet adhesion to collagen and hemostasis (Gruner et al., Blood 102:4021-4027 (2003); Nieswandt and Watson, Blood 102(2):449-461 (2003); Santoro et al., Thromb. Haemost. 74:813-821 (1995); Siljander et al., Blood 15:1333-1341 (2004); Vanhoorelbeke at al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3(2):125-40 (2003)). In addition, platelet .alpha.2.beta.1 may play a role in the regulation of the size of the platelet aggregate (Siljander et al., Blood 103(4):1333-1341 (2004)).

.alpha.2.beta.1 integrin has also been shown as a laminin-binding integrin expressed on endothelial cells (Languino et al., J Cell Bio. 109:2455-2462 (1989)). Endothelial cells are thought to attach to laminin through an integrin-mediated mechanism, however it has been suggested that the .alpha.2 I domain may function as a ligand-specific sequence involved in mediating endothelial cell interactions (Bahou et al., Blood. 84(11):3734-3741 (1994)).

It is anticipated that a therapeutic antibody that binds .alpha.2.beta.1 integrin, including the .alpha.2.beta.1 integrin on platelets, could result in bleeding complications. For example, antibodies targeting other platelet receptors such as GPIb (Vanhoorelbeke et al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3(2):125-40 (2003) or GP IIb/IIIa (Schell at al., Ann. Hematol. 81:76-79 (2002), Nieswandt and Watson, Blood 102(2):449-461 (2003), Merlini et al., Circulation 109:2203-2206 (2004)) have been associated with thrombocytopenia, although the mechanisms behind this are not well understood. It has been hypothesized that binding of an antibody to a platelet receptor can alter its three dimensional structure, and expose normally unexposed epitopes which then leads to platelet elimination (Merlini et al., Circulation 109:2203-2206 (2004). Indeed, the bleeding complications associated with oral doses of GP IIa/IIIb antagonists have been described as the "dark side" of this class of compounds (Bhatt and Topol, Nat. Rev. Drug Discov. 2(1):15-28 (2003)).

The anti-human .alpha.2.beta.1 integrin blocking antibody BHA2.1 was first described by Hangan et al., (Cancer Res. 56:3142-3149 (1996)). Other anti-.alpha.2.beta.1 integrin antibodies are known and have been used in vitro, such as the commercially available antibodies AK7 (Mazurov et al., Thromb. Haemost. 66(4):494-9 (1991), P1E6 (Wayner et al., J. Cell Biol. 107(5):1881-91 (1988)), 10G11 (Giltay et al., Blood 73(5):1235-41 (1989) and A2-11E10 (Bergelson et al., Cell Adhes. Commun. 2(5):455-64 (1994). Hangan et al., (Cancer Res. 56:3142-3149 (1996)) used the BHA2.1 antibody in vivo to study the effects of blocking .alpha.2.beta.1 integrin function on the extravasation of human tumor cells in the liver, and the ability of these tumor cells to develop metastatic foci under antibody treatment. The Ha1/29 antibody (Mendrick and Kelly, Lab Invest. 69(6):690-702 (1993)), specific for rat and murine .alpha.2.beta.1 integrin, has been used in vivo to study the upregulation of .alpha.2.beta.1 integrin on T cells following LCMV viral activation (Andreasen et al., J. Immunol. 171:2804-2811 (2003)), to study SRBC-induced delayed type hypersensitivity and FITC-induced contact type-hypersensitivity responses and collagen-induced arthritis (de Fougerolles et. al., J. Clin. Invest. 105:721-720 (2000)), to study the role of .alpha.2.beta.1 integrin in VEGF regulated angiogenesis (Senger et al., Am. J. Pathol. 160(1):195-204 (2002); Senger et al., PNAS 94(25): 13612-7 (1997)), and to study the role of .alpha.2.beta.1 integrin in PMN locomotion in response to platelet activating factor (PAF) (Werr et al., Blood 95:1804-1809 (2000)).

The use of murine monoclonal antibodies, such as those described above, as human therapeutic agents in non-immunocompromized patients has been limited by the robust immune responses directed against administered murine antibodies, particularly in repeated administration. This response cannot only curtail the effective half-life of the murine antibody in circulation but also can lead to profound injection site and/or anaphylactic responses (Shawler et al., J. Immunol. 135(2):1530 (1985)). In addition, the rodent effector functions associated with the constant regions (Fc) are much less effective than their human counterparts when administered to humans, resulting in a loss of potentially desirable complement activation and antibody-dependent, cell-mediated cytotoxicity (ADCC) activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effect of GBR500 on AsPC-1 human pancreatic cancer xenograft growth in immunodeficient female BALB/c nude (nu/nu) athymic mice.

FIG. 2: Effect of GBR500 on HT29 human colon carcinoma xenograft in nu/nu athymic mice.

FIG. 3: Western Blot analysis of CD49b and GAPDH expression in human cell lines.

FIG. 4: Confocal microscopy images of stained cell line HT1080.

FIG. 5: Confocal microscopy images of stained cell line BxPC-3.

FIG. 6: Confocal microscopy images of stained cell line MIA PaCa2.

FIG. 7: Confocal microscopy images of stained cell line HT-29.

FIG. 8: Confocal microscopy images of stained cell line SW480.

FIG. 9: Effect of GBR500 against the A549 non small cell lung cancer xenograft in nu/nu athymic mice.

FIGS. 10A and 10B: Concentration curves of GBR500 100 mg dose group for male and female monkeys.

SUMMARY OF THE INVENTION

The present invention concerns methods of using humanized anti-alpha 2 (.alpha.2) integrin antibodies for treating cancers. In particular, the invention provides an effective approach for treating cancers selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma. This invention is based on the unexpected results that anti-alpha 2 (.alpha.2) integrin antibodies which bind specifically .alpha.2.beta.1 integrin inhibit tumor growth to a degree comparable to anti-VEGF antibodies. VEGF factors activate or upregulate expression of integrins such as .alpha.1.beta.1, .alpha.2.beta.1, .alpha.4.beta.1, .alpha.5.beta.1 and .alpha.v/.beta.3 on blood vessels and .alpha.4.beta., .alpha.9.beta.1, .alpha.2.beta.1 and .alpha.1/.beta.1 on lymphatic vessels (Avraamides et al., Nat Rev Cancer. 2008 August; 8(8):604-17). It is therefore surprising that antagonism of only .alpha.2.beta.1 leads to a similar outcome as treatment with a VEGF antibody.

Accordingly in one aspect, the invention provides a method of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma, comprising administering to a subject a therapeutically effective amount of a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6).

In another aspect the invention provides a method of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma, comprising administering to a subject a composition comprising a therapeutically effective amount of a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6) and a pharmaceutically acceptable carrier. Compositions for therapeutic uses may be sterile and may be lyophilized.

In another aspect the invention provides a method of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma, comprising administering to a subject a composition comprising a therapeutically effective amount of a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6) and a pharmaceutically acceptable carrier, whereas the dosage regime is once every two weeks.

In yet a further aspect, the invention provides a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6) or a composition comprising said humanized anti-.alpha.2 integrin antibody and a pharmaceutically acceptable carrier for use in a method for the treatment of cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma fibrosarcoma, osteosarcoma, and epidermoid carcinoma.

In a further aspect the invention provides a kit for treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma in a human patient comprising a package comprising a humanized anti-.alpha.2 integrin antibody composition comprising a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6) and instructions for using said humanized anti-.alpha.2 integrin antibody for said treatment.

In a further aspect the invention provides an article of manufacture comprising a humanized anti-.alpha.2 integrin antibody comprising a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40 and/or a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6) a container and a label indicating the use of said humanized anti-.alpha.2 integrin antibody for treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma.

In certain embodiments, the anti-.alpha.2 integrin antibody includes one or more human constant regions (e.g., C.sub.L and/or C.sub.H) and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19 and/or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 or amino acid sequence variants thereof. Various forms of the antibody are contemplated herein. For example, the anti-.alpha.2 integrin antibody may be a full length antibody (e.g., comprising human immunoglobulin constant regions) or an antibody fragment (e.g. Fab or F(ab').sub.2 or Fab' or Fv or scFv fragments). Furthermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (such as a cytotoxic agent).

In an embodiment, the above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185.

In a further embodiment, the above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185 in which position 30 is Thr and/or position 31 is Asn.

In a further embodiment, the above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185 in which (a) position 71 is Lys, (b) position 73 is Asn, (c) position 78 is Val, or (d) any combination of (a)-(c).

In a further embodiment, the above-mentioned heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:70-79 and SEQ ID NOs:109-111.

In a further embodiment, the above-mentioned heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:70-75, SEQ ID NOs:77-79 and SEQ ID NOs:109-111.

In an embodiment, the above-mentioned heavy chain variable region further comprises a FW4 region comprising the amino acid sequence WGQGTLVTVSS (SEQ ID NO:13).

In an embodiment, the above-mentioned heavy chain variable region comprises the amino acid sequence of HCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:2) and HCDR3 (SEQ ID NO:3).

In a further embodiment, the above-mentioned humanized anti-.alpha.2 integrin antibody comprises a heavy chain comprising SEQ ID NO:187.

In an embodiment, the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO:186.

In an embodiment, the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO:186 in which the asparagine (N) at amino acid position 26 is replaced by glutamine (Q).

In an embodiment, the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO:186 in which (a) position 2 is Phe, (b) position 45 is Lys, (c) position 48 is Tyr, or (d) any combination of (a)-(c).

In an embodiment, the above-mentioned light chain variable region comprises an amino acid sequence selected from SEQ ID NO:41, SEQ ID NOs:80-92 and SEQ ID NO:108.

In an embodiment, the above-mentioned light chain variable region comprises an amino acid sequence selected from SEQ ID NOs:90-92.

In an embodiment, the above-mentioned light chain variable region further comprises a FW4 region comprising the amino acids sequence FGQGTKVEIK (SEQ ID NO:38).

In an embodiment, the above-mentioned light chain variable region comprises the amino acid sequence of LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5) and LCDR3 (SEQ ID NO:6).

In an embodiment, the above-mentioned light chain variable region comprises the amino acid sequence of LCDR1 (SEQ ID NO:112), LCDR2 (SEQ ID NO:5) and LCDR3 (SEQ ID NO:6).

In a further embodiment, the above-mentioned humanized anti-.alpha.2 integrin antibody comprises a light chain comprising SEQ ID NO:188.

In a further embodiment the above-mentioned humanized anti-.alpha.2 integrin antibody comprises:

(i) a heavy chain variable region comprising the amino acid sequence of (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (c) SEQ ID NO:40; and

(ii) a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSWNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6).

In a further embodiment the above-mentioned humanized anti-.alpha.2 integrin antibody comprises:

(i) a heavy chain variable region comprising the amino acid sequence of (a) HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3), or (b) SEQ ID NO:40; and

(ii) a light chain variable region comprising the amino acid sequence of (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO:4) or SAQSSVNYIH (SEQ ID NO:112), (b) LCDR2 (DTSKLAS; SEQ ID NO:5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO:6).

In a further embodiment the above-mentioned humanized anti-.alpha.2 integrin antibody comprises:

(i) a heavy chain variable region comprising the amino acid sequence of HCDR1 (GFSLTNYGIH, SEQ ID NO:1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO:2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO:3); and

(ii) a light chain variable region comprising the amino acid sequence of LCDR1 (SAQSSVNYIH, SEQ ID NO:112), LCDR2 (DTSKLAS; SEQ ID NO:5) and LCDR3 (QQWTTNPLT, SEQ ID NO:6).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185, (b) the light chain variable region comprises the amino acid sequence of SEQ ID NO:186, or (c) both (a) and (b).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185 in which position 30 is Thr and/or position 31 is Asn; (b) the light chain variable region comprises the amino acid sequence of SEQ ID NO:186 in which the asparagine (N) at amino acid position 26 is replaced by glutamine (Q); or (c) both (a) and (b).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:185 in which (a) position 71 is Lys, (b) position 73 is Asn, (c) position 78 is Val, or (d) any combination of (a)-(c); (ii) the light chain variable region comprises the amino acid sequence of SEQ ID NO:186 in which (a) position 2 is Phe, (b) position 45 is Lys, (c) position 48 is Tyr, or (d) any combination of (a)-(c); or (iii) both (i) and (ii).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein (a) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:70-79 and SEQ ID NOs:109-111; (b) the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:41, SEQ ID NOs:80-92 and SEQ ID NO:108; or (c) both (a) and (b).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein (a) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:70-75, SEQ ID NOs:77-79 and SEQ ID NOs:109-111; (b) the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs:90-92; or (c) both (a) and (b).

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein the humanized anti-.alpha.2 integrin antibody comprises a heavy chain comprising SEQ ID NO:187 and a light chain comprising SEQ ID NO:188.

Also provided is the above-mentioned method comprising the humanized anti-.alpha.2 integrin antibody, wherein the humanized anti-.alpha.2 integrin antibody comprises a heavy chain comprising SEQ ID NO:174 or SEQ ID NO:176 and a light chain comprising SEQ ID NO:178.

In an embodiment, the above-mentioned anti-.alpha.2 integrin antibody recognizes the I domain of human .alpha.2 integrin.

In an embodiment, the above-mentioned anti-.alpha.2 integrin antibody binds .alpha.2.beta.1 integrin.

In an embodiment, the above-mentioned anti-.alpha.2 integrin antibody binds an epitope of .alpha.2 integrin, the epitope comprising:

(a) a Lys residue corresponding to position 192 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 40 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11;

(b) an Asn residue corresponding to position 225 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 73 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11;

(c) a Gln residue corresponding to position 241 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 89 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11;

(d) a Tyr residue corresponding to position 245 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 93 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11;

(e) an Arg residue corresponding to position 317 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 165 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11;

(f) an Asn residue corresponding to position 318 of the .alpha.2 integrin amino acid sequence set forth in SEQ ID NO:8 or position 166 of the .alpha.2 integrin I domain amino acid sequence set forth in SEQ ID NO:11; or

(g) any combination of (a) to (f).

In an embodiment, the above-mentioned humanized anti-.alpha.2 integrin antibody is a full length antibody.

In an embodiment, the above-mentioned humanized anti-.alpha.2 integrin antibody is an antigen binding fragment.

In an embodiment, the above-mentioned humanized anti-.alpha.2 integrin antibody inhibits binding of .alpha.2 or .alpha.2.beta.1 integrin to an .alpha.2.beta.1 integrin ligand.

In an embodiment, the above-mentioned .alpha.2.beta.1 integrin ligand is selected from collagen, laminin, Echovirus-1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepellin, collectin and C1q complement protein.

In embodiments, the above-mentioned method is not associated with (a) platelet activation, (b) platelet aggregation, (c) a decrease in circulating platelet count, (d) bleeding complications, or (e) any combination of (a) to (d).

In an embodiment, the above-mentioned anti-.alpha.2 integrin antibody competively inhibits the binding of an antibody comprising the UL region of SEQ ID NO:19 and the VH region of SEQ ID NO:21 to human .alpha.2.beta.1 integrin or the I domain thereof.

Preferred antibodies bind to the I-domain of human .alpha.2.beta.1 integrin. In particular, the preferred antibodies are able to block .alpha.2-dependent adhesion of cells to the extracellular matrix (ECM), particularly to at least one or both of collagen and laminin. Humanized antibodies are provided, including antibodies based on an antibody referred to herein as TMC-2206. Anti-.alpha.2 integrin antibodies are provided that are highly specific for human .alpha.2.beta.1 integrin, and whose administration is not associated with undesired effects such as bleeding complications or complications due to cellular activation. The binding specificity (e.g., epitope specificity) of these antibodies is associated with their unexpected non-hemorrhagic profile.

The humanized anti-.alpha.2.beta.1 integrin antibody used in the present invention may have a heavy chain variable region comprising the amino acid sequence of HCDR1 (GFSLTNYGIH; SEQ ID NO:1) and/or HCDR2 (VIWARGFTNYNSALMS; SEQ ID NO:2) and/or HCDR3 (ANDGVYYAMDY; SEQ ID NO:3). The humanized anti-.alpha.2.beta. integrin antibody may have a light chain variable region comprising the amino acid sequence of LCDR1 (SANSSVNYIH; SEQ ID NO:4 or SAQSSVNYIH; SEQ ID NO:112) and/or LCDR2 (DTSKLAS; SEQ ID NO:5) and/or LCDR3 (QQWTTNPLT; SEQ ID NO:6). In certain embodiments, the humanized anti-.alpha.2.beta.1 integrin antibodies have a heavy chain comprising HCDR1 (GFSLTNYGIH; SEQ ID NO:1) and/or HCDR2 (VIWARGFTNYNSALMS; SEQ ID NO:2) and/or HCDR3 (ANDGVYYAMDY; SEQ ID NO:3) and a light chain variable region comprising the amino acid sequence of LCDR1 (SANSSVNYIH; SEQ ID NO:4 or SAQSSVNYIH; SEQ ID NO:112) and/or LCDR2 (DTSKLAS; SEQ ID NO:5) and/or LCDR3 (QQWTTNPLT; SEQ ID NO:6). In other embodiments, the antibody comprises an amino acid sequence variant of one or more of such CDRs, which variant comprises one or more amino acid insertion(s) within or adjacent to a CDR residue and/or deletion(s) within or adjacent to a CDR residue and/or substitution(s) of CDR residue(s) (with substitution(s) being the preferred type of amino acid alteration for generating such variants).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma using antibodies specifically reactive with human alpha 2 (.alpha.2) integrin, including humanized antibodies. The humanized antibodies may have human framework regions (FWs) and complementarity determining regions (CDRs) from a non-human antibody, typically a mouse, specifically reactive with human .alpha.2 integrin. In preferred embodiments, one or more of the CDR regions are derived from or based on the murine antibody secreted by the BHA2.1 hybridoma (Hangan et al., Cancer Res., 56(13): 3142-9 (1996)). This antibody binds to human and rat .alpha.2.beta.1 integrin, but does not bind the murine counterpart. The antibody so produced by the BHA2.1 hybridoma is referred to herein as TMC-2206 and is commercially available from Chemicon (now part of Millipore, catalog number MAB1998). Further provided are methods of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma using antibodies having similar binding properties and antibodies (or other antagonists) having similar functionality as the antibodies disclosed herein. Preferred anti-.alpha.2 integrin antibodies include those that (a) bind to the I domain of .alpha.2 integrin, (b) inhibit the function of .alpha.2 integrin (e.g., collagen or laminin binding), (c) bind to .alpha.2 integrin on resting platelets without inducing platelet activation and (d) recognize the binding epitope of TMC-2206 (e.g., compete with TMC-2206 for the binding to .alpha.2 integrin). Such antibodies may bind preferentially to the inactive or closed conformation of the target .alpha.2 integrin molecule without competing for the ligand binding site. Advantages of anti-.alpha.2 integrin antibodies as described herein that bind preferentially to the closed conformation of the .alpha.2.beta.1 integrin and/or bind to .alpha.2.beta.1 integrin without competing for the ligand binding site (e.g., are not a ligand mimetic) include preventing potential platelet activation, platelet aggregation, decreases in circulating platelet count and/or bleeding complications in a treated subject.

"Bleeding complications" as used herein refers to any adverse effect on blood levels and physiology, including platelet thrombotic responses, thrombocytopenia, increased time to clot, increased bleeding time and blood loss that limit therapeutic use of the anti-.alpha.2 integrin antibody.

.alpha.2.beta.1 integrin is a molecule comprised of an .alpha.2 integrin subunit (see, e.g., SEQ ID NO:7, for DNA sequence and SEQ ID NO:8 for protein sequence of human .alpha.2) from the family of alpha integrins, and a 81 integrin subunit (see, e.g., SEQ ID NO:9 for DNA sequence and SEQ ID NO:10 protein sequence of human .beta.1) from the family of beta integrins, and may be from any subject including a mammal, but preferably is from a human. The .alpha.2.beta.1 integrin may be purified from any natural source, or may be produced synthetically (e.g., by use of recombinant DNA technology). The nucleic acid coding sequences for .alpha.2 integrin and for .beta.1 integrin are described in Takada and Hemler J. Cell Biol. 109(1):397-407 (1989; GenBank submission X17033; subsequently updated to entry NM 002203) and Argraves, W. S, J. Cell. Biol. Sep. 105(3):1183-90 (1987; Genbank submission X07979.1 and related sequences representing alternatively spliced variants), respectively.

The `I` domain of the .alpha.2.beta.1 integrin molecule refers to a region of this .alpha.2.beta.1 integrin molecule within the .alpha.2 subunit, and is described, for example, in Kamata et al., J. Biol. Chem. 269:9659-9663 (1994); Emsley et al., J. Biol. Chem. 272:28512 (1997) and Cell 101:47 (2000). The amino acid sequence of a human I domain of .alpha.2 integrin is shown as SEQ ID NO:11 (see also, e.g., SEQ ID NO: 107). The I domain of .alpha.2 integrin contains a MIDAS type of ligand binding site (Metal Ion Dependent Adhesion Site) which has a requirement and a specificity for a given divalent cation to support ligand binding. The amino acid sequences for an I domain of .alpha.2 integrin in rat is shown as SEQ ID NO:93 (see also, e.g., SEQ ID NO:113) and in mouse is shown as SEQ ID NO:94 (see also, e.g., SEQ ID NO:114). Cynomolgus monkey and rhesus monkey I domain sequences were cloned from the leukocyte fraction derived from whole blood and are provided in SEQ ID NO:103 (DNA), SEQ ID NO:171 (amino acid) for cynomolgus and SEQ ID NO:104 (DNA), SEQ ID NO:172 (amino acid) for rhesus, respectively.

A TMC-2206 (BHA2.1) epitope refers to a region of the I domain of human .alpha.2 integrin to which the TMC-2206 antibody binds. This epitope spans a region of 127 amino acids encompassing amino acid residues, K40, N73, Q89, Y93, R165, and N166, which contribute to binding and optionally, other amino acid residues of the .alpha.2 integrin I domain as described in WO2007/056858.

The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, metastatic cancers as well as adenomas or adenocarcinomas. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Benign tumor" or "benign cancer" refers to a tumor that remains localized at the site of origin and does not have the capacity to infiltrate, invade, or metastasize to a distant site. "Malignant tumor" refers to a tumor that invades and damages other tissues around them. Treatment of cancer refers to both therapeutic use and prophylactic or preventative use of the anti-.alpha.2 integrin antibodies described herein. Those in need of treatment include those already diagnosed with the cancer as well as those in which the onset of the disorder is to be prevented or delayed.

Cancers can be selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, and multiple myeloma. Cancers which are preferably treated using the anti-.alpha.2 integrin antibodies described herein are selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amendable for treatment of the invention include metastatic cancers. Thus even more preferred are cancers selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, multiple myeloma, metastatic colorectal and metastatic breast cancer. Particular preferred are cancers selected from the group consisting of non-small cell lung cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, kidney cancer, prostate cancer, mesothelioma, fibrosarcoma, osteosarcoma, epidermoid carcinoma, metastatic colorectal, metastatic prostate and metastatic breast cancer. More particular preferred are cancers selected from the group consisting of non-small cell lung cancer, pancreatic cancer, glioblastoma, liver cancer, breast cancer, colon cancer, colorectal cancer, kidney cancer, prostate cancer, mesothelioma, fibrosarcoma, metastatic colorectal, metastatic prostate and metastatic breast cancer. Even more particular preferred are cancers selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, colorectal cancer, non-small cell lung cancer, fibrosarcoma, metastatic colorectal, and metastatic breast cancer. Most particular preferred are cancers selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, colorectal cancer, non-small cell lung cancer, and fibrosarcoma. Most preferred are pancreatic cancer, breast cancer or metastatic breast cancer, with a particular preference to pancreatic cancer. "Breast cancer" as referred herein include mammary adenocarcinoma. The method of the present invention is particularly suitable for the treatment of vascularized tumors.

A subject, including for purposes of treatment, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, cats, cows etc. Preferably, the subject is a human.

The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (V.sub.H, V.sub.H-V.sub.H), anticalins, PepBodies.TM., antibody-T-cell epitope fusions (Troybodies) or Peptibodies. "Antibody fragment" and "antigen binding fragment" have the same meaning and are equivalently used herein.

A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (e.g., polyclonal) antibody preparations which typically include different antibodies directed against different determinants (e.g., epitopes) on an antigen, each monoclonal antibody is directed against at least a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). Monoclonal antibodies can also be isolated using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293 (see also, e.g., Lindenbaum, et al., Nucleic Acids Research 32 (21):0177 (2004)).

Monoclonal antibodies can include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984) for mouse-human chimeric antibodies).

A hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a hypervariable loop (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). Framework or FR residues are those variable domain residues other than the hypervariable region residues. For antibodies described herein, the CDR and framework regions are identified based on the Kabat numbering system except that the CDR1 of the heavy chain is defined by Oxford Molecular's AbM definition as spanning residues 26 to 35. The Oxford Molecular's AbM antibody modeling software (Martin et al., Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121-153 (1991); Pedersen et al., Immunomethods, 1, 126 (1992); and Rees et al., In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172. (1996)) combines the Kabat CDR and the Chothia hypervariable region numbering systems to define CDRs.

Humanized forms of non-human (e.g., murine) antibodies may be chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In addition, individual or groups of Fv framework region (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions or domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 (1989), and Foote and Winter, J. Mol. Biol. 224: 487 (1992)).

Single-chain Fv or scFv antibody fragments may comprise the V.sub.H and V.sub.L regions or domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains which enables the scFv to form the desired structure for antigen binding (for a review, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)).

Diabody refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

Linear antibody refers to antibodies such as those described in Zapata et al., Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

An isolated antibody refers to one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

An epitope tagged antibody refers to one wherein the antibody of the invention is fused to an epitope tag. The epitope tag polypeptide has enough residues to provide an epitope against which an antibody thereagainst can be made, yet is short enough such that it does not interfere with activity of the anti-.alpha.261 integrin antibody. The epitope tag preferably is sufficiently unique so that the antibody thereagainst does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3(6): 547-553 (1990)). In certain embodiments, the epitope tag is a salvage receptor binding epitope which is an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

A cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. This can include radioactive isotopes (e.g., .sup.131I .sup.125I, .sup.90Y and .sup.186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent function of cells and/or does not cause destruction of cells. A non-cytotoxic agent may include an agent that can be activated to become cytotoxic. A non-cytotoxic agent may include a bead, liposome, matrix or particle (see, e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Such agents may be conjugated, coupled, linked or associated with an anti-.alpha.2.beta.1 integrin antibody as described herein.

A chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include but are not limited to Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other related nitrogen mustards.

A prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are mot limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, .beta.-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form can be those chemotherapeutic agents described above.

A label refers to a detectable compound or composition which is conjugated or coupled directly or indirectly to the antibody. The label may itself be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

Solid phase refers to a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

The terms "once every two weeks dosis regimen", "once every two weeks dosing", and "once every two weeks administration", as used herein, refer to the time course of administering a substance (e.g., anti-.alpha.2 integrin antibody) to a subject to achieve a therapeutic objective (e.g., the treatment of a cancer). The once every two weeks dosing regimen is not intended to include a weekly dosing regimen. Preferably, the substance is administered every 9-19 days, more preferably, every 11-17 days, even more preferably, every 13-15 days, and most preferably, every 14 days.

A liposome refers to a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the antibodies of the invention and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

An isolated nucleic acid molecule refers to a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

A viral vector refers to a vehicle for the transfer of a nucleic acid (e.g. DNA or RNA) to cells through viral infection or transduction. Examples of viral vectors include retroviruses, adenoviruses, pox viruses, and baculovirus.

A non-viral vector refers to a nucleic acid vehicle such as a CAN, plasmid or chromosome that is delivered to cells by non-viral methods such as electroporation, injections, and cationic reagent mediated transfection.

Expression control sequences refer to those DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Cell, cell line, and cell culture are often used interchangeably and all such designations include progeny. Transformants and transformed cells (e.g., obtained by transfection, transformation or transduction of nucleic acids, vectors, virus, etc.) include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

Humanized antibodies as described herein include antibodies that have variable region frameworks derived from a human acceptor antibody molecule, hypervariable or CDR sequences from a donor murine antibody, and constant regions, if present, derived from human sequences.

Humanized antibodies used in the present invention have been constructed comprising CDRs from both the heavy chain variable and light chain variable regions of the murine monoclonal antibody clone BHA2.1 (Hangan et al., Cancer Res. 56:3142-3149 (1996)). Preferred starting materials for constructing antibodies are anti-.alpha.2 integrin antibodies such as those secreted by the BHA2.1 hybridoma (e.g., TMC-2206) that are function-blocking antibodies directed against human .alpha.2 integrin and are dependent for binding and activity on the presence of an intact I-domain within the targeted .alpha.2 integrin. Preferred are humanized antibodies with the epitope specificity of TMC-2206 (or BHA2.1), including antibodies which bind to the inactive conformation of the .alpha.2 integrin molecule, and/or do not act as ligand mimetics. Preferred are humanized antibodies with the epitope specificity of TMC-2206 (or BHA2.1) that, although they interact with .alpha.2.beta.1 integrin present on both leukocytes and platelets, do not cause platelet activation, impair aggregation of activated platelets on collagen, have minimal or no effect on bleeding and/or are not associated with bleeding complications at administered concentrations, including therapeutic doses in vivo.

Antibodies may be constructed wherein the human acceptor molecule for the light chain variable region is selected based on homology considerations between potential acceptor molecule variable regions and with the light chain variable region of the murine antibody. Germline candidate human acceptor molecules are preferred to reduce potential antigenicity. Germline databases are made up of antibody sequences that read through the end of the heavy chain FW3 region and partially into the CDR3 sequence. For selection of a FW4 region, it is preferred to search databases of mature antibody sequences which have been derived from the selected germline molecule, and also preferred to select a reasonably homologous FW4 region for use in the recombinant antibody molecule. Human acceptor molecules are preferably selected from the same light chain class as the murine donor molecule, and of the same canonical structural class of the variable region of the murine donor molecule. Secondary considerations for selection of the human acceptor molecule for the light chain variable region include homology in CDR length between the murine donor molecule and the human acceptor molecule. Human acceptor antibody molecules are preferably selected by homology searches to the V-BASE database, and other databases such as the Kabat and the public NCBI databases may be used as well. For humanized anti-.alpha.2 integrin antibodies with the same or similar epitope specificity and/or functional properties as TMC-2206, a preferred light chain human acceptor molecule is SEQ ID NO:37 with the germline antibody sequence A14 for the FW 1-3 region and the sequence FGQGTKVEIK for FW4 (SEQ ID NO:38) which represents a common FW-4 of mature kappa 1 light chains (e.g., light chain sequence AAB24132 (NCBI entry gi/259596/gb/AAB24132).

Antibodies may be constructed wherein the human acceptor molecule for the heavy chain variable region is selected based on homology considerations between potential acceptor molecule variable regions and the heavy chain variable region of the murine antibody. Germline candidate human acceptor molecules are preferred to reduce potential antigenicity. Germline databases are made up of antibody sequences that read through the end of the heavy chain FW3 region and partially into the CDR3 sequence. For selection of a FW4 region, it is preferred to search databases of mature antibody sequences which have been derived from the selected germline molecule, and also preferred to select a reasonably homologous FW4 region for use in the recombinant antibody molecule. Human acceptor molecules are preferably selected from the same heavy chain class as the murine donor molecule, and of the same canonical structural class of the variable region of the murine donor molecule. Secondary considerations for selection of the human acceptor molecule for the heavy chain variable region include homology in CDR length between the murine donor molecule and the human acceptor molecule. Human acceptor antibody molecules are preferably selected by homology search to the V-BASE database, although other databases such as the Kabat and the public NCBI databases may be used as well. For anti-.alpha.2 integrin antibodies with the same or similar epitope specificity and/or functional properties as TMC-2206, a preferred heavy chain acceptor molecule is SEQ ID NO:39 with the germline antibody sequence 4-59 for the FW 1-3 region (SEQ ID NO:12) and antibody, CAA48104.1 (NCBI entry, gi/33583/emb/CAA48104.1) a mature antibody derived from the 4-59 germline sequence for the FW 4 region (SEQ ID NO:13).

Methods for humanizing a nonhuman .alpha.2 integrin antibody are known to the skilled person and are described e.g. in WO2007/056858. In order to humanize an anti-.alpha.2 integrin antibody, the nonhuman antibody starting material is obtained, including by preparation from immunization or by purchase of commercially available antibodies. Exemplary techniques for generating antibodies used in the present invention are described in WO2007/056858.

The .alpha.2.beta.1 integrin antigen to be used for production of antibodies may be, for example, a soluble form of .alpha.2.beta.1 integrin or other fragment of .alpha.2.beta.1 integrin (e.g., an .alpha.2.beta.1 integrin fragment comprising a human .alpha.2 integrin I-domain (SEQ ID NO:11); see also, e.g., SEQ ID NO: 107). Other forms of .alpha.2 integrin useful for generating antibodies will be apparent to those skilled in the art based on the sequence of .alpha.2 integrin (e.g., a human .alpha.2 integrin as in SEQ ID NO:8).

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl groups.

Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 .mu.g for rabbits or 5 .mu.g for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293 (see also, e.g., Lindenbaum, et al., Nucleic Acids Research 32 (21):0177 (2004)).

In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and M.C.-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (e.g., Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures including, for example, protein A chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, and/or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, including those that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is described in further detail below.

In certain embodiments, it may be desirable to generate amino acid sequence variants of the humanized antibody, particularly where these improve the binding affinity or other biological properties of the humanized antibody.

Amino acid sequence variants of humanized anti-.alpha.2.beta.1 integrin antibody are prepared by introducing appropriate nucleotide changes into a humanized anti-.alpha.231 integrin antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences shown for the anti-.alpha.2 integrin antibody TMC-2206 (e.g., derived from or based on variable region sequences as shown in SEQ ID NOS: 19 and 21). Any combination of amino acid deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the humanized anti-.alpha.2 integrin antibody, such as changing the number or position of glycosylation sites.

There are a number of methods used to make antibodies human or human-like (e.g., "humanization"). Approaches to humanize antibodies have varied over the years. One approach was to generate murine variable regions fused to human constant regions, so-called murine-human Fc chimeras (see, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); U.S. Pat. No. 5,807,715). Another approach exploited the fact that CDRs could be readily identified based on their hypervariable nature (Kabat et al, J. Biol. Chem. 252:6609-6616 (1977)), Kabat, Adv. Protein Chem. 32:1-75 (1978)) and canonical structure (Chothia and Lesk, J. Mol. Biol. 196(4):901-17 (1987); Lazakani et al., J. Mol. Biol. 272:929 (1997) and humanized by grafting just the non-human CDR regions (referred to as donor CDRs) onto a human framework (referred to as acceptor frameworks) as shown, for example by Jones et al., Nature 321(6069):522-5 (1986); (see, e.g., U.S. Pat. No. 5,225,539; U.S. Pat. No. 6,548,640). The six CDR loops are presented in a cluster, and based on crystallographic analysis, critical framework residues within the so-called "Vernier" zone flanking the CDRs or in the heavy-light chain interface can be readily identified (see, e.g., Chothia and Lesk, J. Mol. Biol. 196(4):901-17 (1987); Chothia et al., J. Mol. Biol. 186(3):651-63 (1985); Chothia et al., Nature 342(6252):877-83 (1989)). These residues can be back-mutated to the murine residue to restore the correct relative orientation of the six CDRs (see, e.g., Verhoyen et al., Science 239(4847):1534-6 (1988); Reichman et al., Nature 332(6162):323-7 (1988); Tempest et al., Biotechnology (NY) 9(3):266-71 (1991)). Since variable regions can be classified in families that bear relatively high homology between mouse and human (reviewed in e.g., Pascual and Capra Adv. Immunol. 49:1-74 (1991)), these early studies also indicated that the potential for loss in affinity could be minimized in the grafted antibody by selecting the human germline sequence with the highest homology to the murine antibody of interest for use as the human acceptor molecule (see, e.g., U.S. Pat. No. 5,225,539; Verhoyen et al., Science 239(4847):1534-6 (1988)).

Family homologies and structural relationships between frameworks that impact correct presentation of a given type of CDR canonical structure have been reported (see, e.g., Al-Lazakani et al., J. Mol. Biol. 273(4):927-48 (1997) and references therein). Preferably, a best fit human or germline sequence is chosen. Available databases of antibody germline sequences may be used to determine the family subtype of a given murine heavy and light chain and to identify best fit sequences useful as human acceptor frameworks within that human subfamily. Both the linear amino acid homology of the donor and acceptor frameworks as well as the CDR canonical structure are preferably taken into account.

Exemplary heavy chain residues which may be substituted in a humanized anti-.alpha.2 integrin antibody include any one or more of the following framework residue numbers: H37, H48, H67, H71, H73, H78 and H91 (Kabat numbering system). Preferably at least four of these framework residues are substituted. A particularly preferable set of substitutions for the heavy chain in humanized anti-.alpha.2 integrin antibodies as exemplified herein is H37, H71, H73 and H78. Similarly, residues in the light chain can also be substituted. Exemplary light chain residues for substitution include any one or more of the following residue numbers: L1, L2, L4, L6, L46, L47, L49 and L71. Preferably at least three of these framework residues are substituted. A particularly preferable set of substitutions for the light chain in humanized anti-.alpha.2 integrin antibodies as exemplified herein is L2, L46 and L49.

A useful method for identification of certain residues or regions of a humanized anti-.alpha.2 integrin antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells Science, 244: 1081-1085 (1989)). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (preferably alanine or polyalanine) to affect the interaction of the amino acids with .alpha.2.beta.1 integrin antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed humanized anti-.alpha.2 integrin antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include a humanized anti-.alpha.2 integrin antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of a humanized anti-.alpha.2 integrin antibody molecule include the fusion to the N- or C-terminus of a humanized anti-.alpha.2 integrin antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody (see below).

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in a humanized anti-.alpha.2 integrin antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable loops, but framework alterations are also contemplated. Hypervariable region residues or framework residues involved in antigen binding are generally substituted in a relatively conservative manner. Such conservative substitutions are shown below under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" or as further described below in reference to amino acid classes, are introduced and the products screened.

TABLE-US-00001 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln; asn Lys Asn (N) gln; his; lys; arg Gln Asp (D) glu Glu Cys (C) ser Ser Gln (Q) asn Asn Glu (E) asp Asp Gly (G) pro; ala Ala His (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; Leu phe; norleucine Leu (L) norleucine; ile; val; Ile met; ala; phe Lys (K) arg; gln; asn Arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr Leu Pro (P) ala Ala Ser (S) thr Thr Thr (T) ser Ser Trp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; Leu ala; norleucine

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper confirmation of a humanized anti-.alpha.2 integrin antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody and/or adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains or lacks one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, substitution by, or deletion of, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Nucleic acid molecules encoding amino acid sequence variants of humanized anti-.alpha.2 integrin antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, or cassette mutagenesis of an earlier prepared variant or a non-variant version of humanized anti-.alpha.2 integrin antibody.

Ordinarily, amino acid sequence variants of a humanized anti-.alpha.2 integrin antibody will have an amino acid sequence having at least 75% amino acid sequence identity with the original humanized antibody amino acid sequences of either the heavy or the light chain (e.g., variable region sequences as in SEQ ID NO:21 or SEQ ID NO:19, respectively), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the humanized anti-.alpha.2 integrin residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions (as described above) as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology. Thus sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp. 3 (1978)) can be used in conjunction with the computer program. For example, the percent identity can the be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences.

Antibodies having the characteristics identified herein as being desirable in a humanized anti-.alpha.2 integrin antibody are screened for by methods as described herein. For example, methods for screening candidate anti-.alpha.2 integrin antibodies for preferred characteristics and functionalities are provided that include screening for antibodies which bind to the epitope on .alpha.2.beta.1 integrin bound by an antibody of interest (e.g., those which compete with, inhibit or block binding of the TMC-2206 antibody to .alpha.2.beta.1 integrin). Cross-blocking assays can be performed and are described, for example, in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). In addition, or alternatively, epitope mapping, for example, as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine whether the antibody binds an epitope of interest.

Immobilized .alpha.2.beta.1 integrin can similarly be used to determine relative binding potencies by measuring K.sub.i values in competition assays. For example, fluorescently labeled Eu-TMC-2206 is used in the presence of varying concentrations of unlabeled candidate antibody, for example, using an assay system similar to that described above. Following a specified incubation time, the amount of bound Eu-TMC-2206 is determined. The inhibition curves are fitted with the "one site competition" model using Prism software (GraphPad, Inc. CA) to obtain IC.sub.50 values and to calculate the K.sub.1 using the equation of Cheng and Prusoff (Biochem, Pharmacol. 22(23):3099-108 (1973)).

It is desirable to prepare, identify and/or select humanized anti-.alpha.2 integrin antibodies which have beneficial binding properties, for example, under conditions as described in Example 2 of WO2007/056858, wherein candidate antibodies are tested for their ability to block .alpha.281-integrin mediated cell adhesion in comparison to TMC-2206 and the mouse-human chimeric antibody derived from TMC-2206. For example, CHO cells expressing human .alpha.2 integrin and endogenous hamster (31 (Symington et al., J. Cell Biol. 120(2):523-35 (1993)) are prepared and labeled with CFSE (Molecule Probes, OR).

Labeled cells are prepared and the cell concentration is adjusted; cells are kept in the dark until used. A collagen-coated plate (rat-tail collagen Type I; BD Biosciences) is prepared and each serially diluted antibody solution is added to the collagen plate. Labeled cells are then added to the well and the plate is incubated. After washing, cells are lysed and the fluorescence intensity (excitation, 485 nm; emission, 535 nm) is read. The inhibitory activity of each antibody is calculated.

Additionally, binding constants of the candidate antibodies for the immobilized .alpha.2.beta.1 integrin ligand can be calculated as described in Example 2 of WO2007/056858. Wells in a 96 well microtiter plate are coated with platelet .alpha.2.beta.1-integrin (custom-coated with human platelet .alpha.2.beta.1 by GTI Inc., WI) and then blocked. For example, to determine the affinity of TMC-2206 for its .alpha.2 integrin antigen, fluorescently labeled TMC-2206 or isotype control IgG antibody are used. The fluorescently labeled antibody, including Eu-TMC-2206 or Eu-isotype control IgG, is applied to the blocked .alpha.2.beta.1-integrin microtiter plates. After incubating the sealed plates to allow the antibody-antigen interaction to reach equilibrium, samples are transferred from each well into a fresh well containing an enhancement solution for the measurement of free (unbound) label. The enhancement solution is also added to the emptied wells for the measurement of bound label. The K.sub.d values of the anti-.alpha.2 integrin antibody is calculated by Scatchard analysis. The relative affinity of TMC-2206 derivatives (including humanized antibodies derived from or based on TMC-2206) can be determined by determining the Ki value in a competition assay. For example, for the competition assay, Eu-labelled TMC-2206 is added to .alpha.2 (31-coated wells in the presence of unlabelled anti-.alpha.2 integrin antibodies, including TMC-2206 or chimeric (including humanized) antibodies derived from or based on TMC-2206, or isotype control IgG antibody at various concentrations. After a period of incubation to reach equilibrium, the wells are washed and the bound labeled antibody levels are measured as retained Eu label in each well. The Ki value can be derived from the EC50 values using the K.sub.d value obtained for the Eu-TMC-2206 antibody by the direct binding studies as described above.

In certain embodiments, the humanized anti-.alpha.2 integrin antibody is an antibody fragment. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). However, these fragments can be produced directly by recombinant host cells, such as bacteria (see, e.g., Better et al., Science 240 (4855)1041-1043 (1988); U.S. Pat. No. 6,204,023. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab').sub.2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab').sub.2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

In some embodiments, it may be desirable to generate multispecific (e.g., bispecific) humanized anti-.alpha.2 integrin antibodies having binding specificities for at least two different epitopes. Exemplary bispecific antibodies (e.g., with two different binding arms) may bind to two different epitopes of the .alpha.2.beta.1 integrin protein. Alternately, an anti-.alpha.2 integrin arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (Fc.gamma.R), such as Fc.gamma.R1 (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms on a cell which has .alpha.2.beta.1 integrin bound to its surface. Bispecific antibodies can be used to localized cytotoxic agents to cells with .alpha.2.beta.1 integrin bound to their surface. These antibodies possess a .alpha.2.beta.1 integrin binding arm and an arm which binds the cytotoxic agent (e.g., gelonin, saporin, anti-interferon alpha, vinca alkaloid, ricin A chain, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific antibodies).

According to another approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or smaller size to the large side chain(s) are created on the interface of the second antibody by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimers over other unwanted end-products such as homodimers (see, e.g., WO96/27011).

Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed, for example, in U.S. Pat. No. 4,676,980 along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. Bispecific antibodies can be prepared using chemical linkage. For example, Brennan et al., (Science 229:81 (1985)) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab').sub.2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vincal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Fab'-SH fragments, recovered from E. coli, can be chemically coupled to form bispecific antibodies. For example, Shalaby et al., (J. Exp. Med. 175:217-225 (1992)) describe the production of a fully humanized bispecific antibody F(ab').sub.2 molecule. Where each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (see, e.g., Kostgelny et al., J. Immunol. 148(5):1547-1553 (1992)). The leucine zipper peptides from the Fos and Jun proteins were linked to Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be utilized for the production of antibody heterodimers. The diabody technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable region (VH) connected to a light-chain variable region (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv or scFv) dimers also has been reported (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994)). Alternatively, the bispecific antibody, may be a linear antibody, for example, produced as described in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared (see, e.g., Tutt et al., J. Immunol. 147:60 (1991)).

Other modifications of the humanized anti-.alpha.2 integrin antibodies are contemplated. For example, it may be desirable to modify the antibody with respect to effector function, so as to enhance or decrease the effectiveness of the antibody, for example, in treating cancer. Cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in the region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement mediated cell killing (CMC) and/or antibody-dependent cellular cytotoxicity (ADCC) (see e.g., Caron et al., J. Exp. Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers (see, e.g., those described in Wolff et al., Cancer Research 53:2560-2565 (1993)). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced CMC and/or ADCC capabilities (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989)).

Immunoconjugates comprising a humanized anti-.alpha.2 integrin antibody conjugated to a moiety, e.g., a molecule, composition, complex, or agent, for example a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate), for the targeting of the agent to an anti-.alpha.2 integrin-expressing cell, tissue or organ. Such an immunoconjugate may be used in a method of targeting the moiety or agent to a particular site of action characterized by the presence of .alpha.2 or .alpha.2.beta.1 integrin.

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin or the tricothecenes. A variety of radionuclides are available for the production of radioconjugated anti-alpha 2 integrin antibodies. Examples include .sup.212Bi, .sup.131In, .sup.90Y or .sup.186Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as gluteraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), or bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclide to the antibody (see, e.g., WO94/11026).

In another embodiment, the antibody may be conjugated to a receptor (such as streptavidin) for utilization in pretargeting .alpha.2 integrin-expressing cell, tissue or organ wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a ligand (e.g., avidin) which is conjugated to an agent, for example a cytotoxic agent (e.g., a radio-nuclide).

The anti-.alpha.2 integrin antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an anti-.alpha.2 integrin antibody can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent (e.g., doxorubicin) is optionally contained within the liposome (see, e.g., Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989)).

Humanized anti-.alpha.2 integrin antibodies may also be used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see, e.g., WO81/01145) to an active drug. (see, e.g., WO88/07378 and U.S. Pat. No. 4,975,278). The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active form. Enzymes that are useful include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as .beta.-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; .beta.-lactamase useful for converting drugs derivatized with .beta.-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known as abzymes, can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein, including for delivery of the abzyme to a .alpha.2 integrin-expressing cell, tissue or organ.

Enzymes may be covalently bound to the anti-.alpha.2 integrin antibodies by techniques well known in the art, including the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an anti-.alpha.2 integrin antibody linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, for example, to increase tissue or tumor penetration. It may also be desirable to modify the antibody fragment in order to increase its serum half-life. This may be achieved by incorporation of a salvage receptor binding epitope into the antibody fragment, for example, by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, for example, by DNA or peptide synthesis (see, e.g., WO96/32478).

Covalent modifications of the humanized anti-.alpha.2 integrin antibodies may be made, for example, by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Cysteinyl residues, for example, most commonly are reacted with .alpha.-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues, for example, are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues, for example, are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate. Arginyl residues, for example, are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK.sub.a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. Tyrosyl residues, for example, are specifically modified with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using .sup.125I or .sup.131I to prepare labeled proteins for use in radioimmunoassay. Carboxyl side groups, for example, aspartyl or glutamyl, are selectively modified by reaction with carbodiimides (R--N.dbd.C.dbd.N--R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine (see, e.g., WO87/05330; Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the antibody may be accomplished, for example, chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact (see, e.g., Hakimuddin, et al., Arch. Biochem. Biophys. 259: 52 (1987); Edge et al., Anal. Biochem., 118: 131 (1981)). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases, (see, e.g., Thotakura et al., Meth. Enzymol. 138: 350 (1987)).

Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes (see, e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337).

For recombinant production of the antibody, the nucleic acid(s) encoding the antibody are isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

An anti-.alpha.2 integrin antibody may be produced recombinantly, including as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a eukaryotic signal sequence (e.g., an immunoglobulin signal sequence), the signal sequence is substituted by a prokaryotic signal sequence including, for example, pectate lysase (such as pelB), alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion, a yeast signal sequence may be utilized, including, for example, the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces .alpha.-factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available and may be utilized. The DNA for such a precursor region (e.g., the signal sequence) is ligated in reading frame to DNA encoding an anti-.alpha.2 integrin antibody.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2.mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (e.g., the SV40 origin may typically be used only because it contains the early promoter).

Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, (e.g., the gene encoding D-alanine racemase for Bacilli).

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs methotrexate, neomycin, histidinol, puromycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-.alpha.2 integrin antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding anti-.alpha.2 integrin antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker, including an aminoglycosidic antibiotic, such as kanamycin, neomycin, or G418 (see e.g., U.S. Pat. No. 4,965,199).

One suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (see, e.g., Jones, Genetics, 85: 12 (1977)). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6.mu. circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis by Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed (see, e.g., Fleer et al., Bio/Technology, 9: 968-975 (1991)).

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-.alpha.2 integrin antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the arabinose promoter (e.g., araB), phoA promoter, .beta.-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding the anti-.alpha.2 integrin antibody.

Promoter sequences are known for eukaryotes. Most eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT (SEQ ID NO:115) region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO:116) sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. Such sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include but are not limited to the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.

Anti-.alpha.2 integrin antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus or Simian Virus 40 (SV40), from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446, and a modification of this system is described in U.S. Pat. No. 4,601,978 (see, also Reyes et al., Nature 297: 598-601 (1982) on expression of human .beta.-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus). Alternatively, the rous sarcoma virus long terminal repeat can be used as the promoter.

Transcription of DNA encoding an anti-.alpha.2 integrin antibody by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin). Often, however, an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers (see, also, e.g., Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters). The enhancer may be spliced into the vector at a position 5' or 3' to the anti-.alpha.2 integrin antibody-encoding sequence, but is preferably located at a site 5' from the promoter. Other gene regulation systems well known in the art (e.g. inducible systems, such as tetracycline inducible systems and GeneSwitch.TM.) can be used to control the transcription of DNA encoding an anti-.alpha.2 integrin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an anti-.alpha.2 integrin antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region (see, e.g., WO94/11026 and the expression vector disclosed therein).

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells as described above. Suitable prokaryotes for this purpose include eubacteria, including gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. Suitable E. coli cloning hosts include E. coli 294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325).

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-alpha 2 integrin antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful, such as Schizosaccharomyces pombe; Kluyveromyces hosts including K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, or K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi including Neurospora, Penicillium, Tolypocladium, or Aspergillus hosts such as A. nidulans or A. niger.

Suitable host cells for the expression of glycosylated anti-.alpha.2 integrin antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells, including a variety of mammalian cells, has become routine procedure. Examples of useful mammalian host cells include: a monkey kidney CV1 line transformed by SV40 (e.g., COS-7, ATCC CRL 1651); a human embryonic kidney line 293 or 293 cells subcloned for growth in suspension culture (see e.g., Graham et at, J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary (CHO) cells, including CHO cells lacking DHFR (see, e.g., DHFR Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse sertoli cells ((e.g., TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); mouse mammary tumor (e.g., MMT 060562, ATCC CCL51); TRI cells (see, e.g., Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; or a human hepatoma line (e.g., Hep G2).

Host cells are transformed with an above-described expression or cloning vectors for anti-.alpha.2 integrin antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants and/or amplifying the genes encoding the desired sequences.

The host cells used to produce an anti-.alpha.2 integrin antibody may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are selected by those skilled in the art, including those culture conditions previously used with the host cell selected for expression.

Anti-.alpha.2 integrin antibodies can be purified from cells, including microbial or mammalian cells using, for example, protein A chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and/or affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains (see, e.g., Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is useful for mouse isotypes and for human .gamma.3 (see, e.g., Guss et al, EMBO J. 5:1516-1517 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM. (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Protein purification can include one or more of the following techniques such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE.TM., chromatography on an anion or cation exchange resin (e.g., a polyaspartic acid column), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and/or hydrophobic interaction chromatography. For example, it may be useful following any purification step(s), to subject a mixture comprising the antibody of interest and contaminants to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

Formulations of an anti-.alpha.2 integrin antibody, including those for therapeutic administration, are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, diluents, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, diluents, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG). For therapeutic uses the anti-.alpha.2 integrin antibody of the present invention may be formulated e.g. in phosphate buffered saline (PBS) containing 0.03% Tween-80.TM.. The antibody formulation may also contain more than one active compound for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. It may be desirable to use anti-.alpha.2 integrin antibody in addition to one or more agents currently used to prevent or treat the disorder in question. In addition, it may be desirable to further provide an immunosuppressive agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles or nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37.degree. C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

The anti-.alpha.2 integrin antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, or intranasal. If desired for local immunosuppressive treatment, intralesional administration of the antibody (including perfusing or otherwise contacting the graft with the antibody before transplantation) is done. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the anti-.alpha.2 integrin antibody is suitably administered by pulse infusion, for example, with declining doses of the antibody. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections. This may depend in part on whether the administration is brief or chronic. More preferably the anti-.alpha.2 integrin antibodies or the compositions as described herein are administered in the methods of the present invention by intravenous infusion, intravenous bolus, subcutaneous administration, subcutaneous infusion or subcutaneous bolus, whereas intravenous infusion or intravenous bolus is most preferred. The term "intravenous infusion" refers to introduction of a drug into the vein of an animal or human patient over a period of time greater than approximately 5 minutes, preferably between approximately 30 to 90 minutes, although, according to the invention, intravenous infusion is alternatively administered for 10 hours or less. The term "intravenous bolus" or "intravenous push" refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, preferably 5 minutes or less. The term "subcutaneous administration" refers to introduction of a drug under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle. The pocket may be created by pinching or drawing the skin up and away from underlying tissue. The term "subcutaneous infusion" refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less. The term "subcutaneous bolus" refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is preferably less than approximately 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds. Administration is preferably within a pocket between the skin and underlying tissue, where the pocket is created, for example,--by pinching or drawing the skin up and away from underlying tissue. Optionally, the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen. Intermittent or periodic dosing is a dosing that is continuous for a certain period of time and is at regular intervals that are preferably separated more than by one day.

"Therapeutically effective amount" or "effective amount" which are used synonymously herein, refer to an amount of the anti-.alpha.2 integrin antibodies described herein effective to ameliorate or prevent the symptoms, or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. The term "therapeutically effective amount" of the anti-.alpha.2 integrin antibodies described herein specifically refers to the amount needed to delay or inhibit tumor growth.

For the prevention or treatment of cancer, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the anti-.alpha.2 integrin antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

The anti-.alpha.2 integrin antibodies can be thus administered to a subject, preferably to human, in the method of the present invention, at a therapeutically effective amount ranging from about 0.1 to about 100 mg/kg. Preferably, a therapeutically effective amount ranging from about 1 to about 20 mg/kg, more preferably a therapeutically effective amount ranging from about 3 to about 10 mg/kg is administered to a subject, preferably to human. A therapeutically effective amount of the humanized antibody or binding fragment thereof can be administered to the subject in one or more therapeutically effective doses.

Depending on the type and severity of the disease from about 0.1 mg/kg to about 100 mg/kg of antibody is an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage to e.g. human might range from 0.1 mg/k to 20 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is readily monitored by those skilled in the art. According to toxicokinetics studies as described in example 6 the anti-.alpha.2 integrin antibodies of the present invention have an estimated half life T1/2 of between 199 and 316 hours. Thus a once every two weeks dosis regimen seems preferable.

Unexpectedly the anti-alpha 2 (.alpha.2) integrin antibodies used in the present invention inhibit tumor growth to a degree comparable to anti-VEGF antibodies. Specifically at a dose of 50 mg/kg of anti-alpha 2 (.alpha.2) integrin antibody administered biweekly for 22 days in a mouse xenograft study the size of the tumor was around 60% of the isotype control on day 27. Thus the invention provides a method of treating cancer selected from the group consisting of squamous cell cancer, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema such as that associated with brain tumors, Meigs' syndrome, melanoma, mesothelioma, multiple myeloma, fibrosarcoma, osteosarcoma, and epidermoid carcinoma, by administering to a subject a therapeutically effective amount of a humanized anti-.alpha.2 integrin antibody, whereas the size of the tumor treated with the humanized anti-.alpha.2 integrin antibody is equal to or less than 90%, preferably equal to or less than 80%, more preferably equal to or less than 70%, most preferably equal to or less than 60%, in particular equal to or less than 50%, more particular equal to or less than 40%, most particular equal to or less than 30% of the size of the tumor treated with the control antibody, whereas the size of the tumor is usually measured as tumor volume or tumor weight.

An anti-.alpha.2 integrin antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, results from pharmacological and toxicity studies and other factors known to medical practitioners. A therapeutically effective amount of the antibody to be administered is determined by consideration of such, and is the minimum amount necessary to prevent, ameliorate, or treat an .alpha.2.beta.1 integrin-associated disorder. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.

The anti-.alpha.2 integrin antibody need not be, but may be optionally formulated, co-administered or used as an adjunct therapy with one or more agents currently used to prevent or treat the disorder in question. For example, the antibody may be given in conjunction with radiotherapy and or one or several cancer medications. These cancer medications may comprise another antibody, chemo-therapeutic agent, cytotoxic agent, anti-angiogenic agent, immunosuppressive agent, prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy, corticosteroid, anti-emetic cancer vaccine, analgesic, anti-vascular agent, or growth-inhibitory agent. More specific agents include, for example, irinotecan (CAMPTOSAR.RTM.), cetuximab (ERBITUX.RTM.), fulvestrant (FASLODEX.RTM.), vinorelbine (NAVELBINE.RTM.), EFG-recep-tor antagonists such as erlotinib (TARCEVA.RTM.) VEGF antagonists such as bevacizumab (AVASTIN.RTM.), vincristine (ONCOVIN.RTM.), inhibitors of mTor (a serine/threonine protein kinase) such as rapamycin and CCl-779, and anti-HER1, HER2, ErbB, and/or EGFR antagonists such as trastuzumab (HERCEPTIN.RTM.), pertuzumab (OMNI-TARG.TM.), or Iapatinib, and other cytotoxic agents including chemotherapeutic agents. Alternatively, or in addition, .alpha.2.beta.1 integrin antagonists may be administered to the mammal suffering from an .alpha.2.beta.1 integrin-associated disorder. The effective amount of such other agents depends on the amount of anti-.alpha.2 integrin antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

An article of manufacture containing materials, including an anti-.alpha.2 integrin antibody, useful for the treatment of the cancer as described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an anti-alpha 2 integrin antibody. The label on, or associated with, the container indicates that the composition is used for treating the cancer as described above. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.

EXAMPLES

Example 1

In Vitro Evaluation of Anti-.alpha..sub.2 Integrin Antibody Potencies in Inhibiting Interaction Between Human .alpha..sub.2 Integrin Expressed on Human Carcinoma Cell Lines and Human Collagen

An in vitro binding assay between human cell line and human collagen has been established to assess the potency of different anti-.alpha..sub.2 integrin antibodies to inhibit the interaction between human VLA-2 (.alpha..sub.2.beta..sub.1) integrin expressed on human carcinoma cell lines and human collagen type I. In this assay, fluorescently labelled human pancreatic cancer cell lines that naturally expresses VLA-2 were distributed in 96 well plates which have been previously coated with human collagen type I. A fluorescently labelled human pancreatic cancer cell line that doesn't express VLA-2 was used as a negative control. Fluorescently-labelled cells were then incubated in the collagen coated 96 well plates in presence of different concentrations of the anti-.alpha..sub.2 integrin (GBR500 or TMC2206) or isotype-matched control antibody (GBR600) for one hour. Plates were gently washed and the remaining fluorescence was measured in each well of the plate. The strength of the fluorescence signal measured in each individual well is proportional to the number of cells that have adhered to the collagen.

Material and Methods

TABLE-US-00002 TABLE 1 Antibodies Clone number or Catalogue antibody name Supplier number Description TMC-2206 Glenmark/ MAB1998 Mouse anti-human Millipore .alpha..sub.2 integrin GBR500 Glenmark NA Humanized anti-human .alpha..sub.2 integrin GBR600 Glenmark NA Humanized IgG.sub.4 control antibody

The humanized anti-human .alpha..sub.2 integrin GBR500 as referred herein comprises a heavy chain comprising SEQ ID NO:187 and a light chain comprising SEQ ID NO:188.

TABLE-US-00003 TABLE 2 Cell lines VLA-2 Name Source Supplier Cat# expression SK-BR-3 Breast Carcinoma ATCC HTB-30 Low AsPC-1 Pancreatic ATCC CRL-1682 High Carcinoma HPAF-II Pancreatic ATCC CRL-1997 High Carcinoma MIA PaCa-2 Pancreatic ATCC CRL-1420 Negative Carcinoma

Flow Cytometry

After incubation with Versene (Gibco, cat#15040), AsPC-1 cells, HPAF-II cells and MIA PaCa-2 cells were collected and resuspended in PBS-2.5% FBS at a concentration of 1.times.10.sup.6 cells/mL. One hundred .mu.l of the cell suspension was incubated with 10 .mu.g/mL of GBR500-FITC or hlgG4-FITC as a control for 20 minutes on ice. Cells were washed twice with PBS-2.5% FBS and analyzed by flow cytometry. Trypsinised SK-BR-3 cell line were treated in the same way as described for the pancreatic cell line. Expression level of VLA-2 molecule was expressed as the Mean Fluorescence Intensity (MFI).

Collagen Binding Assay

Ninety six well ELISA plates (black cliniplate, Thermo Firsher scientific, cat no 9502867) were coated with 100 .mu.l of human collagen type I (SIGMA, cat no C7774) at 50 .mu.g/mL in Acetic Acid 0.02N or in PBS. Collagen was either diluted in acetic acid and incubated for 1 hour at 37.degree. C. or diluted in PBS and incubated overnight at 4.degree. C. Plates were blocked with 150 .mu.l of PBS supplemented with 0.1% BSA or 1% BSA (Sigma, cat no A3059). Cells were first labelled with CFSE (Invitrogen cat no C34554) into serum free DMEM medium (PAA, cat no E15-005). Three .mu.l of a 15 mM CFSE solution was added to 5 ml cells at a concentration between 1.times.10.sup.6 cells/ml to 0.6.times.10.sup.6 cells/ml. Cells were incubated with CFSE for 10 minutes at 37.degree. C. and CFSE excess was removed by centrifugation of the cells at 900 rpm for 3 min. CFSE-labelled cells were resuspended at a concentration between 0.6.times.10.sup.6 to 1.times.10.sup.6 cells/ml in DMEM supplemented with 0.1% BSA. Fifty .mu.l of antibody dilutions in DMEM-0.1% BSA were distributed to the collagen coated plate and fifty .mu.l of CFSE labelled cells were immediately distributed to the plate. GBR600 antibody was used as an isotype control antibody for GBR500. Plates were incubated at room temperature for 1 hour and cells that were not bound to the collagen were removed by dumping the supernatant. Plates were washed four times with PBS buffer either manually or using BioTek washer. Wells in the plates were filled with PBS and fluorescence with excitation at 498 nm and emission at 525 nm was measured using Synerg HT2 fluorometer. Data were analyzed using PRISM software. Activity of the anti-VLA-2 antibody was expressed as EC.sub.50 which is defined as the concentration of antibody that provokes a response halfway between the baseline and the maximum response.

Results:

TABLE-US-00004 TABLE 3 FACS staining MFI Cell line hIgG4 GBR500 AsPC-1 6.8 69.8 HPAF-II 14.9 166.5 MIA PaCa-2 4.9 5.0 SK-BR-3 3.4 6.0

Collagen Binding Assay

Collagen binding assay using the three pancreatic cancer cell lines and the breast cancer cell line were performed twice. Table 4 summarizes the EC-50 values obtained in the 2 experiments performed is depicted below.

TABLE-US-00005 TABLE 4 EC-50 values Experiment I Experiment II EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 Cell line GBR500 TMC2206 GBR500 TMC2206 AsPC-1 0.047 .mu.g/mL 0.074 .mu.g/mL 0.036 .mu.g/mL 0.087 .mu.g/mL HPAF-II 0.224 .mu.g/mL 0.3257 .mu.g/mL 0.103 .mu.g/mL 0.253 .mu.g/mL MIA Paca-2 No cell binding No cell binding SK-BR-3 0.039 .mu.g/mL 0.032 .mu.g/mL

GBR500 and TMC-2206 antibodies inhibited the binding of the VLA-2 positive pancreatic and breast cancer cells to the human collagen. The MIA PaCa-2 cell line which doesn't express VLA-2 doesn't bind to the collagen. This result demonstrates that VLA-2 expression is a prerequisite for cell adherence to collagen type I.

CONCLUSION

1) .alpha.2 integrin expression on the carcinoma cancer cell lines AsPC-1, HPAF-II and SKBR3 cells can be detected using fluorescently-labelled GBR500 antibody. The pancreatic cancer cell line express a higher level of the .alpha.2 integrin compare to the breast cancer cell line. 2) The VLA-2 positive cell lines AsPC-1, HPAF-II and SK-BR-3 cell lines adhered to the collagen whereas the VLA-2 negative cell line MiaPaCA doesn't. 3) Antibody EC-50 values for the inhibition of collagen binding for the different cell lines tested are shown in Table 5:

TABLE-US-00006 TABLE 5 EC-50 values Cell lines GBR500 EC.sub.50 TMC-2206 EC.sub.50 AsPC-1 0.041 .+-. 0.008 (n = 2) 0.080 .+-. 0.009 (n = 2) HPAF-II 0.163 .+-. 0.086 (n = 2) 0.290 .+-. 0.051 (n = 2) MiaPaCa No cell binding No cell binding SK-BR-3 0.036 .+-. 0.005 (n = 2) Not done

The EC.sub.50 value obtained with the HPAF-II cell line was about 4 times (3.9 times for GBR500 and 3.6 for TMC-2206) higher compared to the EC.sub.50 value measured for AsPC-1 cell line. This difference cannot be attributed to VLA-2 expression level, since both pancreatic cell lines expressed similar VLA-2 levels (see FACS staining data). Moreover, the SK-BR-3 cell line expressing a low level of VLA-2, displayed an EC.sub.50 value comparable to AsPC-1 (high VLA-2 expression). However, the collagen coating conditions were different between the pancreatic cell lines and the breast cancer cell line (collagen diluted acid acetic at 37.degree. C. for one hour versus collagen diluted in PBS at 4.degree. C. overnight), therefore EC50 value comparisons between these cell lines should be interpreted with caution.

4) This study identifies .alpha..sub.2.beta..sub.1 integrin mediated adhesion to type I collagen as a potential therapeutic target. Moreover, the anti-VLA-2 antibody GBR500 and TMC2206 displayed a good capacity to inhibit the binding of VLA-2 expressing cell lines to collagen. GBR500 antibody is therefore a potential therapeutic candidate in the treatment of pancreatic and breast cancers.

Example 2

Effect of GBR500 Against the Human Pancreatic Carcinoma Tumour Xenograft AsPC-1 in BALB/c Nude (nu/nu) Athymic Mice

Female BALB/c nude (nu/nu) athymic mice, of at least 6-8 weeks age were used in the xenograft study. Animals obtained from Australian Research Council (ARC) were assigned into treatment groups on day -2 of the study and treatment was being started as per the regime described in Table 6. On day 1 human AsPC-1 pancreatic carcinoma tumour cells (ATCC.RTM. Number: CRL-1682) were harvested from sub-confluent cultures grown in vitro and the number of viable cells determined. Cells were re-suspended in 1.times.PBS at a concentration of 5.times.10.sup.7 cells/ml and animals were injected subcutaneously in the rear right flank with approximately 5.times.10.sup.6 cells in a volume of 0.1 ml.

Animals were examined regularly for the appearance of tumours and dosed biweekly for 22 days starting from day -2 (Total 7 injections on days -2, 2, 6, 9, 13, 16, 20). Antibodies were administered in a volume of 10 ml/kg. At day 20 treatments were stopped and animals were monitored up to day 27.

TABLE-US-00007 TABLE 6 Treatment Groups and Study Design Dose Treatment Route of Number of Group Treatment level Days administration animals 1 Isotype 50 mg/kg biweekly* for IP 14 control 22 days 2 Avastin .RTM. 40 mg/kg biweekly for IP 14 22 days 3 GBR500 5 mg/kg biweekly for IP 14 22 days 4 GBR500 50 mg/kg biweekly for IP 14 22 days 5 Cetuximab 40 mg/kg biweekly for IP 14 22 days *Biweekly for 22 days starting from day -2 (Total 7 injections on days -2, 2, 6, 9, 13, 16, 20)

Tumour measurements were obtained twice weekly using digital callipers for the duration of the study. Tumour dimensions were recorded (length and width), and tumour volumes calculated using the formula W.sup.2.times.L.times.0.536, where W is the widest tumour dimension and L is the longest. The results of the study are shown in FIG. 1. The tumor volumes refer to the mean per group of 14 animals. At a dose of 50 mg/kg GBR500, the size of the tumor was around 60% of the isotype control on day 27.

Example 3

Effect of GBR500 Against the HT29 Human Colon Carcinoma Xenograft in nu/nu Athymic Mice

Nu/Nu male mice, from Harlan, Italy, were used. The animals were maintained in cages using steam autoclaved (sterile) bedding, diet and water were offered ad libitum. Animals were identified by a uniquely numbered ear-tag which appears on the data sheets. Body weight at the day of tumor implantation was: 24-31 g.

Number of Groups-Treatment Schedule:

Number of groups was 5. Number of animals/group was 10. Treatment was being started as per the regime described in Table 7.

TABLE-US-00008 TABLE 7 Treatment Groups and Study Design Dose Group Compound mg/kg Route/Schedule 1 IgG4 Isotype 50 IP* Day 6, 9, 13, 16, 20, 23, 27** 2 Ha1/29 5 IP Day 6, 9, 13, 16, 20, 23, 27** 3 GBR500 50 IP Day 6, 9, 13, 16, 20, 23, 27** 4 Ha1/29 + 50 + 5 IP Day 6, 9, 13, 16, 20, 23, 27** GBR500 5 Avastin .RTM. 40 IP Day 6, 9, 13, 16, 20, 23, 27** *IP: intraperitoneally **Treatment starts at day 6 after tumor implants

Substances:

Test compounds were stored at 4.degree. C. temperature and protected from light until use. Test compounds were dissolved in 0.03% Tween-80.TM. in Phosphate buffered saline and were diluted immediately before use in order to reach the right concentration (IgG4 Isotype, Ha1/29 and GBR500 in PBS; Avastin.RTM. in saline solution). Treatments were administered intraperitoneally (IP) in a volume of 10 ml/kg

Tumor:

HT29 tumor fragment from mice previously inoculated with HT29 cells (ATCC HTB-38.TM.) were implanted subcutaneously into the left flank of athymic nude mice. Animals were examined regularly for the appearance of tumors. When measurable tumors have been established in the majority of mice, animals were assigned into treatment groups, with a target of 10 mice per group (5 mice per cage). When treatment starts the mean tumor volume was 120 mm.sup.3.

Evaluation of Antitumor Activity in the Xenograft Models and Toxicity:

At least twice a week the tumor growth and the net body weight were evaluated. Tumor growth was assessed by caliper. Dimensions of the tumors were measured regularly by calliper during the experiments, and tumor masses were calculated as follows:

.times..times..times..times..times..times..times..times..function. ##EQU00001## assuming density d=1 mg/mm.sup.3 for tumor tissue

Toxicity was evaluated on the basis of the body weight reduction. Mice were sacrificed when the tumors reach a volume that hampers them.

Results and Conclusions:

Ha 1/29 (5 mg/kg) and GBR500 (50 mg/kg) administered as single agents twice a week, gave a tumor weight inhibition at day 28 of 12% and 16%, respectively. Ha1/29 in combination with GBR500 showed a reduction of tumor weight of 19% (Table 8).

TABLE-US-00009 TABLE 8 Dosage scheme and tumor weight inhibition Com- Dose % Tumor Weight Inhibition Group pound mg/kg 6 9 13 16 20 23 28 31 35 1 IgG4 50 -- -- -- -- -- -- -- -- -- Isotype 2 Ha1/29 5 1 14 28 33 23 17 12 3 -4 3 GBR500 50 2 4 11 18 12 15 16 13 0 4 Ha1/29 + 50 + 5 2 17 24 33 23 25 19 7 -1 GBR500 5 Avastin .RTM. 40 1 20 28 43 44 50 50 40 24

The maximal antitumor activity of antibodies treated groups was observed at day 16 with a tumor weight inhibition of 33, 18, 33% (Ha1/29, GBR500 and the combination group respectively). Avastin.RTM., administered at 40 mg/kg, showed a tumor growth inhibition of 50% at day 28. In FIG. 2 the comparison of the average tumor growth observed in the different treatment groups is shown. Treatments were well tolerated and no dead mice in treated groups were found during the experiment. No signs of distress were observed during and after treatments and no significant body weight loss was observed.

Example 4

Detection of Expression Levels of CD49b (Integrin Subunit .alpha.2) in Human Cell Lines

Cell Lines and Culture Conditions

A panel of cell lysates was screened for expression of CD49b. This panel consisted of lysates from four non-transformed human cell lines (BJ, 1407, primary fibroblasts, WRL-68) and 96 human cancer cell lines from different tissues/organs (including colorectal, skin, breast, prostate, pancreas, lung, cervix, kidney, ovary, CNS, bone, liver, thyroid, and blood). The cell lines used are shown in Table 9 A and 9B.

TABLE-US-00010 TABLE 9A Cell lines Tissue tumor origin Lane Cell line Source Type Growth medium 1 fibrosarcoma 1 HT-1080 ECACC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA 2 adenocarcinoma 2 CACO-2 1st. ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA colon Zooprofilattico BS 3 CL-11 DSMZ ADHESION 80% mixture of Ham's F12 + Dulbecco's MEM (at E1) + 20% FBS 4 COLO-205 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 5 COLO-206F DSMZ ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 6 COLO-320 DSMZ ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 7 COLO-678 DSMZ ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 8 COLO-741 ICLC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 9 DLD-1 IEO (ATCC) ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. + 10 mM HEPES, and 1.0 mM sodium pyruvate 10 HCC2998 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 11 HCT-116 ECACC ADHESION McCoy's + 2 mM L-Glutamine + 10% FCS 12 HCT15 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 13 HT-29 ECACC ADHESION McCoy's + 2 mM L-Glutamine + 10% FCS 14 KM12 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 15 LoVo ECACC ADHESION HAM's F12 + 10% FCS + 2 mM Glut. 16 LS-174T ATCC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA + 1% Na Pyruvate 17 LS-180 ICLC ADHESION MEM(EBSS) + 10% FCS + 2 mM glutamine + 1% AAEE + 1% Sodium pyruvate 18 SW1417 ICLC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 19 SW403 DSMZ ADHESION DMEM + 2 mM glutamine + 10% 20 SW48 ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 21 SW480 IEO (ATCC) ADHESION Leibowitz's L-15 + 10% FCS + 2 mM Glutamine or RPMI + 10% fcs 22 SW620 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 23 T84 ATCC ADHESION HAM's F12 + DMEM E1 + 10% FCS 3 non tumoral 24 BJ ADHESION D-MEM/M199 + 10% FCS + 2 mM Glut 25 I407 PHA ADHESION EMEM(EBSS) + 2 mM Glutamine + 1% Non Essential Amino Acids (NEAA) + 10% Foetal Bovine Serum (FBS) 26 NHDF PROMOCELL ADHESION Fibroblast basal medium + bullet kit + 1 ng/ml diFGF + 10% fcs 27 WRL-68 ECACC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA 4 melanoma 28 A375 ECACC ADHESION DMEM + 2 mM Glutamine + 10% FCS 29 Mewo ATCC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA 30 SK-MEL-28 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 31 SK-MEL-5 ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. 32 UACC-257 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 5 adenocarcinoma 33 BT-20 ATCC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine mammary 34 MCF7 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 35 MDA-MB-231 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 36 MDA-MB-435S NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 37 MDA-MB-468 ITM ADHESION RPMI or Leibowitz's L-15 + 10% FCS + 2 mM Glutamine 38 SK-BR-3 ATCC ADHESION McCoy's + 2 mM L-Glutamine + 10% FCS 39 T47D ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 6 carcinoma 40 DU-145 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut prostate 41 LnCap ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. + 10 Mm Hepes + 1 mM Na Pyruvate 42 PC-3 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 7 adenocarcinoma 43 BxPC-3 ECACC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut pancreas 44 CAPAN-1 DSMZ ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 45 MIA-PaCa-2 ECACC ADHESION DMEM + 2 mM Glutamine + 10% FCS 46 PANC-1 ATCC ADHESION DMEM + 4 mM glutamine + 4.5 g/L glucose + 10% FCS 8 non-small cell 47 A549 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut lung cancer 48 CAL-12T IFOM ADHESION DMEM + 2 mM Glutamine + 10% FCS 49 HOP-62 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 50 NCI-H1437 ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 51 NCI-H1770 ATCC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut 52 SK-LU-1 ICLC ADHESION MEM(EBSS) + 10% FCS + 2 mM glutamine + 1% AAEE + 1% Sodium pyruvate

TABLE-US-00011 TABLE 9B Cell lines Tissue tumor origin Lane Cell line Source Type Growth medium 9 adenocarcinoma 53 C-33-A IEO ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. cervix 54 HeLa ECACC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA 10 adenocarcinoma 55 A498 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut kidney 56 ACHN NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 57 SN12C NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 58 TK10 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 59 U031 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 11 adenocarcinoma 60 A2780 ECACC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut ovary 61 IGROV-1 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 62 OVCAR-3 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 63 OVCAR-8 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 12 glioblastoma 64 SNB19 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 65 T98G ATCC ADHESION EMEM + 2 mM glut + 10% FCS + 1% AAEE + 1% (1.0 mM)sodium pyruvate 66 U251 NCI ADHESION RPMI 1640 + 10% FCS + 2 mM Glut 67 U-87-MG ATCC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA 13 osteosarcoma 68 U-2-OS ATCC ADHESION McCoy's + 2 mM L-Glutamine + 10% FCS 14 non-small cell 69 NCI-H1299 ATCC ADHESION RPMI1640 + 10% FCS + 10 mM HEPES + 1 mM lung cancer Na Pyruvate + 2 mM Glut. 70 NCI-H1975 ATCC ADHESION RPMI1640 + 20% FCS 15 small cell 71 NCI-H146 ATCC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut. + 10 mM lung cancer HEPES, and 1.0 mM sodium pyruvate 72 NCI-H1963 ATCC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut 73 NCI-H209 ATCC SUSPENSION RPMI 1640 medium, 90%; fetal bovine serum, 10% 74 NCI-H526 ATCC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut. + 10 mM HEPES, and pyruvate + 1.0 mM sodium pyruvate 75 NCI-H69 ATCC SUSPENSION RPMI 1640 medium with 2 m 10 mM HEPES, and 1.0 mM sodium 10% FCSM L-glutamine 76 NCI-H82 ATCC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut. + 10 mM HEPES, and 1.0 mM sodium pyruvate 77 NCI-N592 NMS SUSPENSION RPMI + 10% FCS + 1% HEPES + 1% Sodio Pyruvato 16 adenocarcinoma 78 HepG2 ECACC ADHESION E-MEM + 10% FCS + 2 mM L-Glutamine + 1% NEAA liver 17 papillary thyroid 79 WRO Istituto ADHESION DMEM + Na piruvato + 10% fcs carcinoma Tumori 18 carcinoma 80 A431 ATCC ADHESION RPMI 1640 + 10% FCS + 2 mM Glut epidermoid 19 mesothelioma 81 MSTO-211H Ospedale S. ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. + Matteo Pavia 10 mM HEPES, and 1.0 mM sodium pyruvate 82 REN Ospedale S. ADHESION RPMI 1640 + 10% FCS + 2 mM Glut. Matteo Pavia 20 leukemia 83 HL-60 ECACC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut leukemia 84 K-562 ECACC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut leukemia T 85 CEM/VM1 W. Beck-St SUSPENSION E-MEM + 10% FCS + 2 mM L-Glutamine + lymphoblastoid Jude 100 nM VM26 Hospital leukemia T 86 KARPAS-299 DSMZ SUSPENSION RPMI 1640 + 10% FCS + 2 mM glutamine lymphoblastoid thrombocythemia 87 SET-2 DSMZ SUSPENSION RPMI 1640 + 20% FBS leukemic multiple myeloma 88 KMS-11 Istituto SUSPENSION RPMI 1640 + 10% FCS + 2 mM glutamine Tumori multiple myeloma 89 RPMI-8226 ECACC SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut. multiple myeloma 90 RPMI-8226 DSMZ SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut. Leukemia B cell 91 697 DSMZ SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut Leukemia B cell 92 MEC-1 DSMZ SUSPENSION 90% Iscove's MDM + 10% FBS Leukemia B cell 93 NALM-6 DSMZ SUSPENSION RPMI 1640 + 10% FCS + 2 mM glutamine leukemia B cell 94 RS4-11 ATCC SUSPENSION RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, + 10% fetal bovine Lymphoma B cell 95 GRANTA-519 DSMZ SUSPENSION DMEM + 2 mM glutamine + 10% Lymphoma B cell 96 SU-DHL-10 DSMZ SUSPENSION 80-90% RPMI 1640 + 10-20% FBS Lymphoma B cell 97 SU-DHL-4 DSMZ SUSPENSION 80-90% RPMI 1640 + 10-20% FBS Lymphoma B cell 98 SU-DHL-6 DSMZ SUSPENSION 80-90% RPMI 1640 + 10-20% FBS Lymphoma B cell 99 SUP-B15 DSMZ SUSPENSION 80% McCoy's 5A + 20% FBS Lymphoma 100 SUP-M2 DSMZ SUSPENSION RPMI 1640 + 10% FCS + 2 mM Glut

Lysate Preparation for Western Blot

The lysates are prepared from sub-confluent cultures of cell lines maintained in appropriate growth medium in the presence of 10% Foetal Bovine Serum (see table 8 for details). Adherent cell lines were seeded in 150 mm plates (cells harvested at approx. 60-70% confluency); suspension cell lines were grown in T-175 flasks (cells harvested at approx. 200,000 cells/ml).

Protocol for Adherent Cell Lines:

1) Wash plate with cold PBS (without Ca2+ and Mg2+). Remove PBS.

2) Allow excess PBS to drain to one side and remove.

3) Add 1 ml cold lysis buffer and put plate on ice. Scrape the cells.

4) Collect the lysate and wash the plate with another 200 .mu.l lysis buffer and add to the lysate, agitate in cold room for 15 min.

6) Spin for 15 min in microfuge (15000 rpm at 4.degree. C.).

7) Recover supernatant and freeze in aliquots in liquid Nitrogen.

8) Determine protein concentration in an aliquot of each lysate using a BSA reference curve.

9) Bring samples to 1 mg/ml with complete Lysis Buffer, 4.times. LDS Sample Buffer, 20.times. Reducing Agent (1M DTT) and boil 10 min.

Protocol for Suspension Cell Lines:

1) Spin cell suspension for 15 min (2000 rpm at 4.degree. C.) and remove medium.

2) Wash with cold PBS (without Ca2+ and Mg2+), spin for 15 min (2000 rpm at 4.degree. C.) and remove PBS.

3) Add 1 ml cold lysis buffer and pipette the cell lysate in ice.

4 Keep cell/lysate agitating in cold room for 15 min.

6) Spin for 15 min (15000 rpm at 4.degree. C.).

7) Recover supernatant and freeze in aliquots in liquid Nitrogen.

8) Determine protein concentration in all lysates in parallel using the same BSA reference curve.

9) Bring samples to 1 mg/ml with Complete Lysis Buffer, 4.times. LDS Sample Buffer, 20.times. Reducing Agent (1M DTT) and boil 10 min.

Complete Lysis Buffer Composition:

TABLE-US-00012 50 mM Hepes pH 7.5 150 mM NaCl 1% Tritonx-100 1% Deoxycholate 0.1% SDS 10 mM EDTA

Add DTT (final 1 mM) and protease/phosphatase inhibitor cocktails (Sigma P-2850, P-5726, P-8340) as required just before use. DTT Reducing Agent (Biorad, cat. #161-0610): 50 mM final concentration.

LDS Sample Buffer Composition (Invitrogen, cat. #02 98 22 201):

TABLE-US-00013 106 mM Tris HCl pH 8.5 150 mM Tris base 1% TritonX-100 2% LDS 10% glycerol 0.51 mM EDTA 0.22 mM Serva Blue G250 0.175 mM Phenol Red

Western Blot

Protein extracts (10 .mu.g proteins/sample) were resolved by SDS-PAGE using 4-12% Bis-Tris Midi gels (Invitrogen) according to manufacturer's instructions. Membranes were stained with Ponceau Red after transfer. Anti-CD49b and anti-GAPDH antibodies were used diluted 1:1000 and 1:2000, respectively, in blocking buffer containing 5% non-fat dry milk. HRP-conjugated secondary antibodies were used 1:5000 in blocking buffer containing 5% non-fat dry milk.

Antibodies Used in the Study

Mouse monoclonal IgG2a anti-CD49b (Integrin .alpha..sub.2 chain) antibody (Becton Dickinson, cat. #611016); Rabbit polyclonal IgG anti-GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) antibody (Santa Cruz Biotechnology, cat. # sc-25778); HRP-conjugated anti-mouse and anti-rabbit antibodies (Pierce); Biotin-GBR500 (lot AT-220208A) recombinant humanized monoclonal antibody; Biotin-IgG4 (lot AT-090108A) recombinant humanized monoclonal antibody (Glenmark Pharmaceuticals S.A.); Fluorolink.TM.Cy.TM.2 goat anti-mouse IgG (GE Healthcare, cat. # PA42002); Streptavidin-FITC (BD Pharmingen, ca. #554060). Sample Preparation for Immunofluorescence

Cells were cultured in Lab-Tek chamber slides (Nunc) for 48 hours (70,000 cells/chamber), and afterwards fixed with formaldehyde 3.7% (v/v) for 20 minutes. Cells were washed twice with PBS, then saturated for 30 minutes with a blocking solution containing 1% (w/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X-100 (Sigma-Aldrich) in PBS. Primary antibodies were added at the recommended dilution in blocking solution. After 1 hour incubation at 37.degree. C., the solution was removed and cells were washed twice with PBS.

Secondary antibodies, or alternatively Streptavidin-FITC, were added in blocking solution at the recommended dilution together with 1 mg/ml DRAQ5.TM. (Alexis, cat. # BOS-889-001-R200). Slides were incubated for 1 hour at 37.degree. C., then the solution was removed and cells washed twice with PBS. PBS was removed and slides were mounted with coverslips using a Mowiol solution (Mowiol 4.88, Calbiochem cat. #475904).

Laser-Scanning Confocal Microscopy

Immunofluorescence pictures were obtained using an Axioplan microscope (Zeiss) coupled with a Radiance 2000 laser scanning system (Bio-Rad, 40.times. objective, oil immersion). Acquisition was performed using Kalman filter (10 iterations); laser power was equal for the same fluorescence channel in different samples.

Results and Conclusions

Western Blot Analysis of CD49b Expression in Human Cell Lines

Ponceau Red staining of membranes confirmed homogeneous transfer of cellular proteins to membrane.

Western Blot analyses for CD49b and GAPDH expression are shown in FIG. 3. Overall, CD49b is ubiquitously expressed in adherent cell lines, but not in suspension ones (leukemias--block 15 and SCLCs--block 20). There are some exceptions of a few adherent cell lines with undetectable CD49b levels (for example, MIA PaCa-2 and U031), and conversely, of cell lines which grow in suspension expressing high CD49b levels (CEM/VM1 and RS4-11). All cell lines show consistent GAPDH expression, used as a loading/WB control.

The highest levels of CD49b were found in the following cell lines: HT-1080 (fibrosarcoma); BxPC-3 (adenocarcinoma pancreas); CAL-12T (non-small cell lung cancer); NCI-H1299 (non-small-cell lung cancer); TK10 (adenocarcinoma kidney); SNB19 and U251 (glioblastomas); A431 (epidermoid carcinoma).

Regarding colorectal carcinomas, the majority of the 22 tested lines exhibited homogenous, moderately high relative expression of CD49b. In only 3/22 tested lines, expression levels were relatively low, but still readily detectable by Western Blot.

CD49b Detection in Selected Cell Lines by Confocal Microscopy Using GBR500 Antibody

Confocal microscopy was used to test whether Western Blot data obtained using Becton-Dickinson anti-CD49b correlated with cell surface expression using GBR500. In the samples studied, cell lines with high expression of the antigen as judged by Western blot were also found to exhibit specific immunoreactivity at the plasma membrane using GBR500. Human tumor cell lines which express CD49b at the plasma membrane and which therefore could be suitable for in vivo studies include HT-29 colorectal carcinoma and BX-PC3 pancreatic carcinoma lines, though many further candidates have been identified in this study.

Five selected cell lines (HT-1080, BxPC-3, MIAPaCa2, HT-29 and SW480) were immunostained with Biotin-GBR500 and biotin-hIgG4 (unrelated isotype) and analysed by laser-scanning confocal microscopy. These tumor cell lines were selected on the basis of results obtained by Western Blotting using BD anti-CD49b antibody:

BxPC-3: pancreatic carcinoma with high level of CD49b

MIAPaCa2: pancreatic carcinoma with undetectable CD49b

HT-29: colorectal carcinoma with moderately high level of CD49b

SW480: colorectal carcinoma with relatively low, but detectable level of CD49b

The fibrosarcoma cell line HT1080, known to express high levels of CD49b, was used as positive control: confocal microscopy confirms strong membrane staining concentrated at lamellipodia-like areas of the plasma membrane (FIG. 4).

As shown in the images reported in FIG. 5 (BxPC-3) and FIG. 6 (MIA PaCa2) there does appear to be correlation between Western Blot data obtained with the BD antibody and immunocytochemistry results obtained with GBR500: BxPC-3 are strongly positive, while MIAPaCa2 are negative for staining with GBR500. In particular, confocal microscopy revealed strong biotin-GRB500, but not biotin-hIgG4, immunostaining of cell membrane within areas of cell-to-cell contact for BxPC-3. This is a distinct staining pattern compared to HT-1080 cells, where GBR500 staining is confined to possibly lamellipodia-like regions of the plasma membrane.

The colon adenocarcinoma cell line HT-29 showed intense biotin-GRB500 immunostaining (FIG. 7), with a distribution similar to that observed in BX-PC-3 cells.

In SW480 cells, another line with low CD49b expression as judged by Western Blot, biotin-GBR500 staining was nearly indistinguishable from biotin-IgG4 staining (FIG. 8), confirming a low CD49b expression level detected by Western Blot.

In conclusion, CD49b expression was detected in the majority of colon carcinoma cell lines, as well as several other solid tumor types, where it is commonly detected. CD49b expression is relatively rare in Small Cell Lung Carcinoma, and leukemias/lymphomas. Highest expressing cell lines as judged by Western Blot are HT-1080 (fibrosarcoma), BxPC-3 (pancreatic adenocarcinoma), CAL-12T (non-small cell lung cancer), NCI-H1299 (non-small-cell lung cancer), TK10 (renal adenocarcinoma), SNB19 and U251 (glioblastomas).

Example 5

Effect of GBR500 Against the A549 Non Small Cell Lung Cancer Xenograft in nu/nu Athymic Mice

Nu/Nu male mice, from Harlan, Italy, were used. The animals were maintained in cages using steam autoclaved (sterile) bedding, diet and water were offered ad libitum. Animals were identified by a uniquely numbered ear-tag which appears on the data sheets. Body weight at the day of tumor implantation was: 24-31 g.

Number of groups-treatment schedule:

Number of groups was 4. Number of animals/group was 10. Treatment was being started at day 6 after tumor implantation until day 27 as per the regime described in Table 10.

TABLE-US-00014 TABLE 10 Treatment Groups and Study Design Dose Group Compound mg/kg Route/Schedule 1 IgG4 Isotype 50 IP* Day 6, 9, 13, 16, 20, 23, 27** 2 Ha1/29 + 5 + 5 IP Day 6, 9, 13, 16, 20, 23, 27** GBR500 3 Ha1/29 + 5 + 50 IP Day 6, 9, 13, 16, 20, 23, 27** GBR500 4 Avastin .RTM. 40 IP Day 6, 9, 13, 16, 20, 23, 27** *IP: intraperitoneally **Treatment starts at day 6 after tumor implants

Substances:

Test compounds were stored at 4.degree. C. temperature and protected from light until use. Test compounds were dissolved in 0.03% Tween-80.TM. in Phosphate buffered saline and were diluted immediately before use in order to reach the right concentration (IgG4 Isotype, Ha1/29 and GBR500 in PBS; Avastin.RTM. in saline solution). Treatments were administered intraperitoneally (IP) in a volume of 10 ml/kg

Tumor:

The A549 epitelial lung carcinoma cell line (ATCC.RTM. Number: CCL-185) was used as a representative for non small lung cell cancer. Tumor fragments from a A549 xenograft were implanted subcutaneously into the left flank of athymic nude mice. Animals were examined regularly for the appearance of tumors. When measurable tumors have been established in the majority of mice, animals were assigned into treatment groups, with a target of 10 mice per group (5 mice per cage). When treatment starts the mean tumor volume was 120 mm.sup.3.

Evaluation of Antitumor Activity in the Xenograft Models and Toxicity:

At least twice a week the tumor growth and the net body weight were evaluated. Tumor growth was assessed by caliper. Dimensions of the tumors were measured regularly by calliper during the experiments, and tumor masses were calculated as follows:

.times..times..times..times..times..times..times..times..function. ##EQU00002## assuming density d=1 mg/mm.sup.3 for tumor tissue

Toxicity was evaluated on the basis of the body weight reduction. Mice were sacrificed when the tumors reach a volume that hampers them.

Results and Conclusions:

As can be seen from Table 11 the maximal antitumor activity of antibodies treated groups was observed at day 17 with a tumor weight inhibition of 18% and 11% (Ha1/29+GBR500 combination groups respectively). Avastin.RTM., administered at 40 mg/kg, showed a tumor growth inhibition of 20% at day 28. In FIG. 9 the comparison of the average tumor growth observed in the different treatment groups is shown. Treatments were well tolerated and no dead mice in treated groups were found during the experiment. No signs of distress were observed during and after treatments and no significant body weight loss was observed.

TABLE-US-00015 TABLE 11 Treatment Groups and Study Design Dose % Tumor Weight Inhibition Group Compound mg/kg 7 10 14 17 21 24 28 1 IgG4 Isotype 50 -- -- -- -- -- -- -- 2 Ha1/29 + 5 + 5 -3 3 15 18 14 3 0% GBR500 3 Ha1/29 + 5 + 50 -4 5 14 11 11 11 9 GBR500 4 Avastin .RTM. 40 -4 3 10 12 17 18 20

Example 6

GBR500 Toxicokinetics in Cynomolgus Monkeys

As part of a 6-week toxicity study with toxicokinetic endpoints Cynomolgus monkeys were dosed via slow intravenous infusion of GBR 500 over approximately 60 minutes. The animals were dosed once per week for six weeks. (days 1, 8, 15, 22, 29, and 36). Dose Group Assignment and Dose Levels are summarized in Table 12 below:

TABLE-US-00016 TABLE 12 Dose Group Assignment and Dose Levels Group Number of Males/Females Dose Level (mg/kg) 1 5/5 0 (control) 2 3/3 10 3 3/3 30 4 3/3 100

Blood samples of 1 ml were taken from animals on Day 1 (predose, 15 minutes, 4, 8, 24, 48 and 120 hours post infusion, Day 8 (predose, 15 minutes post infusion), Day 15 (predose, 15 minutes post infusion), Day 22 (predose, 15 minutes post infusion), Day 29 (predose, 15 minutes post infusion), Day 36 (predose, 15 minutes, 4, 8, 24, 48 and 120 hours post infusion), and Days 50, 57, 64, 71, 78, 84, 91, and 98. GBR 500 concentration was determined with a validated ELISA assay.

The toxicokinetic (TK) profile of each animal was characterized by non-compartmental analysis of the GBR 500 serum concentration using validated computer software (WinNonlin, version 3.2, Pharsight Corp., Mountain View, Calif., USA). A model was selected based on the vascular route of administration and the serum matrix. The concentration at time zero on Day 1 was assumed to be 0 for the purpose of parameter estimation. Serum concentration values obtained at the predose time-point were used to estimate the concentration at time zero on Day 36.

For the Group 4 recovery animals, the half life T1/2 was estimated between 199 and 316 hours, the volume of distribution Vz was estimated between 10.1-23.6 mL/kg, and the clearance CL between 0.03-0.60 mL/hr/kg. Vz and CL estimates indicated that GBR500 was not distributed beyond the plasma and was very slowly cleared from it. FIGS. 10A and 10B show the concentration curves of the 100 mg dose group for male and female monkeys.

Example 7

In vitro Evaluation of Anti-.alpha..sub.2 Integrin Antibody Potencies in Inhibiting Interaction Between Human .alpha..sub.2 Integrin Expressed on Human Fibrosarcoma Cell Line and Human Collagen

Material and Methods

Flow cytometry and collagen binding inhibition assays were performed as described in Example 1. A fibrosarcoma cell line HT-1080 was used in the experiments (Table 13). HT-1080 was trypsinized to prepare cells for flow cytometry as described for SK-BR-3

TABLE-US-00017 TABLE 13 Cell lines VLA-2 Name Source Supplier Cat# expression HT-1080 Fibrosarcoma ATCC CCL-121 High

Results

TABLE-US-00018 TABLE 14 FACS staining MFI Cell line hIgG4 GBR500 HT-1080 4.9 165.1 VLA-2 expression by HT-1080 was high.

Collagen Binding Assay

TABLE-US-00019 TABLE 15 EC-50 values Cell lines GBR500 EC.sub.50 TMC-2206 EC.sub.50 HT-1080 0.076 .+-. 0.045 (n = 3) 0.097 .+-. 0.038 (n = 3) GBR 500 and TMC-2206 inhibited binding of HT-1080 cells to human collagen I.

CONCLUSION

.alpha.2 integrin expression on the fibrosarcoma cell line HT-1080 was detected using fluorescently-labelled GBR500 antibody. The level of expression VLA-2 is high. The VLA-2 positive cell line HT-1080 adhered to human collagen type I. This binding was inhibited by VLA-2 antibodies GBR 500 and TMC-2206.

Example 8

Effect of GBR500 Against the HT-1080 Fibrosarcoma Xenograft in nu/nu Athymic Mice

Nu/Nu male mice, from Harlan, Italy, were used. The animals were maintained in cages using steam autoclaved (sterile) bedding, diet and water were offered ad libitum. Animals were identified by a uniquely numbered ear-tag which appears on the data sheets. Body weight at the day of tumor implantation was: 24-31 g.

Number of Groups-Treatment Schedule:

Number of groups was 4. Number of animals/group was 10. Treatment was being started as per the regime described in Table 16.

TABLE-US-00020 TABLE 16 Treatment Groups and Study Design Dose Group Compound mg/kg Route/Schedule 1 IgG4 Isotype 50 IP* Day 6, 9, 13, 16, 20, 23, 27** 2 Ha1/29 + 5 + 5 IP Day 6, 9, 13, 16, 20, 23, 27** GBR500 3 Ha1/29 + 5 + 50 IP Day 6, 9, 13, 16, 20, 23, 27** GBR500 4 Avastin .RTM. 40 IP Day 6, 9, 13, 16, 20, 23, 27** *IP: intraperitoneally **Treatment starts at day 6 after tumor implants

Substances:

Test compounds were stored at 4.degree. C. temperature and protected from light until use. Test compounds were dissolved in 0.03% Tween-80.TM. in Phosphate buffered saline and were diluted immediately before use in order to reach the right concentration (IgG4 Isotype, Ha1/29 and GBR500 in PBS; Avastin.RTM. in saline solution). Treatments were administered intraperitoneally (IP) in a volume of 10 ml/kg.

Tumor:

The HT-1080 fibrosarcoma cell line (ATCC.RTM. Number: CCL-121) was used. Tumor fragments from a HT-1080 xenograft were implanted subcutaneously into the left flank of athymic nude mice. Animals were examined regularly for the appearance of tumors.

When measurable tumors have been established in the majority of mice, animals were assigned into treatment groups, with a target of 10 mice per group (5 mice per cage). When treatment starts the mean tumor volume was about 300 mm.sup.3.

Evaluation of Antitumor Activity in the Xenograft Models and Toxicity:

At least twice a week the tumor growth and the net body weight were evaluated. Tumor growth was assessed by caliper. Dimensions of the tumors were measured regularly by calliper during the experiments, and tumor masses were calculated as follows:

.times..times..times..times..times..times..times..times..function. ##EQU00003## assuming density d=1 mg/mm.sup.3 for tumor tissue

Toxicity was evaluated on the basis of the body weight reduction. Mice were sacrificed when the tumors reach a volume that hampers them.

Results and Conclusions:

At day 11, after two doses of the antibody, Avastin led to a reduction of tumor weight of 31.6% relative to control. The Ha 1/29 5 mg/GBR500 5 mg combination led to a tumor weight reduction of 3.5% and the Ha 1/29 5 mg/GBR500 50 mg combination led to a tumor weight reduction of 27.7% (Table 17).

TABLE-US-00021 TABLE 17 Treatment Groups and Study Design Dose % Tumor Weight Inhibition Day 11 Group Compound mg/kg Day 11 1 IgG4 Isotype 50 0 2 Ha1/29 + 5 + 5 3.5 GBR500 3 Ha1/29 + 5 + 50 27.7 GBR500 4 Avastin .RTM. 40 31.6

Treatments were well tolerated and no dead mice in treated groups were found during the experiment. No signs of distress were observed during and after treatments and no significant body weight loss was observed.

SEQUENCE LISTINGS

1

190110PRTHomo SapiensMISC_FEATUREhCDR1 [CDR1 of heavy chain variable region] 1Gly Phe Ser Leu Thr Asn Tyr Gly Ile His1 5 10216PRTHomo SapiensMISC_FEATUREhCDR2 [CDR2 of heavy chain variable region] 2Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met Ser1 5 10 15311PRTHomo SapiensMISC_FEATUREhCDR3 [CDR3 of heavy chain variable region] 3Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr1 5 10410PRTHomo SapiensMISC_FEATURElCDR1 [CDR1 of light chain variable region] 4Ser Ala Asn Ser Ser Val Asn Tyr Ile His1 5 1057PRTHomo SapiensMISC_FEATURElCDR2 [CDR2 of light chain variable region] 5Asp Thr Ser Lys Leu Ala Ser1 569PRTHomo SapiensMISC_FEATURElCDR3 [CDR3 of light chain variable region] 6Gln Gln Trp Thr Thr Asn Pro Leu Thr1 575361DNAHomo Sapiensmisc_featureHuman alpha2 integrin DNA 7ctgcaaaccc agcgcaacta cggtcccccg gtcagaccca ggatggggcc agaacggaca 60ggggccgcgc cgctgccgct gctgctggtg ttagcgctca gtcaaggcat tttaaattgt 120tgtttggcct acaatgttgg tctcccagaa gcaaaaatat tttccggtcc ttcaagtgaa 180cagtttgggt atgcagtgca gcagtttata aatccaaaag gcaactggtt actggttggt 240tcaccctgga gtggctttcc tgagaaccga atgggagatg tgtataaatg tcctgttgac 300ctatccactg ccacatgtga aaaactaaat ttgcaaactt caacaagcat tccaaatgtt 360actgagatga aaaccaacat gagcctcggc ttgatcctca ccaggaacat gggaactgga 420ggttttctca catgtggtcc tctgtgggca cagcaatgtg ggaatcagta ttacacaacg 480ggtgtgtgtt ctgacatcag tcctgatttt cagctctcag ccagcttctc acctgcaact 540cagccctgcc cttccctcat agatgttgtg gttgtgtgtg atgaatcaaa tagtatttat 600ccttgggatg cagtaaagaa ttttttggaa aaatttgtac aaggccttga tataggcccc 660acaaagacac aggtggggtt aattcagtat gccaataatc caagagttgt gtttaacttg 720aacacatata aaaccaaaga agaaatgatt gtagcaacat cccagacatc ccaatatggt 780ggggacctca caaacacatt cggagcaatt caatatgcaa gaaaatatgc ctattcagca 840gcttctggtg ggcgacgaag tgctacgaaa gtaatggtag ttgtaactga cggtgaatca 900catgatggtt caatgttgaa agctgtgatt gatcaatgca accatgacaa tatactgagg 960tttggcatag cagttcttgg gtacttaaac agaaacgccc ttgatactaa aaatttaata 1020aaagaaataa aagcgatcgc tagtattcca acagaaagat actttttcaa tgtgtctgat 1080gaagcagctc tactagaaaa ggctgggaca ttaggagaac aaattttcag cattgaaggt 1140actgttcaag gaggagacaa ctttcagatg gaaatgtcac aagtgggatt cagtgcagat 1200tactcttctc aaaatgatat tctgatgctg ggtgcagtgg gagcttttgg ctggagtggg 1260accattgtcc agaagacatc tcatggccat ttgatctttc ctaaacaagc ctttgaccaa 1320attctgcagg acagaaatca cagttcatat ttaggttact ctgtggctgc aatttctact 1380ggagaaagca ctcactttgt tgctggtgct cctcgggcaa attataccgg ccagatagtg 1440ctatatagtg tgaatgagaa tggcaatatc acggttattc aggctcaccg aggtgaccag 1500attggctcct attttggtag tgtgctgtgt tcagttgatg tggataaaga caccattaca 1560gacgtgctct tggtaggtgc accaatgtac atgagtgacc taaagaaaga ggaaggaaga 1620gtctacctgt ttactatcaa aaagggcatt ttgggtcagc accaatttct tgaaggcccc 1680gagggcattg aaaacactcg atttggttca gcaattgcag ctctttcaga catcaacatg 1740gatggcttta atgatgtgat tgttggttca ccactagaaa atcagaattc tggagctgta 1800tacatttaca atggtcatca gggcactatc cgcacaaagt attcccagaa aatcttggga 1860tccgatggag cctttaggag ccatctccag tactttggga ggtccttgga tggctatgga 1920gatttaaatg gggattccat caccgatgtg tctattggtg cctttggaca agtggttcaa 1980ctctggtcac aaagtattgc tgatgtagct atagaagctt cattcacacc agaaaaaatc 2040actttggtca acaagaatgc tcagataatt ctcaaactct gcttcagtgc aaagttcaga 2100cctactaagc aaaacaatca agtggccatt gtatataaca tcacacttga tgcagatgga 2160ttttcatcca gagtaacctc cagggggtta tttaaagaaa acaatgaaag gtgcctgcag 2220aagaatatgg tagtaaatca agcacagagt tgccccgagc acatcattta tatacaggag 2280ccctctgatg ttgtcaactc tttggatttg cgtgtggaca tcagtctgga aaaccctggc 2340actagccctg cccttgaagc ctattctgag actgccaagg tcttcagtat tcctttccac 2400aaagactgtg gtgaggatgg actttgcatt tctgatctag tcctagatgt ccgacaaata 2460ccagctgctc aagaacaacc ctttattgtc agcaaccaaa acaaaaggtt aacattttca 2520gtaacactga aaaataaaag ggaaagtgca tacaacactg gaattgttgt tgatttttca 2580gaaaacttgt tttttgcatc attctcccta ccggttgatg ggacagaagt aacatgccag 2640gtggctgcat ctcagaagtc tgttgcctgc gatgtaggct accctgcttt aaagagagaa 2700caacaggtga cttttactat taactttgac ttcaatcttc aaaaccttca gaatcaggcg 2760tctctcagtt tccaagcctt aagtgaaagc caagaagaaa acaaggctga taatttggtc 2820aacctcaaaa ttcctctcct gtatgatgct gaaattcact taacaagatc taccaacata 2880aatttttatg aaatctcttc ggatgggaat gttccttcaa tcgtgcacag ttttgaagat 2940gttggtccaa aattcatctt ctccctgaag gtaacaacag gaagtgttcc agtaagcatg 3000gcaactgtaa tcatccacat ccctcagtat accaaagaaa agaacccact gatgtaccta 3060actggggtgc aaacagacaa ggctggtgac atcagttgta atgcagatat caatccactg 3120aaaataggac aaacatcttc ttctgtatct ttcaaaagtg aaaatttcag gcacaccaaa 3180gaattgaact gcagaactgc ttcctgtagt aatgttacct gctggttgaa agacgttcac 3240atgaaaggag aatactttgt taatgtgact accagaattt ggaacgggac tttcgcatca 3300tcaacgttcc agacagtaca gctaacggca gctgcagaaa tcaacaccta taaccctgag 3360atatatgtga ttgaagataa cactgttacg attcccctga tgataatgaa acctgatgag 3420aaagccgaag taccaacagg agttataata ggaagtataa ttgctggaat ccttttgctg 3480ttagctctgg ttgcaatttt atggaagctc ggcttcttca aaagaaaata tgaaaagatg 3540accaaaaatc cagatgagat tgatgagacc acagagctca gtagctgaac cagcagacct 3600acctgcagtg ggaaccggca gcatcccagc cagggtttgc tgtttgcgtg catggatttc 3660tttttaaatc ccatattttt tttatcatgt cgtaggtaaa ctaacctggt attttaagag 3720aaaactgcag gtcagtttgg atgaagaaat tgtggggggt gggggaggtg cggggggcag 3780gtagggaaat aatagggaaa atacctattt tatatgatgg gggaaaaaaa gtaatcttta 3840aactggctgg cccagagttt acattctaat ttgcattgtg tcagaaacat gaaatgcttc 3900caagcatgac aacttttaaa gaaaaatatg atactctcag attttaaggg ggaaaactgt 3960tctctttaaa atatttgtct ttaaacagca actacagaag tggaagtgct tgatatgtaa 4020gtacttccac ttgtgtatat tttaatgaat attgatgtta acaagagggg aaaacaaaac 4080acaggttttt tcaatttatg ctgctcatcc aaagttgcca cagatgatac ttccaagtga 4140taattttatt tataaactag gtaaaatttg ttgttggttc cttttatacc acggctgccc 4200cttccacacc ccatcttgct ctaatgatca aaacatgctt gaataactga gcttagagta 4260tacctcctat atgtccattt aagttaggag agggggcgat atagagacta aggcacaaaa 4320ttttgtttaa aactcagaat ataacattta tgtaaaatcc catctgctag aagcccatcc 4380tgtgccagag gaaggaaaag gaggaaattt cctttctctt ttaggaggca caacagttct 4440cttctaggat ttgtttggct gactggcagt aacctagtga atttttgaaa gatgagtaat 4500ttctttggca accttcctcc tcccttactg aaccactctc ccacctcctg gtggtaccat 4560tattatagaa gccctctaca gcctgacttt ctctccagcg gtccaaagtt atcccctcct 4620ttacccctca tccaaagttc ccactccttc aggacagctg ctgtgcatta gatattaggg 4680gggaaagtca tctgtttaat ttacacactt gcatgaatta ctgtatataa actccttaac 4740ttcagggagc tattttcatt tagtgctaaa caagtaagaa aaataagcta gagtgaattt 4800ctaaatgttg gaatgttatg ggatgtaaac aatgtaaagt aaaacactct caggatttca 4860ccagaagtta cagatgaggc actggaaacc accaccaaat tagcaggtgc accttctgtg 4920gctgtcttgt ttctgaagta ctttttcttc cacaagagtg aatttgacct aggcaagttt 4980gttcaaaagg tagatcctga gatgatttgg tcagattggg ataaggccca gcaatctgca 5040ttttaacaag caccccagtc actaggatgc agatggacca cactttgaga aacaccaccc 5100atttctactt tttgcacctt attttctctg ttcctgagcc cccacattct ctaggagaaa 5160cttagattaa aattcacaga cactacatat ctaaagcttt gacaagtcct tgacctctat 5220aaacttcaga gtcctcatta taaaatggga agactgagct ggagttcagc agtgatgctt 5280tttagtttta aaagtctatg atctgatctg gacttcctat aatacaaata cacaatcctc 5340caagaatttg acttggaaaa g 536181181PRTHomo SapiensMISC_FEATUREHuman alpha2 integrin 8Met Gly Pro Glu Arg Thr Gly Ala Ala Pro Leu Pro Leu Leu Leu Val1 5 10 15Leu Ala Leu Ser Gln Gly Ile Leu Asn Cys Cys Leu Ala Tyr Asn Val 20 25 30Gly Leu Pro Glu Ala Lys Ile Phe Ser Gly Pro Ser Ser Glu Gln Phe 35 40 45Gly Tyr Ala Val Gln Gln Phe Ile Asn Pro Lys Gly Asn Trp Leu Leu 50 55 60Val Gly Ser Pro Trp Ser Gly Phe Pro Glu Asn Arg Met Gly Asp Val65 70 75 80Tyr Lys Cys Pro Val Asp Leu Ser Thr Ala Thr Cys Glu Lys Leu Asn 85 90 95Leu Gln Thr Ser Thr Ser Ile Pro Asn Val Thr Glu Met Lys Thr Asn 100 105 110Met Ser Leu Gly Leu Ile Leu Thr Arg Asn Met Gly Thr Gly Gly Phe 115 120 125Leu Thr Cys Gly Pro Leu Trp Ala Gln Gln Cys Gly Asn Gln Tyr Tyr 130 135 140Thr Thr Gly Val Cys Ser Asp Ile Ser Pro Asp Phe Gln Leu Ser Ala145 150 155 160Ser Phe Ser Pro Ala Thr Gln Pro Cys Pro Ser Leu Ile Asp Val Val 165 170 175Val Val Cys Asp Glu Ser Asn Ser Ile Tyr Pro Trp Asp Ala Val Lys 180 185 190Asn Phe Leu Glu Lys Phe Val Gln Gly Leu Asp Ile Gly Pro Thr Lys 195 200 205Thr Gln Val Gly Leu Ile Gln Tyr Ala Asn Asn Pro Arg Val Val Phe 210 215 220Asn Leu Asn Thr Tyr Lys Thr Lys Glu Glu Met Ile Val Ala Thr Ser225 230 235 240Gln Thr Ser Gln Tyr Gly Gly Asp Leu Thr Asn Thr Phe Gly Ala Ile 245 250 255Gln Tyr Ala Arg Lys Tyr Ala Tyr Ser Ala Ala Ser Gly Gly Arg Arg 260 265 270Ser Ala Thr Lys Val Met Val Val Val Thr Asp Gly Glu Ser His Asp 275 280 285Gly Ser Met Leu Lys Ala Val Ile Asp Gln Cys Asn His Asp Asn Ile 290 295 300Leu Arg Phe Gly Ile Ala Val Leu Gly Tyr Leu Asn Arg Asn Ala Leu305 310 315 320Asp Thr Lys Asn Leu Ile Lys Glu Ile Lys Ala Ile Ala Ser Ile Pro 325 330 335Thr Glu Arg Tyr Phe Phe Asn Val Ser Asp Glu Ala Ala Leu Leu Glu 340 345 350Lys Ala Gly Thr Leu Gly Glu Gln Ile Phe Ser Ile Glu Gly Thr Val 355 360 365Gln Gly Gly Asp Asn Phe Gln Met Glu Met Ser Gln Val Gly Phe Ser 370 375 380Ala Asp Tyr Ser Ser Gln Asn Asp Ile Leu Met Leu Gly Ala Val Gly385 390 395 400Ala Phe Gly Trp Ser Gly Thr Ile Val Gln Lys Thr Ser His Gly His 405 410 415Leu Ile Phe Pro Lys Gln Ala Phe Asp Gln Ile Leu Gln Asp Arg Asn 420 425 430His Ser Ser Tyr Leu Gly Tyr Ser Val Ala Ala Ile Ser Thr Gly Glu 435 440 445Ser Thr His Phe Val Ala Gly Ala Pro Arg Ala Asn Tyr Thr Gly Gln 450 455 460Ile Val Leu Tyr Ser Val Asn Glu Asn Gly Asn Ile Thr Val Ile Gln465 470 475 480Ala His Arg Gly Asp Gln Ile Gly Ser Tyr Phe Gly Ser Val Leu Cys 485 490 495Ser Val Asp Val Asp Lys Asp Thr Ile Thr Asp Val Leu Leu Val Gly 500 505 510Ala Pro Met Tyr Met Ser Asp Leu Lys Lys Glu Glu Gly Arg Val Tyr 515 520 525Leu Phe Thr Ile Lys Lys Gly Ile Leu Gly Gln His Gln Phe Leu Glu 530 535 540Gly Pro Glu Gly Ile Glu Asn Thr Arg Phe Gly Ser Ala Ile Ala Ala545 550 555 560Leu Ser Asp Ile Asn Met Asp Gly Phe Asn Asp Val Ile Val Gly Ser 565 570 575Pro Leu Glu Asn Gln Asn Ser Gly Ala Val Tyr Ile Tyr Asn Gly His 580 585 590Gln Gly Thr Ile Arg Thr Lys Tyr Ser Gln Lys Ile Leu Gly Ser Asp 595 600 605Gly Ala Phe Arg Ser His Leu Gln Tyr Phe Gly Arg Ser Leu Asp Gly 610 615 620Tyr Gly Asp Leu Asn Gly Asp Ser Ile Thr Asp Val Ser Ile Gly Ala625 630 635 640Phe Gly Gln Val Val Gln Leu Trp Ser Gln Ser Ile Ala Asp Val Ala 645 650 655Ile Glu Ala Ser Phe Thr Pro Glu Lys Ile Thr Leu Val Asn Lys Asn 660 665 670Ala Gln Ile Ile Leu Lys Leu Cys Phe Ser Ala Lys Phe Arg Pro Thr 675 680 685Lys Gln Asn Asn Gln Val Ala Ile Val Tyr Asn Ile Thr Leu Asp Ala 690 695 700Asp Gly Phe Ser Ser Arg Val Thr Ser Arg Gly Leu Phe Lys Glu Asn705 710 715 720Asn Glu Arg Cys Leu Gln Lys Asn Met Val Val Asn Gln Ala Gln Ser 725 730 735Cys Pro Glu His Ile Ile Tyr Ile Gln Glu Pro Ser Asp Val Val Asn 740 745 750Ser Leu Asp Leu Arg Val Asp Ile Ser Leu Glu Asn Pro Gly Thr Ser 755 760 765Pro Ala Leu Glu Ala Tyr Ser Glu Thr Ala Lys Val Phe Ser Ile Pro 770 775 780Phe His Lys Asp Cys Gly Glu Asp Gly Leu Cys Ile Ser Asp Leu Val785 790 795 800Leu Asp Val Arg Gln Ile Pro Ala Ala Gln Glu Gln Pro Phe Ile Val 805 810 815Ser Asn Gln Asn Lys Arg Leu Thr Phe Ser Val Thr Leu Lys Asn Lys 820 825 830Arg Glu Ser Ala Tyr Asn Thr Gly Ile Val Val Asp Phe Ser Glu Asn 835 840 845Leu Phe Phe Ala Ser Phe Ser Leu Pro Val Asp Gly Thr Glu Val Thr 850 855 860Cys Gln Val Ala Ala Ser Gln Lys Ser Val Ala Cys Asp Val Gly Tyr865 870 875 880Pro Ala Leu Lys Arg Glu Gln Gln Val Thr Phe Thr Ile Asn Phe Asp 885 890 895Phe Asn Leu Gln Asn Leu Gln Asn Gln Ala Ser Leu Ser Phe Gln Ala 900 905 910Leu Ser Glu Ser Gln Glu Glu Asn Lys Ala Asp Asn Leu Val Asn Leu 915 920 925Lys Ile Pro Leu Leu Tyr Asp Ala Glu Ile His Leu Thr Arg Ser Thr 930 935 940Asn Ile Asn Phe Tyr Glu Ile Ser Ser Asp Gly Asn Val Pro Ser Ile945 950 955 960Val His Ser Phe Glu Asp Val Gly Pro Lys Phe Ile Phe Ser Leu Lys 965 970 975Val Thr Thr Gly Ser Val Pro Val Ser Met Ala Thr Val Ile Ile His 980 985 990Ile Pro Gln Tyr Thr Lys Glu Lys Asn Pro Leu Met Tyr Leu Thr Gly 995 1000 1005Val Gln Thr Asp Lys Ala Gly Asp Ile Ser Cys Asn Ala Asp Ile 1010 1015 1020Asn Pro Leu Lys Ile Gly Gln Thr Ser Ser Ser Val Ser Phe Lys 1025 1030 1035Ser Glu Asn Phe Arg His Thr Lys Glu Leu Asn Cys Arg Thr Ala 1040 1045 1050Ser Cys Ser Asn Val Thr Cys Trp Leu Lys Asp Val His Met Lys 1055 1060 1065Gly Glu Tyr Phe Val Asn Val Thr Thr Arg Ile Trp Asn Gly Thr 1070 1075 1080Phe Ala Ser Ser Thr Phe Gln Thr Val Gln Leu Thr Ala Ala Ala 1085 1090 1095Glu Ile Asn Thr Tyr Asn Pro Glu Ile Tyr Val Ile Glu Asp Asn 1100 1105 1110Thr Val Thr Ile Pro Leu Met Ile Met Lys Pro Asp Glu Lys Ala 1115 1120 1125Glu Val Pro Thr Gly Val Ile Ile Gly Ser Ile Ile Ala Gly Ile 1130 1135 1140Leu Leu Leu Leu Ala Leu Val Ala Ile Leu Trp Lys Leu Gly Phe 1145 1150 1155Phe Lys Arg Lys Tyr Glu Lys Met Thr Lys Asn Pro Asp Glu Ile 1160 1165 1170Asp Glu Thr Thr Glu Leu Ser Ser 1175 118093700DNAHomo Sapiensmisc_featureHuman beta1 integrin DNA 9agccgccgcc acccgccgcg cccgacaccc gggaggcccc gccagcccgc gggagaggcc 60cagcgggagt cgcggaacag caggcccgag cccaccgcgc cgggccccgg acgccgcgcg 120gaaaagatga atttacaacc aattttctgg attggactga tcagttcagt ttgctgtgtg 180tttgctcaaa cagatgaaaa tagatgttta aaagcaaatg ccaaatcatg tggagaatgt 240atacaagcag ggccaaattg tgggtggtgc acaaattcaa catttttaca ggaaggaatg 300cctacttctg cacgatgtga tgatttagaa gccttaaaaa agaagggttg ccctccagat 360gacatagaaa atcccagagg ctccaaagat ataaagaaaa ataaaaatgt aaccaaccgt 420agcaaaggaa cagcagagaa gctcaagcca gaggatatta ctcagatcca accacagcag 480ttggttttgc gattaagatc aggggagcca cagacattta cattaaaatt caagagagct 540gaagactatc ccattgacct ctactacctt atggacctgt cttactcaat gaaagacgat 600ttggagaatg taaaaagtct tggaacagat ctgatgaatg aaatgaggag gattacttcg 660gacttcagaa ttggatttgg ctcatttgtg gaaaagactg tgatgcctta cattagcaca 720acaccagcta agctcaggaa cccttgcaca agtgaacaga actgcaccag cccatttagc 780tacaaaaatg tgctcagtct tactaataaa ggagaagtat ttaatgaact tgttggaaaa 840cagcgcatat ctggaaattt ggattctcca gaaggtggtt tcgatgccat catgcaagtt 900gcagtttgtg gatcactgat tggctggagg aatgttacac ggctgctggt gttttccaca 960gatgccgggt ttcactttgc tggagatggg aaacttggtg gcattgtttt accaaatgat 1020ggacaatgtc acctggaaaa taatatgtac acaatgagcc attattatga ttatccttct 1080attgctcacc ttgtccagaa actgagtgaa aataatattc agacaatttt tgcagttact 1140gaagaatttc agcctgttta caaggagctg aaaaacttga tccctaagtc agcagtagga 1200acattatctg caaattctag caatgtaatt cagttgatca ttgatgcata caattccctt 1260tcctcagaag tcattttgga aaacggcaaa ttgtcagaag gagtaacaat aagttacaaa 1320tcttactgca agaacggggt gaatggaaca ggggaaaatg gaagaaaatg ttccaatatt 1380tccattggag atgaggttca atttgaaatt agcataactt caaataagtg tccaaaaaag 1440gattctgaca gctttaaaat taggcctctg ggctttacgg aggaagtaga ggttattctt 1500cagtacatct gtgaatgtga

atgccaaagc gaaggcatcc ctgaaagtcc caagtgtcat 1560gaaggaaatg ggacatttga gtgtggcgcg tgcaggtgca atgaagggcg tgttggtaga 1620cattgtgaat gcagcacaga tgaagttaac agtgaagaca tggatgctta ctgcaggaaa 1680gaaaacagtt cagaaatctg cagtaacaat ggagagtgcg tctgcggaca gtgtgtttgt 1740aggaagaggg ataatacaaa tgaaatttat tctggcaaat tctgcgagtg tgataatttc 1800aactgtgata gatccaatgg cttaatttgt ggaggaaatg gtgtttgcaa gtgtcgtgtg 1860tgtgagtgca accccaacta cactggcagt gcatgtgact gttctttgga tactagtact 1920tgtgaagcca gcaacggaca gatctgcaat ggccggggca tctgcgagtg tggtgtctgt 1980aagtgtacag atccgaagtt tcaagggcaa acgtgtgaga tgtgtcagac ctgccttggt 2040gtctgtgctg agcataaaga atgtgttcag tgcagagcct tcaataaagg agaaaagaaa 2100gacacatgca cacaggaatg ttcctatttt aacattacca aggtagaaag tcgggacaaa 2160ttaccccagc cggtccaacc tgatcctgtg tcccattgta aggagaagga tgttgacgac 2220tgttggttct attttacgta ttcagtgaat gggaacaacg aggtcatggt tcatgttgtg 2280gagaatccag agtgtcccac tggtccagac atcattccaa ttgtagctgg tgtggttgct 2340ggaattgttc ttattggcct tgcattactg ctgatatgga agcttttaat gataattcat 2400gacagaaggg agtttgctaa atttgaaaag gagaaaatga atgccaaatg ggacacgggt 2460gaaaatccta tttataagag tgccgtaaca actgtggtca atccgaagta tgagggaaaa 2520tgagtactgc ccgtgcaaat cccacaacac tgaatgcaaa gtagcaattt ccatagtcac 2580agttaggtag ctttagggca atattgccat ggttttactc atgtgcaggt tttgaaaatg 2640tacaatatgt ataattttta aaatgtttta ttattttgaa aataatgttg taattcatgc 2700cagggactga caaaagactt gagacaggat ggttattctt gtcagctaag gtcacattgt 2760gcctttttga ccttttcttc ctggactatt gaaatcaagc ttattggatt aagtgatatt 2820tctatagcga ttgaaagggc aatagttaaa gtaatgagca tgatgagagt ttctgttaat 2880catgtattaa aactgatttt tagctttaca aatatgtcag tttgcagtta tgcagaatcc 2940aaagtaaatg tcctgctagc tagttaagga ttgttttaaa tctgttattt tgctatttgc 3000ctgttagaca tgactgatga catatctgaa agacaagtat gttgagagtt gctggtgtaa 3060aatacgtttg aaatagttga tctacaaagg ccatgggaaa aattcagaga gttaggaagg 3120aaaaaccaat agctttaaaa cctgtgtgcc attttaagag ttacttaatg tttggtaact 3180tttatgcctt cactttacaa attcaagcct tagataaaag aaccgagcaa ttttctgcta 3240aaaagtcctt gatttagcac tatttacata caggccatac tttacaaagt atttgctgaa 3300tggggacctt ttgagttgaa tttattttat tatttttatt ttgtttaatg tctggtgctt 3360tctatcacct cttctaatct tttaatgtat ttgtttgcaa ttttggggta agactttttt 3420atgagtactt tttctttgaa gttttagcgg tcaatttgcc tttttaatga acatgtgaag 3480ttatactgtg gctatgcaac agctctcacc tacgcgagtc ttactttgag ttagtgccat 3540aacagaccac tgtatgttta cttctcacca tttgagttgc ccatcttgtt tcacactagt 3600cacattcttg ttttaagtgc ctttagtttt aacagttcac tttttacagt gctatttact 3660gaagttattt attaaatatg cctaaaatac ttaaatcgga 370010798PRTHomo SapiensMISC_FEATUREHuman beta1 integrin 10Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys1 5 10 15Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala 20 25 30Lys Ser Cys Gly Glu Cys Ile Gln Ala Gly Pro Asn Cys Gly Trp Cys 35 40 45Thr Asn Ser Thr Phe Leu Gln Glu Gly Met Pro Thr Ser Ala Arg Cys 50 55 60Asp Asp Leu Glu Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile65 70 75 80Glu Asn Pro Arg Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr 85 90 95Asn Arg Ser Lys Gly Thr Ala Glu Lys Leu Lys Pro Glu Asp Ile Thr 100 105 110Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro 115 120 125Gln Thr Phe Thr Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp 130 135 140Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu145 150 155 160Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met Arg Arg Ile 165 170 175Thr Ser Asp Phe Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr Val 180 185 190Met Pro Tyr Ile Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr 195 200 205Ser Glu Gln Asn Cys Thr Ser Pro Phe Ser Tyr Lys Asn Val Leu Ser 210 215 220Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly Lys Gln Arg225 230 235 240Ile Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met 245 250 255Gln Val Ala Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg 260 265 270Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala Gly Asp Gly 275 280 285Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu 290 295 300Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala305 310 315 320His Leu Val Gln Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala 325 330 335Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile 340 345 350Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser Asn Val Ile 355 360 365Gln Leu Ile Ile Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu 370 375 380Glu Asn Gly Lys Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr385 390 395 400Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser 405 410 415Asn Ile Ser Ile Gly Asp Glu Val Gln Phe Glu Ile Ser Ile Thr Ser 420 425 430Asn Lys Cys Pro Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu 435 440 445Gly Phe Thr Glu Glu Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys 450 455 460Glu Cys Gln Ser Glu Gly Ile Pro Glu Ser Pro Lys Cys His Glu Gly465 470 475 480Asn Gly Thr Phe Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg Val 485 490 495Gly Arg His Cys Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met 500 505 510Asp Ala Tyr Cys Arg Lys Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn 515 520 525Gly Glu Cys Val Cys Gly Gln Cys Val Cys Arg Lys Arg Asp Asn Thr 530 535 540Asn Glu Ile Tyr Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys545 550 555 560Asp Arg Ser Asn Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Lys Cys 565 570 575Arg Val Cys Glu Cys Asn Pro Asn Tyr Thr Gly Ser Ala Cys Asp Cys 580 585 590Ser Leu Asp Thr Ser Thr Cys Glu Ala Ser Asn Gly Gln Ile Cys Asn 595 600 605Gly Arg Gly Ile Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys 610 615 620Phe Gln Gly Gln Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys625 630 635 640Ala Glu His Lys Glu Cys Val Gln Cys Arg Ala Phe Asn Lys Gly Glu 645 650 655Lys Lys Asp Thr Cys Thr Gln Glu Cys Ser Tyr Phe Asn Ile Thr Lys 660 665 670Val Glu Ser Arg Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val 675 680 685Ser His Cys Lys Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr 690 695 700Tyr Ser Val Asn Gly Asn Asn Glu Val Met Val His Val Val Glu Asn705 710 715 720Pro Glu Cys Pro Thr Gly Pro Asp Ile Ile Pro Ile Val Ala Gly Val 725 730 735Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys 740 745 750Leu Leu Met Ile Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys 755 760 765Glu Lys Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys 770 775 780Ser Ala Val Thr Thr Val Val Asn Pro Lys Tyr Glu Gly Lys785 790 79511226PRTHomo SapiensMISC_FEATUREHuman alpha2 I domain 11Ser Pro Asp Phe Gln Leu Ser Ala Ser Phe Ser Pro Ala Thr Gln Pro1 5 10 15Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser 20 25 30Ile Tyr Pro Trp Asp Ala Val Lys Asn Phe Leu Glu Lys Phe Val Gln 35 40 45Gly Leu Asp Ile Gly Pro Thr Lys Thr Gln Val Gly Leu Ile Gln Tyr 50 55 60Ala Asn Asn Pro Arg Val Val Phe Asn Leu Asn Thr Tyr Lys Thr Lys65 70 75 80Glu Glu Met Ile Val Ala Thr Ser Gln Thr Ser Gln Tyr Gly Gly Asp 85 90 95Leu Thr Asn Thr Phe Gly Ala Ile Gln Tyr Ala Arg Lys Tyr Ala Tyr 100 105 110Ser Ala Ala Ser Gly Gly Arg Arg Ser Ala Thr Lys Val Met Val Val 115 120 125Val Thr Asp Gly Glu Ser His Asp Gly Ser Met Leu Lys Ala Val Ile 130 135 140Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Ala Val Leu145 150 155 160Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu 165 170 175Ile Lys Ala Ile Ala Ser Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val 180 185 190Ser Asp Glu Ala Ala Leu Leu Glu Lys Ala Gly Thr Leu Gly Glu Gln 195 200 205Ile Phe Ser Ile Glu Gly Thr Val Gln Gly Gly Asp Asn Phe Gln Met 210 215 220Glu Met2251271PRTHomo SapiensMISC_FEATUREFW1-3 4-59 [FW=1-25; FW2=26-39; FW3=40-71] 12Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro Pro Gly 20 25 30Lys Gly Leu Glu Trp Ile Gly Arg Val Thr Ile Ser Val Asp Thr Ser 35 40 45Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 50 55 60Ala Val Tyr Tyr Cys Ala Arg65 701311PRTHomo SapiensMISC_FEATUREFW4 4-59 [NCBI entry gi/33583] 13Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 101427DNAHomo Sapiensmisc_featureVHL-for [forward primer] 14ccatggctgt cttggggctg ctcttct 271517DNAHomo Sapiensmisc_featureHC-rev [reverse primer] 15ggggccagtg gatagac 171628DNAHomo Sapiensmisc_featureVLL-for [forward primer] 16ccatggattt tcaagtgcag attttcag 281717DNAHomo Sapiensmisc_featureLCkappa-rev [reverse primer] 17gttggtgcag catcagc 1718318DNAHomo Sapiensmisc_featureTMC-2206 VL 18caa ttt gtt ctc acc cag tct cca gca ttc ttg tct gct tct cca ggg 48Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Ala Ser Pro Gly1 5 10 15gag aag gtc acc atg acc tgc agt gcc aac tca agt gtg aat tac att 96Glu Lys Val Thr Met Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30cac tgg tac cag cag aag tca ggc acc tcc ccc aaa aaa tgg att tat 144His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Lys Trp Ile Tyr 35 40 45gac act tcc aaa ctg gct tct gga gtc cct gtt cgc ttc agt ggc agt 192Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60gga tct ggg acc tct tac tct ctc aca atc agc agc atg gag act gag 240Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu65 70 75 80gat gct gcc act tat tac tgc cag cag tgg act act aac cca ctc acg 288Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95ttc ggt gct ggg acc agg gtg gag ctg aaa 318Phe Gly Ala Gly Thr Arg Val Glu Leu Lys 100 10519106PRTHomo Sapiens 19Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Ala Gly Thr Arg Val Glu Leu Lys 100 10520357DNAHomo sapiensCDS(1)..(357)misc_feature(1)..(357)TMC-2206 VH 20cag gtg cag ttg aag gag tca gga cct ggc ctg gtg gcg ccc tca cag 48Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15agc ctg tcc atc act tgt act gtc tct gga ttt tca tta acc aac tat 96Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30ggt att cac tgg gtt cgc cag cct cca gga aag ggt ctg gag tgg ctg 144Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45gga gtg ata tgg gct cgt gga ttc aca aat tat aat tcg gct ctc atg 192Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60tcc aga ctg atc atc aca aaa gac aat tcc cag agt caa gtc ttc tta 240Ser Arg Leu Ile Ile Thr Lys Asp Asn Ser Gln Ser Gln Val Phe Leu65 70 75 80aaa atg aac agt cta caa cct gat gac tca gcc act tac ttc tgt gcc 288Lys Met Asn Ser Leu Gln Pro Asp Asp Ser Ala Thr Tyr Phe Cys Ala 85 90 95aga gcg aac gac ggg gtc tat tat gct atg gac tac tgg ggt cag gga 336Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110acc tca gtc acc gtc tcc tca 357Thr Ser Val Thr Val Ser Ser 11521119PRTHomo sapiens 21Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Ile Ile Thr Lys Asp Asn Ser Gln Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Pro Asp Asp Ser Ala Thr Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser 1152231DNAHomo Sapiensmisc_featureTMC-2206-r5' [forward primer] 22cccgaattca caggtgcagt tgaaggagtc a 312335DNAHomo Sapiensmisc_featureTMC-2206-r3' [reverse primer] 23cgggatcctt aggatcattt accaggagag tggga 352431DNAHomo Sapiensmisc_featureTMC-2206-k5' [forward primer] 24cccgaattca caatttgttc tcacccagtc t 312535DNAHomo Sapiensmisc_featureTMC-2206-k3' [reverse primer] 25cgggatcctt atctctaaca ctcattcctg ttgaa 352622PRTHomo SapiensMISC_FEATUREIgkappa (Igk) leader sequence 26Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Gly Ser Thr Gly Asp 202776DNAHomo Sapiensmisc_featureIgkappa-S Oligonucleotides [Primer] 27tcgagccacc atggagacag acacactcct gctatgggta ctgctgctct gggttccagg 60ttccactgga gacgcg 762876DNAHomo Sapiensmisc_featureIgkappa-AS oligonucleotides [Primer] 28aattcgcgtc tccagtggaa cctggaaccc agagcagcag tacccatagc aggagtgtgt 60ctgtctccat ggtggc 762933DNAHomo Sapiensmisc_featureTMC2206VH-hIgG1/4Fc-SalI 29cttggtcgac gctgaggaga cggtgactga ggt 333030DNAHomo Sapiensmisc_featurehIgG1/4Fc-SalI-F [forward primer] 30tcagcgtcga ccaagggccc atcsgtcttc 303148DNAHomo Sapiensmisc_featurehIgG1/4Fc-NotI-R [reverse primer] 31aagggaagcg gccgcttatc atttacccyg agacagggag aggctctt 483239DNAHomo Sapiensmisc_featureTMC2206VL-hKc-SalI [reverse primer] 32tcgtttgatg tcgaccttgg tcccagcacc gaacgtgag 393335DNAHomo Sapiensmisc_featurehKc-SalI-F [forward primer] 33accaaggtcg acatcaaacg aactgtggct gcacc 353445DNAHomo Sapiensmisc_featurehKc-NotI-R [reverse primer] 34aagggaagcg gccgcttatc arcactctcc cctgttgaag ctctt 453529DNAHomo

Sapiensmisc_featureTMC-2206VLwt-hKc-F [forward primer] 35agggtggagc tgaaacgaac tgtggctgc 293624DNAHomo Sapiensmisc_featureTMC-2206VLwt-hKc-R [reverse primer] 36tcgtttcagc tccaccctgg tccc 2437107PRTHomo SapiensMISC_FEATUREA14 VL germline protein 37Asp Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Gln Ala Ser Glu Gly Ile Gly Asn Tyr 20 25 30Leu Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 1053810PRTHomo SapiensMISC_FEATUREFW 4 of mature kappa light chain 38Phe Gly Gln Gly Thr Lys Val Glu Ile Lys1 5 1039121PRTHomo SapiensMISC_FEATURE4-59 VH germline protein 39Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg His Asn Ser Ser Ser Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12040119PRTHomo SapiensMISC_FEATUREhVH1.0 40Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11541106PRTHomo SapiensMISC_FEATUREhVL1.0 41Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 1054248DNAHomo Sapiensmisc_featurehVH3.0-F [forward primer] 42agcgtggaca ccagcaagaa ccagttcagc ctgaagctga gcagcgtg 484351DNAHomo Sapiensmisc_featurehVH3.0-R [reverse primer] 43gttcttgctg gtgtccacgc tgatggtcac gcgggacatg agagcgctgt t 514448DNAHomo Sapiensmisc_featurehVH4.0-F [forward primer] 44cctccaggca agggcctgga gtggatcggc gtgatatggg ctcgcggc 484551DNAHomo Sapiensmisc_featurehVH4.0-R [reverse primer] 45ctccaggccc ttgcctggag gctggcgtat ccagtggatg ccatagttgg t 514633DNAHomo Sapiensmisc_featurehVL3.0-F [forward primer] 46cccaagctcc tgatctatga cacttccaag ctg 334742DNAHomo Sapiensmisc_featurehVL3.0-R [reverse primer] 47agtgtcatag atcaggagct tgggggcctg gtcgggcttc tg 424845DNAHomo Sapiensmisc_featurehVL4.0-F [forward primer] 48gacgcgaatt cagacgtggt gatgacccag tctccagcat tcctg 454924DNAHomo Sapiensmisc_featurehVH2.0-F [forward primer] 49gtgaccatca gcaaggacaa cagc 245045DNAHomo Sapiensmisc_featurehVH2.0-R [reverse primer] 50gctgttgtcc ttgctgatgg tcacgcggga catgagagcg ctgtt 455127DNAHomo Sapiensmisc_featurehVH5.0-F [forward primer] 51atcggcgtga tatgggctcg cggcttc 275245DNAHomo Sapiensmisc_featurehVH5.0-R [reverse primer] 52gccgcgagcc catatcacgc cgatccactc caggcccttg cctgg 455324DNAHomo Sapiensmisc_featurehVH6.0-F [forward primer] 53atatgggctc gcggcttcac aaac 245424DNAHomo Sapiensmisc_featurehVH6.0-R [reverse primer] 54gtttgtgaag ccgcgagccc atat 245545DNAHomo Sapiensmisc_featurehVH7.0-F [forward primer] 55gccgcggaca ccgccgtgta ctactgcgcc agagccaacg acggg 455627DNAHomo Sapiensmisc_featurehVH7.0-R [reverse primer] 56gtagtacacg gcggtgtccg cggcggt 275727DNAHomo Sapiensmisc_featurehVH8.0-F [forward primer] 57atatccaact atggcatcca ctgggtt 275848DNAHomo Sapiensmisc_featurehVH8.0-R [reverse primer] 58ccagtggatg ccatagttgg atatgctaaa tccagagacg gtacaggt 485945DNAHomo Sapiensmisc_featurehVL2.0-R [reverse primer] 59cagcttggaa gtgtcataga tcaatttctt gggggcctgg tcggg 456045DNAHomo Sapiensmisc_featurehVL5.0-F [forward primer] 60gacgcgaatt cagacttcgt gctgacccag tctccagcat tcctg 456145DNAHomo Sapiensmisc_featurehVL6.0-F [forward primer] 61gacgcgaatt cacagttcgt gatgacccag tctccagcat tcctg 456245DNAHomo Sapiensmisc_featurehVL7.0-F [forward primer] 62gacgcgaatt cagacttcgt gatgacccag tctccagcat tcctg 456327DNAHomo Sapiensmisc_featurehVL8.0-F [forward primer] 63ttcaccttca ccatcagcag cctggag 276448DNAHomo Sapiensmisc_featurehVL8.0-R [reverse primer] 64ctccaggctg ctgatggtga aggtgaagtc ggtgccgctg ccgctgcc 486528DNAHomo Sapiensmisc_featurehLCQ3-F [forward primer] 65ccaatcaagc gtgaactaca ttcactgg 286648DNAHomo Sapiensmisc_featurehLCQ3-R [reverse primer] 66ccagtgaatg tagttcacgc ttgattgggc gctgcaggtg atggtcac 486723DNAHomo Sapiensmisc_featureIgk-For [forward primer] 67actcctgcta tgggtactgc tgc 236822DNAHomo Sapiensmisc_featurehIgG1Fc-CH1-R [reverse primer] 68gaagtagtcc ttgaccaggc ag 226922DNAHomo Sapiensmisc_featureCI-neo-msc3' [Primer] 69tttcactgca ttctagttgt gg 2270119PRTHomo SapiensMISC_FEATUREhVH2.0 70Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11571119PRTHomo SapiensMISC_FEATUREhVH3.0 71Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11572119PRTHomo SapiensMISC_FEATUREhVH4.0 72Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11573119PRTHomo SapiensMISC_FEATUREhVH5.0 73Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11574119PRTHomo SapiensMISC_FEATUREhVH6.0 74Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11575119PRTHomo SapiensMISC_FEATUREhVH7.0 75Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11576119PRTHomo sapiensMISC_FEATUREhVH8.0 76Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11577119PRTHomo SapiensMISC_FEATUREhVH9.0 Position 48 can be Leucine or Isoleucine 77Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Val Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11578119PRTHomo SapiensMISC_FEATUREhVH10.0 78Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11579119PRTHomo SapiensMISC_FEATUREhVH11.0 Position 48 can be Leucine or Isoleucine 79Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11580106PRTHomo SapiensMISC_FEATUREhVL2.0 80Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10581106PRTHomo SapiensMISC_FEATUREhVL3.0 81Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25

30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10582106PRTHomo SapiensMISC_FEATUREhVL4.0 82Asp Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10583106PRTHomo SapiensMISC_FEATUREhVL5.0 83Asp Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10584106PRTHomo SapiensMISC_FEATUREhVL6.0 84Gln Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10585106PRTHomo SapiensMISC_FEATUREhVL7.0 85Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10586106PRTHomo SapiensMISC_FEATUREhVL8.0 86Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10587106PRTHomo SapiensMISC_FEATUREhVL9.0 87Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Lys 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10588106PRTHomo SapiensMISC_FEATUREhVL10.0 88Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10589106PRTHomo SapiensMISC_FEATUREhVL11.0 89Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10590106PRTHomo SapiensMISC_FEATUREhVL1.0Q 90Gln Phe Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10591106PRTHomo SapiensMISC_FEATUREhVL10.0Q 91Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10592106PRTHomo SapiensMISC_FEATUREhVL12.0Q 92Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10593222PRTRattus rattusMISC_FEATURERat alpha2 integrin I domain protein 93Ser Pro Asp Phe Gln Ser Leu Thr Ser Phe Ser Pro Ala Val Gln Asp1 5 10 15Val Val Val Val Cys Asp Glu Ser Asn Ser Ile Tyr Pro Trp Glu Ala 20 25 30Val Lys Asn Phe Leu Glu Lys Phe Val Gln Gly Leu Asp Ile Gly Pro 35 40 45Lys Lys Thr Gln Val Ala Leu Ile Gln Tyr Ala Asn Asp Pro Arg Val 50 55 60Val Phe Asn Leu Thr Thr Tyr Lys Asn Lys Glu Asp Met Val Gln Ala65 70 75 80Thr Ser Glu Thr Arg Gln Tyr Gly Gly Asp Leu Thr Asn Thr Phe Lys 85 90 95Ala Ile Gln Phe Ala Arg Asp Ile Ala Tyr Leu Pro Glu Ser Gly Gly 100 105 110Arg Pro Gly Ala Thr Lys Val Met Val Val Val Thr Asp Gly Glu Ser 115 120 125His Asp Gly Ser Lys Leu Gln Thr Val Ile Gln Gln Cys Asn Asp Asp 130 135 140Glu Ile Leu Arg Phe Gly Ile Ala Val Leu Gly Tyr Leu Asn Arg Asn145 150 155 160Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu Ile Lys Ala Ile Ala Ser 165 170 175Thr Pro Thr Glu Arg Tyr Phe Phe Asn Val Ala Asp Glu Ala Ala Leu 180 185 190Leu Glu Lys Ala Gly Thr Leu Gly Glu His Ile Phe Ser Ile Glu Gly 195 200 205Thr Val Gln Gly Gly Asp Asn Phe Gln Met Glu Met Ala Gln 210 215 22094228PRTMus musculusMISC_FEATUREMouse alpha2 integrin I domain protein 94Ser Pro Asp Phe Gln Phe Leu Thr Ser Phe Ser Pro Ala Val Gln Ala1 5 10 15Cys Pro Ser Leu Val Asp Val Val Val Val Cys Asp Glu Ser Asn Ser 20 25 30Ile Tyr Pro Trp Glu Ala Val Lys Asn Phe Leu Val Lys Phe Val Thr 35 40 45Gly Leu Asp Ile Gly Pro Lys Lys Thr Gln Val Ala Leu Ile Gln Tyr 50 55 60Ala Asn Glu Pro Arg Ile Ile Phe Asn Leu Asn Asp Phe Glu Thr Lys65 70 75 80Glu Asp Met Val Gln Ala Thr Ser Glu Thr Arg Gln His Gly Gly Asp 85 90 95Leu Thr Asn Thr Phe Arg Ala Ile Glu Phe Ala Arg Asp Tyr Ala Tyr 100 105 110Ser Gln Thr Ser Gly Gly Arg Pro Gly Ala Thr Lys Val Met Val Val 115 120 125Val Thr Asp Gly Glu Ser His Asp Gly Ser Lys Leu Lys Thr Val Ile 130 135 140Gln Gln Cys Asn Asp Asp Glu Ile Leu Arg Phe Gly Ile Ala Val Leu145 150 155 160Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu 165 170 175Ile Lys Ala Ile Ala Ser Thr Pro Thr Glu Arg Tyr Phe Phe Asn Val 180 185 190Ser Asp Glu Ala Ala Leu Leu Glu Lys Ala Gly Thr Leu Gly Glu Gln 195 200 205Ile Phe Ser Ile Glu Gly Thr Val Gln Gly Gly Asp Asn Phe Gln Met 210 215 220Glu Met Ser Gln2259526DNAHomo Sapiensmisc_featureKappa-F 95cgaactgtgg ctgcaccatc tgtctt 269641DNAHomo Sapiensmisc_featureKappa-BamHI-R 96aattcggatc cttactaaca ctctcccctg ttgaagctct t 419740DNAHomo Sapiensmisc_featureVH12.0-(K71V)-F 97gcctgaccat cagcgtggac aacagcaaga accaggtgag 409840DNAHomo Sapiensmisc_featureVH12.0(K71V)-R 98ctcacctggt tcttgctgtt gtccacgctg atggtcaggc 409940DNAHomo Sapiensmisc_featureVH13.0-(N73T)-F 99ctgaccatca gcaaggacac cagcaagaac caggtgagcc 4010040DNAHomo Sapiensmisc_featureVH13.0-(N73T)R 100ggctcacctg gttcttgctg gtgtccttgc tgatggtcag 4010141DNAHomo Sapiensmisc_featureVH14.0-(V78F)-F 101gcaaggacaa cagcaagaac cagtttagcc tgaagctgag c 4110241DNAHomo Sapiensmisc_featureVH14.0 (V78F)-R 102gctcagcttc aggctaaact ggttcttgct gttgtccttg c 41103648DNAMacaca fascicularismisc_featureCynomolgus alpha2 I domain 103agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60atagatgttg tggttgtgtg tgatgaatca aatagtattt atccttggga tgcagtagac 120aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240gaagaaatga ttgtagcaac atcccagaca tcccaatatg gtggggacct cacaaacaca 300ttcggagcaa ttcaatatgc aagaaaatat gcctattcag cagcttctgg tgggcgacga 360agtgctacga aagtaatggt agttgtaact gacggtgaat cacatgatgg ttcaatgttg 420aaagctgtga ttgatcaatg caaccatgac aatatactga ggtttggcat agcagttctt 480gggtacttaa acagaaacgc ccttgatact aaaaatttaa taaaagaaat aaaagcgatc 540gctagtattc caacagaaag atactttttc aatgtgtctg atgaagcagc tctactagaa 600aaggctggga cattaggaga acaaattttc agcattgaag gtactgtt 648104648DNAMacaca mulattamisc_featureRhesus alpha2 I domain 104agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60atagatgttg tggttgtgtg tgatgaatca aatagtattt atccttggga tgcagtaaag 120aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240gaagaaatga ttgtagcaac atcccagaca tcccaatatg gtggggacct cacaaacaca 300ttcggagcaa ttcaatatgc aagaaaatat gcctattcag cagcttctgg tgggcgacga 360agtgctacga aagtaatggt agttgtaact gacggtgaat cacatgatgg ttcaatgttg 420aaagctgtga ttgatcaatg caaccatgac aatatactga ggtttggcat agcagttctt 480gggtacttaa acagaaacgc ccttgatact aaaaatttaa taaaagaaat aaaagcgatc 540gctagtattc caacagaaag atactttttc aatgtgtctg atgaagcagc tctactagaa 600aaggctggga cattaggaga acaaattttc agcattgaag gtactgtt 648105997DNAHomo Sapiensmisc_featureIgG4 constant region 105tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg agc 48Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser1 5 10 15acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc 96Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc 144Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc 192Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc tac 240Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr65 70 75 80acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag aga 288Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95gtt gag tcc aaa tat ggt ccc cca tgc cca tca tgc cca gca cct gag 336Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu 100 105 110ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag gac 384Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg gac 432Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat ggc 480Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc aac 528Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 576Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185

190ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc ccg 624Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gag 672Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag aac 720Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn225 230 235 240cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc 768Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 816Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc agg 864Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca tgc 912Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 290 295 300tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc ctc 960Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320tcc ctg tct ctg ggt aaa tgataggatc cgcggccgc 997Ser Leu Ser Leu Gly Lys 325106326PRTHomo Sapiens 106Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser1 5 10 15Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr65 70 75 80Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 85 90 95Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu 100 105 110Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Leu Gly Lys 325107648DNAHOMO SAPIENSmisc_featureHuman alpha2 I Domain 107agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60atagatgttg tggtggtgtg tgatgaatca aatagtattt atccttggga tgcagtaaag 120aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240gaagaaatga ttgtagcaac atcccagaca tcccaatatg gtggggacct cacaaacaca 300ttcggagcaa ttcaatatgc aagaaaatat gcctattcag cagcttctgg tgggcgacga 360agtgctacga aagtaatggt agttgtaact gacggtgaat cacatgatgg ttcaatgttg 420aaagctgtga ttgatcaatg caaccatgac aatatactga ggtttggcat agcagttctt 480gggtacttaa acagaaacgc ccttgatact aaaaatttaa taaaagaaat aaaagcgatc 540gctagtattc caacagaaag atactttttc aatgtgtctg atgaagcagc tctactagaa 600aaggctggga cattaggaga acaaattttc agcattgaag gtactgtt 648108106PRTHomo SapiensMISC_FEATUREhVL12.0 108Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105109119PRTHomo SapiensMISC_FEATUREhVH12.0 Position 48 can be Leucine or Isoleucine 109Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115110119PRTHomo SapiensMISC_FEATUREhVH13.0 110Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115111119PRTHomo SapiensMISC_FEATUREhVH14.0 Position 48 can be Leucine or Isoleucine 111Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11511210PRTHomo SapiensMISC_FEATURELCDR1 [N26Q] 112Ser Ala Gln Ser Ser Val Asn Tyr Ile His1 5 10113681DNARattus rattusmisc_featureRat alpha 2 I domain 113agtccagact ttcagtcgtt gacaagcttc tcacctgcag ttcaagcttg cccttccctc 60gtagatgtcg tagttgtctg tgatgaatca aacagtattt atccctggga agcagtaaag 120aattttttgg aaaagtttgt gcaaggcctg gatataggac ctaaaaagac acaggtggcg 180ttaattcaat atgccaacga cccaagagtt gtctttaact tgaccactta caaaaacaaa 240gaagatatgg ttcaggccac atccgagacg cgccagtatg gtggggacct cacaaacacc 300ttcaaggcta tccaatttgc aagagacatt gcttatttac cggagtctgg cgggcgccca 360ggtgctacaa aagtcatggt agttgtgact gatggggaat cccatgatgg gtcgaagctg 420caaactgtga tccagcaatg caatgatgac gagatactga ggtttggcat agcggttctt 480ggatatttaa acagaaatgc tcttgatact aaaaatctaa tcaaagaaat taaagcaatc 540gctagcactc caacggagag gtactttttc aatgtggccg atgaggcggc tcttctggag 600aaagctggca ctctagggga gcacatattc agcattgaag gcactgttca aggaggagac 660aacttccaga tggaaatggc a 681114648DNAMus musculusmisc_featureMouse alpha 2 I domain clone 114agtccagact ttcagttctt gaccagcttt tcacctgcag ttcaggcttg cccttccctc 60gtggatgttg tagttgtatg tgatgaatca aacagtattt atccttggga agcagtaaag 120aactttttgg taaagtttgt gacaggcctg gatataggac ctaaaaagac acaggtggcg 180ttaattcaat atgccaatga gccgagaatt atatttaact tgaacgattt cgaaaccaaa 240gaggatatgg tccaggccac atctgagacg cgccaacatg gtggggacct cacaaacacc 300ttcagagcta tcgaattcgc aagagactac gcttattcac agacttctgg cgggcgcccg 360ggtgctacaa aagtcatggt agttgtgacc gatggcgagt cccatgatgg gtcgaagctg 420aaaactgtga tccagcaatg caatgatgac gagatactga ggttcggcat agcagttctt 480gggtatttaa acagaaatgc tcttgatact aaaaatttaa tcaaagaaat aaaagcaatt 540gctagtactc caaccgagag atactttttc aatgtggccg acgaagcggc tcttctggag 600aaggctggaa ctctagggga gcaaatattc agcattgaag gcactgtt 6481156DNAHomo sapiensmisc_featureExemplary upstream sequence from the start of gene transcription 115cncaat 61166DNAHomo sapiensmisc_featureExemplary sequence at 3' end of gene 116aataaa 611731DNAHomo Sapiensmisc_featurehalphaI F 117ggggatccag tcctgaattt tcagctctca g 3111827DNAHomo Sapiensmisc_featurehalphaI R 118gggaattcaa cagtaccttc aatgctg 2711919DNAHomo sapiensmisc_featureHuman I domain forward primer 119ggggatccag tcctgattt 1912018DNAHomo sapiensmisc_featureHuman I domain reverse primer 120ggaattcaac agtacctt 1812129DNAHomo Sapiensmisc_featuremalphaI F 121ggggatccag tccagacttt cagttcttg 2912228DNAHomo Sapiensmisc_featuremalphaI R 122tgggaattca acagtgcctt caatgctg 2812331DNAHomo Sapiensmisc_featureralphaI F 123ggggatccag tccagacttt cagtcgttga c 3112431DNAHomo Sapiensmisc_featureralphaI R 124tgggaattct gccatttcca tctggaagtt g 3112534DNAHomo Sapiensmisc_featurehalphaI I21V F 125cagccctgcc cttccctcgt agatgttgtg gttg 3412634DNAHomo Sapiensmisc_featurehalphaI I21V R 126caaccacaac atctacgagg gaagggcagg gctg 3412733DNAHomo Sapiensmisc_featurehalphaI E44V F 127cagtaaagaa ttttttggta aaatttgtca agg 3312833DNAHomo Sapiensmisc_featurehalphaI E44V R 128ccttgacaaa ttttaccaaa aaattcttta ctg 3312935DNAHomo Sapiensmisc_featurehalphaI Q48T F 129ttttggaaaa atttgtaaca ggcctggata taggc 3513035DNAHomo Sapiensmisc_featurehalphaI Q48T R 130gcctatatcc aggcctgtta caaatttttc cgggg 3513133DNAHomo Sapiensmisc_featurehalphaI N67E F 131cagtatgcca atgagccaag agttgtgttt aac 3313233DNAHomo Sapiensmisc_featurehalphaI N67E R 132gttaaacaca actcttggct cattggcata ctg 3313334DNAhomo Sapiensmisc_featurehalphaI V70I F 133tgccaataat ccaagaattg tgtttaactt gaac 3413433DNAHomo Sapiensmisc_featurehalphaI V70I R 134gttcaagtta acacaattct tggattattg gca 3313534DNAHomo Sapiensmisc_featurehalphaI V71I F 135ccaataatcc aagagttatc tttaacttga acac 3413634DNAHomo Sapiensmisc_featurehalphaI V71I R 136gtgttcaagt taaagataac tcttggatta ttgg 3413733DNAHomo Sapiensmisc_featurehalphaI T76D F 137gtgtttaact tgaacgacta taaaaccaaa gaa 3313833DNAHomo Sapiensmisc_featurehaplhaI T76D R 138ttctttggtt ttatagtcgt tcaagttaaa cac 3313933DNAHomo Sapiensmisc_featurehalphaI Y77F F 139tttaacttga acacatttaa aaccaaagaa gaa 3314033DNAHomo Sapiensmisc_featurehalphaI Y77F R 140ttcttctttg gttttaaatg tgttcaagtt aaa 3314133DNAHomo Sapiensmisc_featurehalphaI K78E F 141aacttgaaca catatgaaac caaagaagaa atg 3314233DNAHomo Sapiensmisc_featurehalphaI K78E R 142catttcttct ttggtttcat atgtgttcaa gtt 3314333DNAHomo Sapiensmisc_featurehalphaI Y93H F 143tcccagacat cccaacatgg tggggacctc aca 3314433DNAHomo Sapiensmisc_featurehalphaI Y93H R 144tgtgaggtcc ccaccatgtt gggatgtctg gga 3314539DNAHomo Sapiensmisc_featurehalphaI Y93F F 145acatgggaga catcccaatt tggtggggac ctcacaaac 3914639DNAHomo Sapiensmisc_featurehalphaI Y93F R 146gtttgtgagg tccccaccaa attgggatgt ctcccatgt 3914733DNAHomo Sapiensmisc_featurehalphaI Q105E F 147ttcggagcaa ttgaatatgc aagaaaatat gcc 3314833DNAHomo Sapiensmisc_featurehalphaI Q105E R 148ggcatatttt cttgcatatt caattgctcc gaa 3314939DNAHomo Sapiensmisc_featurehalphaI A114Q F 149aaatatgcct attcacaagc ttctggtggg cgacgaagt 3915039DNAHomo Sapiensmisc_featurehalphaI A114Q R 150acttcgtcgc ccaccagaag cttgtgaata ggcatattt 3915139DNAHomo Sapiensmisc_featurehalphaI A115T F 151aaatatgcct attcagcaac ttctggtggg cgacgaagt 3915239DNAHomo Sapiensmisc_featurehalphaI A115T R 152acttcgtcgc ccaccagaag ttgctgaata ggcatattt 3915339DNAHomo Sapiensmisc_featurehalphaI A115Q F 153aaatatgcct attcagcaca gtctggtggg cgacgaagt 3915439DNAHomo Sapiensmisc_featurehalphaI A115Q R 154acttcgtcgc ccaccagact gtgctgaata ggcatattt 3915539DNAHomo Sapiensmisc_featurehalphaI R165D F 155gttcttgggt acttaaacga caacgccctt gatactaaa 3915639DNAHomo Sapiensmisc_featurehalphaI R165D R 156tttagtatca agggcgttgt cgtttaagta cccaagaac 3915739DNAHomo Sapiensmisc_featurehalphaI N166D F 157cttgggtact taaacaggga cgcccttgat actaaaaat 3915839DNAHomo Sapiensmisc_featurehalphaI N166D R 158atttttagta tcaagggcgt ccctgtttaa gtacccaag 3915940DNAHomo Sapiensmisc_featurehalphaI E195W F 159ttcaatgtgt ctgattgggc agctctacta gaaaaggctg 4016040DNAHomo Sapiensmisc_featurehalphaI E195W R 160cagccttttc tagtagagct gcccaatcag acacattgaa 4016138DNAHomo Sapiensmisc_featurehalphaI K40D F 161atccttggga tgcagtagac aattttttgg aaaaattt 3816238DNAHomo Sapiensmisc_featurehalphaI K40D R 162aaatttttcc aaaaaattgt ctactgcatc ccaaggat 3816339DNAHomo Sapiensmisc_featurehalphaI R69D F 163cagtatgcca ataatccaga cgttgtgttt aacttgaac 3916439DNAHomo Sapiensmisc_featurehalphaI R69D R 164gttcaagtta aacacaacgt ctggattatt ggcatactg 3916536DNAHomo Sapiensmisc_featurehalphaI N73D F 165aatccaagag ttgtgtttga cttgaacaca tataaa 3616636DNAHomo Sapiensmisc_featurehalphaI N73D R 166tttatatgtg ttcaagtcaa acacaactct tggatt 3616739DNAHomo Sapiensmisc_featurehalphaI Q89H F 167atgattgtag caacatccca cacatcccaa tatggtggg 3916839DNAHomo Sapiensmisc_featurehalphaI Q89H R 168atgattgtag caacatccca cacatcccaa tatggtggg 3916940DNAHomo

Sapiensmisc_featuremalphaI H93Y F 169cacatctgag acgcgccaat atggtgggga cctcacaaac 4017040DNAHomo Sapiensmisc_featuremalphaI H93Y R 170gtttgtgagg tccccaccat attggcgcgt ctcagatgtg 40171216PRTMacaca fascicularisMISC_FEATURECynomologus 171Ser Pro Asp Phe Gln Leu Ser Ala Ser Phe Ser Pro Ala Thr Gln Pro1 5 10 15Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser 20 25 30Ile Tyr Pro Trp Asp Ala Val Asp Asn Phe Leu Glu Lys Phe Val Gln 35 40 45Gly Leu Asp Ile Gly Pro Thr Lys Thr Gln Val Gly Leu Ile Gln Tyr 50 55 60Ala Asn Asn Pro Arg Val Val Phe Asn Leu Asn Thr Tyr Lys Thr Lys65 70 75 80Glu Glu Met Ile Val Ala Thr Ser Gln Thr Ser Gln Tyr Gly Gly Asp 85 90 95Leu Thr Asn Thr Phe Gly Ala Ile Gln Tyr Ala Arg Lys Tyr Ala Tyr 100 105 110Ser Ala Ala Ser Gly Gly Arg Arg Ser Ala Thr Lys Val Met Val Val 115 120 125Val Thr Asp Gly Glu Ser His Asp Gly Ser Met Leu Lys Ala Val Ile 130 135 140Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Ala Val Leu145 150 155 160Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu 165 170 175Ile Lys Ala Ile Ala Ser Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val 180 185 190Ser Asp Glu Ala Ala Leu Leu Glu Lys Ala Gly Thr Leu Gly Glu Gln 195 200 205Ile Phe Ser Ile Glu Gly Thr Val 210 215172216PRTMacaca mulattaMISC_FEATURERhesus 172Ser Pro Asp Phe Gln Leu Ser Ala Ser Phe Ser Pro Ala Thr Gln Pro1 5 10 15Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser 20 25 30Ile Tyr Pro Trp Asp Ala Val Lys Asn Phe Leu Glu Lys Phe Val Gln 35 40 45Gly Leu Asp Ile Gly Pro Thr Lys Thr Gln Val Gly Leu Ile Gln Tyr 50 55 60Ala Asn Asn Pro Arg Val Val Phe Asn Leu Asn Thr Tyr Lys Thr Lys65 70 75 80Glu Glu Met Ile Val Ala Thr Ser Gln Thr Ser Gln Tyr Gly Gly Asp 85 90 95Leu Thr Asn Thr Phe Gly Ala Ile Gln Tyr Ala Arg Lys Tyr Ala Tyr 100 105 110Ser Ala Ala Ser Gly Gly Arg Arg Ser Ala Thr Lys Val Met Val Val 115 120 125Val Thr Asp Gly Glu Ser His Asp Gly Ser Met Leu Lys Ala Val Ile 130 135 140Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Ala Val Leu145 150 155 160Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu 165 170 175Ile Lys Ala Ile Ala Ser Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val 180 185 190Ser Asp Glu Ala Ala Leu Leu Glu Lys Ala Gly Thr Leu Gly Glu Gln 195 200 205Ile Phe Ser Ile Glu Gly Thr Val 210 2151731435DNAHomo Sapiensmisc_featurehvH14.0 IgG4 Heavy Chain 173tctcgagaag cttccaccat g gag aca gac aca ctc ctg cta tgg gta ctg 51 Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 1 5 10ctg ctc tgg gtt cca ggt tcc act gga cag gtg cag ttg cag gag tca 99Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser 15 20 25ggc cct ggc ctg gtg aag ccc agc gag acc ctg agc ctg acc tgt acc 147Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr 30 35 40gtc tct gga ttt agc tta acc aac tat ggc atc cac tgg ata cgc cag 195Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln 45 50 55cct cca ggc aag ggc ctg gag tgg ctg ggc gtg ata tgg gct cgc ggc 243Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Arg Gly 60 65 70ttc aca aac tat aac agc gct ctc atg tcc cgc gtg acc atc agc aag 291Phe Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys75 80 85 90gac aac agc aag aac cag gtg agc ctg aag ctg agc agc gtg acc gcc 339Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala 95 100 105gcg gac acc gcc gtg tac tac tgc gcc aga gcc aac gac ggg gtc tac 387Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr 110 115 120tat gcc atg gac tac tgg ggc cag gga acc ctg gtc acc gtc agc tca 435Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 125 130 135gcg tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 483Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 140 145 150agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 531Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr155 160 165 170ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 579Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 175 180 185ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 627Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 190 195 200ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 675Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 205 210 215tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 723Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 220 225 230aga gtt gag tcc aaa tat ggt ccc cca tgc cca tca tgc cca gca cct 771Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro235 240 245 250gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 819Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 255 260 265gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 867Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 270 275 280gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 915Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 285 290 295ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 963Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 300 305 310aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 1011Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp315 320 325 330tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 1059Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 335 340 345ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1107Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 350 355 360gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 1155Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 365 370 375aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 1203Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 380 385 390atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1251Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys395 400 405 410acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 1299Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 415 420 425agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 1347Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 430 435 440tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 1395Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 445 450 455ctc tcc ctg tct ctg ggt aaa tgataggatc cgcggccgc 1435Leu Ser Leu Ser Leu Gly Lys 460 465174465PRTHomo Sapiens 174Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly1 5 10 15Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60Glu Trp Leu Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser65 70 75 80Ala Leu Met Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln 85 90 95Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460Lys4651751435DNAHomo Sapiensmisc_featurehvH12.0 IgG4 Heavy Chain 175tctcgagaag cttccaccat g gag aca gac aca ctc ctg cta tgg gta ctg 51 Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 1 5 10ctg ctc tgg gtt cca ggt tcc act gga cag gtg cag ttg cag gag tca 99Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser 15 20 25ggc cct ggc ctg gtg aag ccc agc gag acc ctg agc ctg acc tgt acc 147Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr 30 35 40gtc tct gga ttt agc tta acc aac tat ggc atc cac tgg ata cgc cag 195Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln 45 50 55cct cca ggc aag ggc ctg gag tgg ctg ggc gtg ata tgg gct cgc ggc 243Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Arg Gly 60 65 70ttc aca aac tat aac agc gct ctc atg tcc cgc ctg acc atc agc aag 291Phe Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Leu Thr Ile Ser Lys75 80 85 90gac aac agc aag aac cag gtg agc ctg aag ctg agc agc gtg acc gcc 339Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala 95 100 105gcg gac acc gcc gtg tac tac tgc gcc aga gcc aac gac ggg gtc tac 387Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr 110 115 120tat gcc atg gac tac tgg ggc cag gga acc ctg gtc acc gtc agc tca 435Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 125 130 135gcg tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 483Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 140 145 150agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 531Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr155 160 165 170ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 579Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 175 180 185ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 627Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 190 195 200ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 675Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 205 210 215tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 723Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 220 225 230aga gtt gag tcc aaa tat ggt ccc cca tgc cca tca tgc cca gca cct 771Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro235 240 245 250gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 819Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 255 260 265gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 867Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 270 275 280gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 915Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 285 290 295ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 963Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 300 305 310aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 1011Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp315 320 325 330tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 1059Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 335 340 345ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1107Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 350 355 360gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 1155Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 365 370 375aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 1203Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 380 385 390atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1251Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys395 400 405 410acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 1299Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 415 420 425agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 1347Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 430 435 440tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 1395Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 445 450 455ctc tcc

ctg tct ctg ggt aaa tgataggatc cgcggccgc 1435Leu Ser Leu Ser Leu Gly Lys 460 465176465PRTHomo Sapiens 176Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly1 5 10 15Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60Glu Trp Leu Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser65 70 75 80Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln 85 90 95Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460Lys465177744DNAHomo Sapeinsmisc_featureVL10.0Q light chain 177ctatctcgag aagcttccac c atg gag aca gac aca ctc ctg cta tgg gta 51 Met Glu Thr Asp Thr Leu Leu Leu Trp Val 1 5 10ctg ctg ctc tgg gtt cca ggt tcc act gga gac ttc gtg atg acc cag 99Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Phe Val Met Thr Gln 15 20 25tct cca gca ttc ctg agc gtg acc ccc ggc gag aag gtg acc atc acc 147Ser Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys Val Thr Ile Thr 30 35 40tgc agc gcc caa tca agc gtg aac tac att cac tgg tac cag cag aag 195Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile His Trp Tyr Gln Gln Lys 45 50 55ccc gac cag gcc ccc aag aaa ttg atc tat gac act tcc aag ctg gcc 243Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr Asp Thr Ser Lys Leu Ala 60 65 70agc ggc gtg ccc agc cgc ttc agc ggc agc ggc agc ggc acc gac tac 291Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr75 80 85 90acc ttc acc atc agc agc ctg gag gcc gag gac gct gcc acc tat tac 339Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 95 100 105tgc cag cag tgg acc act aac cca ctg acc ttc ggc cag ggc acc aag 387Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys 110 115 120gtc gaa atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 435Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 125 130 135cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 483Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 140 145 150ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 531Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp155 160 165 170aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 579Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 175 180 185agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 627Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 190 195 200gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 675Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 205 210 215ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt 720Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 220 225 230tgataggatc cgcggccgca tagg 744178233PRTHomo Sapeins 178Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser 20 25 30Val Thr Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser 35 40 45Val Asn Tyr Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys 50 55 60Lys Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser 85 90 95Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr 100 105 110Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr Lys Ser Phe Asn Arg Gly Glu Cys225 2301791460DNAHomo Sapiensmisc_featureh2206-VH14 IgG1 Heavy Chain (DANS); Positions -24 to -5 is the leader sequence; Position -4 to -1 are extra amino acids; DANS at positions -4 to -1 is preferably deleted 179ataggctagc ctcgagccac c atg gag aca gac aca ctc ctg cta tgg gta 51 Met Glu Thr Asp Thr Leu Leu Leu Trp Val -20 -15ctg ctg ctc tgg gtt cca ggt tcc act gga gac gcg aat tca cag gtg 99Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Ala Asn Ser Gln Val -10 -5 -1 1cag ttg cag gag tca ggc cct ggc ctg gtg aag ccc agc gag acc ctg 147Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu 5 10 15agc ctg acc tgt acc gtc tct gga ttt agc tta acc aac tat ggc atc 195Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Ile 20 25 30cac tgg ata cgc cag cct cca ggc aag ggc ctg gag tgg ctg ggc gtg 243His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val35 40 45 50ata tgg gct cgc ggc ttc aca aac tat aac agc gct ctc atg tcc cgc 291Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg 55 60 65gtg acc atc agc aag gac aac agc aag aac cag gtg agc ctg aag ctg 339Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu 70 75 80agc agc gtg acc gcc gcg gac acc gcc gtg tac tac tgc gcc aga gcc 387Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala 85 90 95aac gac ggg gtc tac tat gcc atg gac tac tgg ggc cag gga acc ctg 435Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110gtc acc gtc agc tca gcg tcg acc aag ggc cca tcg gtc ttc ccc ctg 483Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 130gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc 531Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 135 140 145ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tca tgg aac tca 579Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 150 155 160ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc 627Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc 675Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac 723Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205 210acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac 771Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 215 220 225aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc 819Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 230 235 240ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc 867Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag 915Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag 963Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys275 280 285 290aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc 1011Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 295 300 305gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag 1059Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 310 315 320tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc 1107Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc 1155Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg 1203Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu355 360 365 370gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat 1251Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 375 380 385ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc 1299Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 390 395 400gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg 1347Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg 1395Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1440His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys435 440 445tgataagcgg ccgcttccct 1460180473PRTHomo Sapiens 180Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10Gly Ser Thr Gly Asp Ala Asn Ser Gln Val Gln Leu Gln Glu Ser Gly -5 -1 1 5Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val 10 15 20Ser Gly Phe Ser Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro25 30 35 40Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Arg Gly Phe 45 50 55Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys Asp 60 65 70Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala 75 80 85Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr Tyr 90 95 100Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala105 110 115 120Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 125 130 135Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 140 145 150Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 155 160 165Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 170 175 180Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr185 190 195 200Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 205 210 215Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 220 225 230Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 235 240 245Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 250 255 260Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr265 270 275 280Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 285 290 295Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 300 305 310Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 315 320 325Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 330 335 340Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu345 350 355 360Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 365 370 375Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 380 385 390Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 395 400 405Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 410 415 420Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln425 430 435 440Lys Ser Leu Ser Leu Ser Pro Gly Lys 445181469PRTHomo SapiensMISC_FEATUREhVH14.0 IgG1 Heavy Chain [DANS-Deleted] 181Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser 35 40 45Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly 50 55 60Leu Glu Trp Leu Gly Val

Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn65 70 75 80Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn 85 90 95Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Lys465182469PRTHomo SapeinsMISC_FEATUREhVH12.0 IgG1 Heavy Chain [DANS-deleted] 182Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 20 25 30Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser 35 40 45Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly 50 55 60Leu Glu Trp Leu Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn65 70 75 80Ser Ala Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn 85 90 95Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly Lys46518324PRTHomo sapiens 183His Asn Ser Ser Ser Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp Gly Gln1 5 10 15Gly Thr Leu Val Thr Val Ser Ser 2018419PRTHomo sapiens 184Glu Glu Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val1 5 10 15Glu Ile Lys185119PRTArtificial sequencehumanized VH region 185Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Xaa Xaa Tyr 20 25 30Gly Ile His Trp Xaa Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Xaa 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Xaa Thr Ile Ser Xaa Asp Xaa Ser Lys Asn Gln Xaa Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Xaa Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115186106PRTArtificial sequencehumanized VL region 186Xaa Xaa Val Xaa Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Xaa Xaa Ile Xaa 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Xaa Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105187446PRTartificialanti-human alpha2 integrin GBR500 (heavy chain) 187Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ala Arg Gly Phe Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445188213PRTartificialanti-human alpha2 integrin GBR500 (light chain) 188Asp Phe Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Ile Thr Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Lys Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Thr Asn Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 2101891338DNAartificialanti-human alpha2 integrin GBR500 (heavy chain nucleo.) 189caggtgcagt tgcaggagtc aggccctggc ctggtgaagc ccagcgagac cctgagcctg 60acctgtaccg tctctggatt tagcttaacc aactatggca tccactggat acgccagcct 120ccaggcaagg gcctggagtg gctgggcgtg atatgggctc gcggcttcac aaactataac 180agcgctctca tgtcccgcct gaccatcagc aaggacaaca gcaagaacca ggtgagcctg 240aagctgagca gcgtgaccgc cgcggacacc gccgtgtact actgcgccag agccaacgac 300ggggtctact atgccatgga ctactggggc cagggaaccc tggtcaccgt cagctcagcg 360tccaccaagg gcccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 420acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 600acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 660tatggtcccc catgcccatc atgcccagca cctgagttcc tggggggacc atcagtcttc 720ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 780gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 840gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 960aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1080caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1260gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1320tccctgtctc tgggtaaa 1338190639DNAartificialanti-human alpha2 integrin GBR500 (light chain nucleo.) 190gacttcgtga tgacccagtc tccagcattc ctgagcgtga cccccggcga gaaggtgacc 60atcacctgca gcgcccaatc aagcgtgaac tacattcact ggtaccagca gaagcccgac 120caggccccca agaaattgat ctatgacact tccaagctgg ccagcggcgt gcccagccgc 180ttcagcggca gcggcagcgg caccgactac accttcacca tcagcagcct ggaggccgag 240gacgctgcca cctattactg ccagcagtgg accactaacc cactgacctt cggccagggc 300accaaggtcg aaatcaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgt 639