BRPI0620469A2 - humanized anti-integrin alpha2 antibody, method for determining whether a sample contains alpha2 integrin, kit, isolated nucleic acid, vector, host cell, process for producing a humanized anti-integrin alpha2 antibody, screening method, composition, method for treatment of alpha2beta1 integrin-associated disorders in patients, method for inhibiting leukocyte binding to collagen, method of targeting a molecule, use of a humanized anti-integrin alpha2 antibody, packaging and epitope of alpha2 integrin that binds an anti-integrin antibody alpha 2 integrin - Google Patents

Abstract

ANTI-INTEGRINE HUMANIZED ANTIBODY <244> 2, METHOD FOR DETERMINING IF A SAMPLE CONTAINS INTEGRIN <244> 2, KIT, NUCLEIC ACID, VECTOR, HOST CELL, PROCESS FOR ANTI-ANYPRODUCTION224 Screening Method, Composition, Method for the Treatment of Integral Associated Disorders <244> 2 <225> 1 IN PATIENTS, METHOD FOR INHIBITION OF LEUKOCYTE CONNECTION, ANCULAR DENOUS USE, -INTEGRIN <244> 2, PACKAGE AND EPITOPE OF INTEGRIN <244> L2 CONNECTING ANTI-INTEGRIN ANTIBODY <244> 2. This invention relates to anti-integrin? 2 antibodies and their uses. Humanized antibodies that bind to the β2 integrin β domain and inhibit the interaction of β2β1 integrin with collagen are described. Also described are the therapeutic uses of anti-β2 integrin antibodies in the treatment of β2 <225> mediated disorders, including anti-β2 integrin antibodies that bind to β2 integrin without activating the platelets.

Description

"HUMANIZED ANTIBODY ΑΝΤΙ-INTEGRIN α2, METHOD FOR DETERMINING IF A SAMPLE CONTAINS INTEGRINE (X2, KIT, NUCLEIC ACID, VECTOR, HOST CELL, PROCESS FOR PRODUCTION OF AN ANTIODY-HUMANIZED ANTIDOTE, METHOD FOR

TREATMENT OF α2β1 INTEGRIN-ASSOCIATED DISORDERS IN PATIENTS, METHOD FOR INHIBITION OF COLLAGEN LEUKOCYTE CONNECTION, METHOD OF DIRECTING AN INTEGRUM AND ANTI-INTEGRUM HUMANIZED ANTIPIN, ANTI-INPRO2 HUMANIZED ANTIBODY ANTI-INTEGRINE a2 ". TECHNICAL FIELD

The present invention relates generally to α2β1 integrin-directed antibodies and their uses, including humanized anti-integrin alpha 2 (a2) antibodies and methods of treatment with anti-α2 integrin antibodies.

BACKGROUND OF THE INVENTION

Α2β1 integrin (very late antigen 2; VLA-2) is expressed in various 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 common ligands for α2β1 are collagen and laminin, both found in the extracellular matrix. In general, the domain

I of integrin a2 binds to collagen in a divalent cation dependent manner, whereas the same domain binds to laminin through divalent cation dependent and independent mechanisms. (Dickeson et al., Cell Adhesion and Communication. 5: 273- 281 (1998)). Α2β1 integrin specificity varies by cell type and functions as a collagen and / or laminin receptor for certain cell types. Α2β1 integrin, for example, is known as a platelet collagen receptor and as a laminin receptor for endothelial cells. (Dickeson et al, J Biol. Chem. 272: 7661-7668 (1997)). Ecovirus-1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, and various collectins and Clq complement proteins are also α2β1 integrin ligands. (Dickeson et al, J Biol. Chem. 107: 143-50 (2006)). Α2β1 integrin has been implicated in a number of biological and pathological processes including collagen-induced platelet aggregation, collagen cell migration, cell-dependent collagen fiber reorganization, as well as collagen-dependent cellular responses that result in increased expression and proliferation of cytokines, (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 hyersensitivity. contact hypersensitivity and collagen-induced arthritis (from 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. al., Am. J. Pathol. 160 (1): 195-204 (2002)). Integrins are heterodimers composed of an α subunit and a β subunit and constitute a large family of cell surface proteins that mediate cell adhesion to extracellular matrix (ECM) as well as plasma proteins, and are essential for some types of cellular interaction. Integrins interact with their extracellular matrix components through their extracellular domains. (Pozzi & Zent, Exp Nephrol. 94: 77-84 (2003)). By binding to ligands, integrins transduce intracellular signals to the cytoskeleton, which modify cellular activity in response to these cell adhesion events, a process called outside-in signaling (see for example: Hemler, Annu Rev Immunol 8: 365: 365-400 (1999); Hynes, Cell. 110 (6): 673-87 (2002)). This signaling can also activate other integrin subtypes expressed in the same cell, called inside-out signaling. Inside-out signaling also occurs through regulatory signals originating from the cellular cytoplasm, such as clasp disruption between an α and β subunit, which are then transmitted to the receptor's external ligand binding domain. Integrins can play an important role in cell adhesion events that control organ development, morphogenesis, physiology, and pathology, as well as normal tissue homeostasis and immune and thrombotic responses, and serve as external media sensors for the cell. These proteins are characterized by a closed conformation under normal conditions and undergo a rapid conformation change upon activation, which exposes the ligand binding site. X-ray crystal microscopy is a recent tool that has been used to study the structure and mechanisms of integrin activation. Understanding the structural characteristics of integrin facilitates the understanding of binding sites, differentiated states and their active and inactive formations. In general, the linker / counter-receptor binding site for all integrins resides within the α domain and is composed of a metal ion dependent binding site known as the MIDAS domain (Dembo et al, J Biol.Chem. 274, 32108-32111 (1988); Feuston et al., J. Med. Chem. 46: 5316-5325 (2003); Gadek et al., Science 295 (5557): 1086-9 (2002)); Gurrath et al. , Eur. J. Biochem. 210: 911-921 (1992)). In α-subunits of collagen-linked integrins, which include αϊ, a2, alO, and therein integrins, the MIDAS site is located in an extra domain inserted into the N-terminal known as domain I, A, or I / A, a shared feature. with α subunits of the integrin β2 leukocyte family (Randi and Hogg, J Biol Chem. 269: 12395-8 (1994), Larson et al J Cell Biol. 108 (2): 703-12 (1989), Lee et al., J. Biol. Chem. 269: 12395-8 (1995); Emsley et al., J. Biol. Chem. 272: 28512-28517 (1997) and Cell 100: 47-56 (2000)). Domains I are structurally homologous to the von Willebrandt Factor Al domain, with a six-chain Rossman-fold topology Beta Sheet (β-sheet) surrounded by seven α-helices (Colombatti and Bonaldo, Blood 77 (11): 2305 -15 (1991); Larson et al., J Cell Biol. 108 (2): 703-712 (1989); Emsley et al., J. Biol. Chem. 272: 28512-28517 (1997); Noite et al; FEBS Letters, 452 (3): 379-385 (1999)). Collagen-binding integrins have an additional α-helix known as the BC helix (Emsley et al., J. Biol. Chem. 272: 28512-28517 (1997) and Cell 100: 47-56 (2000); Noite et al FEBS Letters, 452 (3): 379-385 (1999)).

Integrin / ligand interactions may facilitate leukocyte extravasation into tissues with inflammatory process (Jackson et al., J. Med. Chem. 40: 3359-3368 (1997); Gadek et al., Science 295 (5557): 1086 -9 (2002), Sircar et al., Bioorg. Med. Chem. 10: 2051-2066 (2002)), and play an important role in downstream events following initial leukocyte leakage from the circulation to tissues in response to stimuli. inflammatory, including migration, recruitment, and activation of proinflammatory cells at the site of inflammation (Eble JA, Curr. Phar. Des. 11 (7): 867-880 (2005)). Some α2β1 integrin blocking antibodies have been reported to have an impact on late 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); by Fougerolles et al., J. Clin Invest., 105: 721-720 (2000) and attenuating endothelial cell proliferation and in vitro migration (Senger et al. , Am. J. Pathol, 160 (1): 195-204 (2002), suggesting that α2β1 integrin blockade may prevent / inhibit anomalous or more exacerbated than normal angiogenesis seen in various cancers. circulate in the blood in an inactive state of rest, yet are prepared to respond rapidly to injury sites to a wide variety of agonists.When stimulated, platelets change shape and become highly reactive to plasma proteins such as fibrin. inogen and von Willebrand factor (vWf), other platelets and the endothelial layer of the vessel wall. All of these interactions facilitate rapid formation of platelet / fibrin hemostatic buffer (Cramer, 2002 in Hemostasis and Thrombosis, 4th edition). With ligand binding, platelet receptors transduce outside-in signal pathways, which in turn trigger inside-out signaling which results in activation of secondary receptors such as the fibrinogen platelet receptor and allbp3 integrin, causing platelet aggregation. Antibodies or peptide ligand mimetics that bind or interact with platelet receptors are believed to induce a similar signaling cascade that leads to platelet activation.

Even minor platelet activation can trigger thrombotic platelet responses, thrombocytopenia, and bleeding complications.

Α2β1 integrin 1 is the only collagen-binding integrin expressed in platelets and is thought to play a 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 et al. al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3 (2): 125-40 (2003)). In addition, platelet α2β1 may play a role in regulating platelet aggregate size (Siljander et al., Blood 103 (4): 1333-1341 (2004)).

Α2β1 integrin has also been shown to be a laminin-binding integrin expressed in endothelial cells (Languino et al., J Cell Bio. 109: 2455-2462 (1989)). Endothelial cells are believed to bind to laminin by an integrin-mediated mechanism. However, it has been suggested that the oc2 I domain may function as a specific ligand sequence involved in mediating endothelial cell interactions (Bahou et al., Blood. 84 (11): 3734-3741 (1994)).

Binding of a therapeutic antibody to α2β1 integrin, including platelet α2β1 integrin, is believed to result in bleeding complications. For example, antibodies that target other platelet receptors such as GPIb (Vanhoorelbeke et al., Curr. Drug Targets Cardiovasc. Haematol. Disord. 3 (2): 125-40 (2003) or GP Ilb / lIIa (Schell et al. 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. thrombocytopenia, although the mechanisms of this association are not well understood. It has been hypothesized that binding of an antibody to a platelet receptor may alter its three-dimensional structure and expose normally unexposed epitopes that subsequently cause platelet clearance ( Merlini et al., Circulation 109: 2203-2206 (2004) In fact, hemorrhagic complications associated with oral doses of GP Ila / 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)) If α2β1 integrin has a important role in leukocyte movement in inflammatory tissue, it would be desirable to develop therapeutic agents that could act on α2β1 for diseases associated with α2β1 integrin and / or cellular processes associated with disorders such as cancer, inflammatory diseases and autoimmune diseases, if these agents did not activate platelets. Therefore, there is a need to develop compounds that target α2β integrin, such as α2β1 integrin in leukocytes, which are not associated with adverse bleeding complications.

Human α2β1 anti-integrin, which blocks the BHA2.1 antibody, was first described by Hangan et al. (Cancer Res. 56: 3142-3149 (1996)). Other anti-integrin α2β1 antibodies are known and have been used in vitro, such as 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)), IOG11 (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 α2β1 integrin blockade. on the extravasation of human tumor cells into the liver and the ability of these tumor cells to develop metastatic foci under antibody treatment Hal / 29 antibody (Mendrick and Kelly, Lab Invest. 69 (6): 690-702 (1993)) , specific for mouse and murine α2β1 integrin, was used in vivo to study α2β1 integrin overregulation in T cells following LCMV viral activation (Andreasen et al., J. Immunol. 171: 2804-28 11 (2003)), to study SRBC-induced late type hypersensitivity and contact-induced hypersensitivity responses with FITC and collagen-induced arthritis (de Fougerolles et. al., J. Clin. Invest. 105: 721-720 (2000)), to study the function of α2βΐ 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 function of α2β1 integrin in locomotion of polymorphonuclear leukocytes (PMN) in response to platelet activation factor (FAP) (Werr et al., Blood 95: 1804 -1809 (2000)).

The use of murine monoclonal antibodies, such as those described above, as therapeutic agents for use in non-immunocompromised patients has been limited by significant immune responses directed against murine antibodies, especially following repeated administrations. This response may not only shorten the effective half-life of circulating murine antibodies, but may also provoke deep injection site reactions and / or anaphylactic reactions (Shawler et al., J. Immunol. 135 (2): 1530 (1985). )). In addition, rodent effector functions associated with constant regions (Fc) are much less effective than human ones when administered, resulting in loss of complement-dependent antibody-dependent cell-mediated cytotoxic activity (ADCC). ) potentially desirable. Therefore, there is a need to develop antibodies against α2β1 integrin, including for the treatment of α2β1 integrin-associated disorders, cellular mechanisms and processes, including inflammatory and autoimmune diseases. Additionally, it would be desirable to develop α2β1 anti-integrin antibodies that were not associated with the development of reactions observed with murine antibodies in patients.

BRIEF DESCRIPTION OF GRAPHS

Figure 1: Graphical results of studies on the effects of anti-integrin oi2 antibodies on paralytic disease in mouse EAE models when administered at the first sign of disease (See Example 7). Figure 2: Graphical results from studies of the effects of integrin ai on paralytic disease when administered during the induction phase (See Example 7).

SUMMARY OF THE INVENTION

The present invention provides anti-integrin alpha 2 (α2) antibodies and methods for their use, especially humanized anti-integrin alpha 2 (α2) antibodies and methods for using them. In certain uses, the anti-integrin α2 antibody includes one or more human constant regions (e.g., Cl and / or CH) and a light chain variable region composed of the amino acid sequence SEQ ID NO: 19 and / or a variable region of heavy chain composed of amino acid sequence SEQ ID NO: 21 or variants of these amino acid sequences. Various forms of antibodies are addressed in this document. For example, the anti-integrin α2 antibody may be a complete antibody (eg containing human immunoglobulin constant regions) or an antibody fragment (e.g. Fab or F (ab ') 2 fragments or Fab1 or Fv or scFv). In addition, the antibody may be labeled with a detectable marker, immobilized on the solid phase and / or conjugated to a heterologous compound (such as a cytotoxic agent).

The diagnostic and therapeutic uses of anti-integrin α2 antibodies, as well as prophylactic and prevention are addressed. For diagnostic uses, a method for determining the presence of α2β1 integrin protein is presented by exposing a suspect sample (which may contain α2β1 integrin protein) and an anti-integrin α.2 antibody, identifying the antibody binding to the sample. For this use, the kit provided consists of an anti-integrin a2 antibody and instructions on how to use the antibody to detect α2β1 integrin protein. Therapeutic uses are not limited to the treatment of cellular disorders, mechanisms and processes associated with α2β1 integrin, but also include inflammatory and autoimmune diseases, in particular multiple sclerosis. The applications of anti-integrin a2 antibodies in gene therapy are addressed. Multiple vectors (eg vectors

retrovirals, chromosomes) encoding the anti-0β / β1 light and heavy chain gene sequences can be transferred to cells (eg, fibroblasts, stem cells) and generate populations of anti-0-secreting cells (2/31 MAb These cells may have specific homing properties for different cell types, tissues and / or organs These antibody-producing cells may be introduced into a patient for localized release of anti-α2β1 monoclonal antibody (MAb). mesenchymal stem cells modified with an anti-o; 2 /? MAb vector could be injected into the brain of a patient with multiple sclerosis.Stem cells differentiate from neural cells and secrete anti-a2jSl MAb to treat sclerosis-associated inflammation. In addition, anti-o; 2 / 3l may be conjugated to viruses encoding therapeutic genes (eg ricin). Modified viruses would specifically bind to cells expressing am α2β1 on the cell surface, enabling increased efficiency of transgene transfer.

Immunoconjugates composed of anti-o; 2 / 3l antibody-liposome complexes that encapsulate nucleic acids and encode therapeutic genes may be administered to the patient intravenously. The anti-α2β1 immunoconjugate would bind to β2β1 integrin-expressing cells and facilitate efficient uptake of therapeutic genes. In addition, an isolated nucleic acid encoding the α2 integrin antibody is provided; a vector composed of this nucleic acid, optionally and operably linked to control sequences recognized by the host cell transformed with the vector; a host cell containing this vector; A process for the production of anti-α 2 integrin antibody which consists of culturing the host cell for nucleic acid to be expressed and optionally recovering the antibody from the host cell culture (eg from the cell culture medium of the host cell). host).

Also provided is a composition containing a humanized anti-integrin α2 antibody and a pharmaceutically acceptable carrier or diluent. Compositions for therapeutic uses may be sterile and may be lyophilized. Also provided is a method for treating α2β1 integrin-associated disorders consisting of administering a pharmacologically effective amount of α2-integrin antibody to a patient, such as humanized α2-integrin antibody to a mammal. For such therapeutic uses, other agents (eg, another α2β1 integrin antagonist) may be administered concomitantly with the mammal before, after or simultaneously with the anti-α2 integrin antibody. Similarly, a humanized anti-integrin α2 antibody is provided which has a heavy chain variable region composed of the amino acid sequence (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO : 1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2) and HCDR3 (ANDGVY YAMD Y, SEQ ID NO: 3), or (c) SEQ ID NO: 40. In one application, the above mentioned heavy chain variable region is formed by the amino acid sequence SEQ ID NO: 185.

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

In yet another application, the above mentioned heavy chain variable region is formed by the selected amino acid sequence of SEQ ID NOs: 70-79 and SEQ ID NOs: 10 9-111.

In one application, the α2 integrin antibody mentioned above contains an FW4 region formed by the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 13). In one application, the a2 anti-integrin antibody mentioned above corresponds to the amino acid sequence HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3).

In one application, the a2 anti-integrin antibody mentioned above also contains a light chain. The invention also provides a humanized anti-integrin α2 antibody composed of a light chain variable region formed by 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 one application, the above mentioned light chain variable region corresponds to the amino acid sequence SEQ ID NO: 186.

In another application, the light chain variable region mentioned above consists of the amino acid sequence 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 one application, the above mentioned light chain variable region consists of the amino acid sequence selected from SEQ ID NO: 41, SEQ ID NOs: 80-92 and SEQ ID NO: 108.

In one application, the humanized anti-integrin a2 antibody mentioned above consists of an FW4 region with the amino acid sequence FGQGTKVEIK of SEQ ID NO: 38). In one application, the above-mentioned humanized anti-integrin α2 antibody consists of the amino acid sequence LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO: 6).

In one application, the humanized anti-integrin α2 antibody mentioned above also consists of a heavy chain.

The invention also provides a humanized anti-integrin oc2 antibody consisting of:

(i) a heavy chain variable region composed of the amino acid sequence (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 composed of the amino acid sequence by (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).

Also provided is the humanized anti-integrin ct2 antibody mentioned above, where: (a) the heavy chain variable region is composed of the amino acid sequence SEQ ID NO: 185, (b) the light chain variable region is composed of the amino acids SEQ ID NO: 186, or (c) both (a) and (b).

Also provided is the humanized anti-integrin a2 antibody mentioned above, where: (i) the heavy chain variable region is composed of the amino acid sequence SEQ ID NO: 185 in which (a) position 71 is Lys, (b) a position 73 is Asn, (c) position 78 is Val, or (d) any combination of (a) - (c); (ii) the light chain variable region is composed of the amino acid sequence 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 humanized anti-integrin α2 antibody, where: (a) the heavy chain variable region is composed of the selected amino acid sequence of SEQ ID NOs: 70-79 and SEQ ID NOs: 109-111; (b) the light chain variable region is composed of the 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). In one application, the a.2 anti-integrin antibody mentioned above recognizes the domain of human a2 integrin.

In one application, the above-mentioned anti-integrin antibody2 binds to α2β1 integrin. In one application, the a2 anti-integrin antibody mentioned above binds to an a2 integrin epitope comprising:

(a) a Lys residue corresponding to position 192 of the a2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 40 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(b) an Asn residue corresponding to position 225 of the cx2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(c) a Gln residue corresponding to position 241 of the ct2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 89 of the a.2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(d) a Tyr residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 93 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

e) an Arg residue corresponding to position 317 of the a2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 165 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(f) an Asn residue corresponding to position 318 of the a2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 166 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or

(g) any combination of (a) to (f). An α2 integrin antibody is also provided, where the antibody binds to an α2 integrin epitope, an epitope composed of:

(a) a Lys residue corresponding to position 192 of the a2 integrin amino acid sequence expressed in SEQ

ID NO: 8 or position 40 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(b) an Asn residue corresponding to position 225 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(c) a Gln residue corresponding to position 241 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 89 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(d) a Tyr residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 93 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(e) an Arg residue corresponding to position 317 of the α2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 165 of the α2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11;

(f) an Asn residue corresponding to position 318 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 166 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or

(g) any combination of (a) to (f). In one application, the humanized anti-integrin a.2 antibody mentioned above is a complete antibody. In one application, the humanized anti-integrin α2 antibody mentioned above is an antibody fragment.

In one application, the above-mentioned humanized anti-integrin α2 antibody binds to a detectable marker.

In one application, the above-mentioned humanized anti-integrin α2 antibody is immobilized on the solid phase. In one application, the above-mentioned humanized anti-integrin α2 antibody inhibits the binding of α2 or α2βΐ integrin to an α2β1 integrin ligand.

In one application, the α2β1 integrin ligand is selected from collagen, laminin, Ecovirus 1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, collectin, and Clq complement protein.

The invention also provides a method for determining whether a sample contains α2 integrin, α2β1 integrin, or both, by contacting the sample with the above-mentioned humanized anti-integrin a2 antibody and determining whether the antibody binds to the sample. binding is an indication that the sample contains α2 integrin, α2β1 integrin or both. The invention also provides a kit containing the humanized α2 integrin antibody, optionally containing instructions on how to use it to detect cx2 or α2β1 integrin protein.

The invention also provides an isolated nucleic acid encoding the humanized anti-integrin α2β1 antibody mentioned above.

The invention also provides a vector composed of the nucleic acid mentioned above.

The invention also provides a host cell containing the nucleic acid or vector mentioned above.

The invention also includes a process for producing a humanized anti-integrin α2 antibody comprising culturing the host cell mentioned above under conditions that allow expression of the antibody. In one application, the method further comprises recovering the humanized anti-integrin α2 antibody from the host cell. In another application, the method comprises recovering the humanized anti-integrin cc2 antibody from a host cell culture medium.

The invention also provides a screening method comprising: detecting binding of α2 or β2 integrin to an antibody containing the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21 in the presence versus absence of a test antibody and selection of test antibody if their presence correlates with reduced binding of a2 or α2β1 integrin to the antibody containing the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21. In one application, the integrin c * 2 or α2β1 is immobilized on a solid support.

The invention also provides a screening method comprising: detecting binding of α2β1 integrin to collagen in the presence of a test antibody where the antibody binds to an I α2 domain; detecting the binding of a test antibody to domain I a2 in the presence of Mg ++ ions; detecting binding of the test antibody to domain I a2 in the presence of Ca ++ ions; detecting binding of the test antibody to domain I α2 in the presence of a cation-free medium; and selection of a test antibody if inhibition of α2β1 integrin binding to collagen and domain I α2 binding is identified in the presence of Mg ++ and Ca ++ ions and cation-free medium.

The invention also provides a composition containing the humanized anti-integrin α2 antibody mentioned above and a pharmaceutically acceptable carrier.

The invention also provides a method for treating disorders associated with α2β1 integrin in patients, the method comprising administering to the patient either a therapeutically effective amount of the above mentioned anti-integrin antibody or a composition.

The invention also includes a method for inhibiting leukocyte binding to collagen, which consists in administering to the patient a sufficient amount of the above mentioned anti-integrin α2β1 antibody to inhibit binding of leukocytes to collagen.

The invention also provides for the use of the humanized anti-integrin a2 antibody mentioned above as a medicament.

The invention also provides the use of the above-mentioned humanized anti-integrin α2 antibody or a composition for the treatment of α2β1 integrin-associated disorders.

The invention also provides the use of the above-mentioned humanized α2-integrin antibody or composition for the manufacture of a medicament for the treatment of α2β1 integrin-associated disorders.

The invention also provides a composition for the treatment of α2β1 integrin-associated disorders, comprising the above-mentioned humanized anti-integrin α2 antibody and a pharmaceutically acceptable carrier or diluent.

The invention also includes a package containing the above-mentioned humanized anti-integrin α2 antibody or a composition, together with instructions, for treating an α2β1 integrin-associated disorder. For applications, α2β1 integrin-associated disorder is differentiated from inflammatory disease, autoimmune disease, and diseases characterized by altered or increased angiogenesis.

For applications, α2β1 integrin-associated disorder is differentiated from inflammatory bowel disease, Crohn's disease, ulcerative colitis, transplant rejection, optic neuritis, spinal trauma, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, juvenile diabetes, diabetic retinopathy, age-related macular degeneration, cardiovascular disease, psoriasis, cancer, and infections that induce an inflammatory response. For applications, α2β1 integrin-associated disorder is differentiated from multiple sclerosis (eg, characterized by relapse, acute treatment, delayed treatment), rheumatoid arthritis, optic neuritis, and spinal trauma.

For applications, the above mentioned method is not associated with: (a) platelet activation, (b) platelet aggregation, (c) reduction in circulating platelet count, (d) hemorrhagic complications, or (e) any combination of (a) ) to (d).

In one application, the above-mentioned anti-integrin α2 antibody contains a heavy chain composed of SEQ ID NO: 174 or SEQ ID NO: 176, and a light chain composed of SEQ ID NO: 178).

In one application, the above-mentioned anti-integrin α2 antibody competitively inhibits the binding of an antibody composed of a UL region of SEQ ID NO: 19 and a VH region of SEQ ID NO: 21 to the human α2β1 integrin or domain I of the same.

In one application, the method mentioned above is associated with the relief of a neurological exacerbation or sequelae due to multiple sclerosis.

In one application, the a2 anti-integrin antibody mentioned above inhibits the binding of α2β1 integrin to collagen and is not a ligand mimetic. It also includes a target method for a given fraction, such as a molecule, protein, nucleic acid, vector, composition, complex, etc., for a site characterized by the presence of an α2β1 integrin ligand, the method being to attach or bind a fraction to the humanized α2 integrin antibody mentioned above.

It also includes an α2 integrin epitope that binds an anti-α2 integrin antibody, and the epitope is not composed of the α2 integrin ligand binding site: For applications, epitope binding is not associated with: (a) platelet activation, (b) platelet aggregation, (c) decreased circulating platelet count, (d) hemorrhagic complications, (e) a2 integrin activation, or (f) any combination of (a) to (e). Preferred antibodies bind to domain I of human α2β1 integrin. Specifically, the preferred antibodies are capable of blocking cell-dependent oc2-dependent adhesion to extracellular matrix (ECM), specifically collagen or laminin, or both. Humanized antibodies are provided, including antibodies based on an antibody called TMC-2206. The anti-integrin α2 antibodies provided are highly specific for human α2β1 integrin, and their administration is not associated with undesirable effects such as bleeding complications or complications due to cellular activation. The binding specificity (eg, epitope specificity) of these antibodies is associated with their unexpected nonhemorrhagic profile.

The humanized anti-integrin α2β1 antibody may have a heavy chain variable region containing the amino acid sequence 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-integrin α2β1 antibody may have a light chain variable region containing the amino acid sequence 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 applications, the humanized anti-integrin α2β1 antibody may have a heavy chain containing the amino acid sequence 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 containing the amino acid sequence of LCDR1 (SANSSVNYIH; SEQ ID NO: 4) and / or LCDR2 (DTSKLAS; SEQ ID NO: 5) and / or LCDR3 (QQWTTNPLT; SEQ ID NO: 6). In other applications, the antibody is composed of an amino acid sequence variant of one or more of these CDRs, which is composed of one or more amino acid insertions within or adjacent to a CDR residue and / or deletion (s) within. or adjacent to a CDR residue and / or residue (s) substitution (s) with substitution (s) being the preferred type of amino acids for the production of such variants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes antibodies specifically reactive with human integrin alpha 2 (a2), including humanized antibodies and methods for their use. Humanized antibodies have human framework regions (FWs framework) and complementarity determining regions (CDRs) of a non-human antibody, typically mouse, specifically reactive with human α2 integrin. Nucleotide sequence and amino acid sequence encodings composed of heavy and light chain antibodies are provided. In preferred applications, one or more of the CDR regions are derived from or based on the murine antibody secreted by the hybridoma clone BHA2.1 [referred to herein as the TMC-2206 antibody]. Antibodies with similar binding properties and antibodies (or other antagonists) with functionality similar to the antibodies discussed herein are also included. Preferred anti-integrin α2 antibodies include those that: (a) bind to α2 integrin domain I, (b) inhibit α2 integrin function (eg, collagen or laminin binding), (c) bind integrin a2 on resting platelets without inducing platelet activation and (d) recognize the binding epitope of TMC-22 06 (eg, compete with TMC-22 06 for α 2 integrin binding). Such antibodies may preferentially bind to the inactive or closed conformation of the α 2 integrin molecule without competing for the ligand binding site. Unexpected advantages of α2 integrin antibodies, as described herein, which preferentially bind to the closed conformation of α2β1 integrin and / or bind α2βΐ integrin without competing for the ligand binding site (eg, are not a mimetic ligand) include the potential for prevention of platelet activation, platelet aggregation, reduction in circulating platelet count and / or bleeding complications in treated subjects.

The "haemorrhagic complications" mentioned in this document refer to any adverse effects occurring at the blood level or its physiology, including platelet thrombotic reactions, thrombocytopenia, increased clotting time, increased bleeding time, and blood loss that limit therapeutic use. anti-integrin antibody α2.

Α2β1 integrin is a molecule composed of a cc2 integrin subunit (see eg SEQ ID NO: 7 for the DNA sequence and SEQ ID NO: 8 for the human a2 protein sequence) of the alpha integrin family, and a subunit of the integrin al (see eg SEQ ID NO: 9 for the DNA sequence and the human β1 SEQ ID NO: 10 protein sequence) of the beta integrin family, which can be obtained from any individual and also from a mammal, but is preferably obtained from humans. Α2β1 integrin can be purified from any natural source or can be synthetically produced (eg using recombinant DNA technology). The encoded nucleic acid sequences for α2 integrin and integrin / 31 are described by Takada and Hemler J. Cell Biol. 109 (1): 397-407 (1989; GenBank submission X17033; subsequently updated to NM 002203) and Argraves, W.S, J. Cell. Biol. Sep 105 (3): 1183-90 (1987; Genbank submission X07979.1 and related sequences alternatively representing conjugated variants), respectively.

The 'I' domain of the α2β1 integrin molecule refers to a region of such α2β1 integrin molecule within the α2 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 Î ± 2 integrin domain I is shown as SEQ ID NO: 11 (see also ex: SEQ ID NO: 107). The Î ± 2 integrin domain I contains a MIDAS type of ligand binding site (Metal Ion Dependent Adhesion Site) that requires a specific divalent cation to promote ligand binding. Amino acid sequences for an α2 integrin domain I in mice shown as SEQ ID NO: 93 (see also, eg, SEQ ID NO: 113) and in mice shown as SEQ ID NO: 94 (see also, eg: SEQ ID NO: 114) are shown in Table 28. Domain I sequences of Cynomolgus and rhesus monkeys were cloned from a whole blood derived leukocyte fraction and are presented as SEQ ID NO: 103 (DNA), SEQ ID NO: 171 (amino acid ) for cynomolgus monkeys and as SEQ ID NO: 104 (DNA), SEQ ID NO: 172 (amino acid) for rhesus monkeys, respectively. A TMC-2206 epitope (BHA2.1) refers to a region I Î ± 2 integrin domain I region to which the TMC-2206 antibody binds. This epitope encompasses a region that encompasses amino acid residues K40, N73, Q89, Y93, R165, and N166 and optionally other amino acid residues of the Î ± 2 integrin domain I.

An a2 integrin-associated disorder refers to a disorder, disease or condition involving a2 integrin-dependent processes / functions (eg, binding, activity) that mediate aberrant cellular reactions within target tissue. Examples of a2 integrin-dependent processes involved in disease include collagen-dependent cellular responses such as responses involved in increased cytokine expression and proliferation, aspects of T-cell, mast cell and neutrophil function, inflammatory diseases, mammary duet morphogenesis, epidermal wound healing and angiogenesis. Examples of a2 integrin-associated disorders include, but are not limited to, inflammatory disorders or diseases, which in turn include, but are not limited to, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), transplant reactions (including rejection). optic neuritis, spinal trauma, rheumatoid arthritis, multiple sclerosis (including treatment of associated neurological sequelae as well as relapsing multiple sclerosis), autoimmune diseases or disorders (including systemic lupus erythematosus (SLE ), diabetes mellitus, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma), juvenile diabetes and disorders associated with above-normal angiogenesis alteration or elevation (such as diabetic retinopathy, age-related macular degeneration, cardiovascular disease, psoriasis, rheumatoid arthritis and cancer), in addition to conditions that induce an inflammatory response.

Treatment of α2β1 integrin-associated disorders refers to the therapeutic and prophylactic / preventive use of the anti-integrin ct2 antibodies described in this instrument. Individuals in need of treatment include those who have already been diagnosed with the disorder, as well as those in whom the onset of the disorder should be prevented or delayed.

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

Intermittent or periodic administration is continuous administration for a certain period of time, at regular intervals, preferably more than one day apart.

The term antibody or immunoglobulin is used in the broadest sense and encompasses monoclonal antibodies (full monoclonal antibodies), polyclonal antibodies, muitiespecific antibodies, and antibody fragments provided they have the desired biological activity. Antibody fragments are composed of a portion of a complete antibody, usually an antigen binding or a variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, single domain (e.g., camelid) antibodies, NAR single domain antibodies shark and multispecific antibodies formed by antibody fragments. Antibody fragments may also refer to binding moieties composed of CDRs or antigen binding domains that include but are not limited to VH (VH, Vh-Vh) regions, anticalins, PepBodies ™, T cell epitope fusions (Troybodies). ) or Peptibodies. A monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, eg, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike conventional (eg polyclonal) antibody preparations which generally include different antibodies directed against different determinants (eg epitopes) on an antigen, each monoclonal antibody is directed against at least one single determinant on an antigen. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as a need for antibody production 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., US Patent No. 4,816,567). . Monoclonal antibodies may also be isolated from antibody phage 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 may also be isolated using the techniques described in U.S. 6,025,155 and 6,077,677 and also in U.S. Patent Application Publications no. 2002/0160970 and 2003/0083293 (see also for ex: Lindenbaum, et al., Nucleic Acids Research 32 (21): 0177 (2004)).

Monoclonal antibodies may include chimeric antibodies in which the heavy and / or light chain portion is identical or homologous to the corresponding antibody sequences derived from a specific species or belonging to an antibody class or subclass, while the remainder of the chain (s) (a) is identical or homologous to corresponding antibody sequences derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (see for example: US Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad Sci. USA 81: 6851-6855 (1984) for chimeric mouse-human antibodies). A hypervariable region refers to amino acid residues of an antibody that is responsible for antigen binding. A hypervariable region is composed of amino acid residues from a complementarity determining region or CDR (eg residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31- 35 (Hl), 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 residues of a hypervariable Ioop (eg 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 residues or RF are variable domain residues and not hypervariable region residues. For the antibodies described herein, the CDR and framework regions are identified based on the Kabat numbering system, with the exception of the heavy chain CDR1 which is defined by Oxford Molecular's AbM as residues of the range 26 to 35. The program Oxford Molecular's AbM antibody modeling software (http://people.cryst.cck.ac.uk/~ubc07s/) (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 MJE (ed.), Protein Structure Prediction. University Press, Oxford, 141-172 (1996)) combines the Kabat CDR and Chothia hypervariable region numbering systems to define CDRs.

Humanized forms of non-human antibodies (eg, murine) may be chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody or acceptor) in which residues from the container hypervariable region are replaced by residues from the hypervariable region from non-human species (donor antibody) such as mice, rats, rabbits or primates. humans that have the desired specificity, affinity, and ability. In addition, individual residue groups or residues from the human immunoglobulin framework region may be replaced by corresponding non-human residues. Humanized antibodies may be composed of residues that are not found in a recipient antibody or a donor antibody. These modifications are made to refine antibody performance. In general, the humanized antibody will be substantially composed of all, at least one, and generally two, variable regions or domains, in which all hypervariable loops correspond to those of non-human immunoglobulin and all or substantially all regions of the framework. ) are those of the human immunoglobulin sequence. The humanized antibody will optionally be composed of at least a portion of an immunoglobulin constant region (e.g., Fc), generally that of 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 can make up the Vh and Vl regions or domains of the antibody, and these domains are present in a single peptide chain. Generally, the Fv polypeptide is also composed of a polypeptide linker between the Vh and Vl domains that allows scFv to form the desired antigen binding structure (for review, see for example, 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, composed of a heavy chain variable domain (Vh) connected to a light chain variable domain (V1) in the same polypeptide chain (VH - V1). Because it uses a linker that is too short to allow pairing between the two domains in the same chain, domains are forced to pair with complementary domains from another chain and create antigen binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al. , Proc. Natl. Acad. Know. 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 are composed of a pair of tandem Fd segments (Vh -ChI-Vh -ChI) that form a pair of antigen binding regions. Linear antibodies may be bispecific or monospecific. An isolated antibody refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that may interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred applications, the antibody will be purified: (1) by more than 95% by weight of the antibody as determined by the Lowry method, and preferably by more than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a spinning cup sequencer, or (3) for SDS-PAGE homogeneity under reducing or non-reducing conditions using Coomassie blue or preferably , silver staining. Isolated antibody includes the antibody in situ within the recombinant cells, since at least one component of the antibody's natural environment will not be present. In general, however, isolated antibody will be prepared with at least one purification step.

An labeled epitope antibody refers to cases where the antibody of the invention is fused to a labeled epitope. The tagged epitope polypeptide contains

enough residues to provide an epitope against which an antibody can be produced, but at the same time short enough not to interfere with the activity of the α2β1 anti-integrin antibody. Preferably, the labeled epitope is sufficiently unique that no actual cross-reaction of the antibody with other epitopes occurs. Suitable tag polypeptides generally have at least 6 amino acid residues and generally between 8-50 amino acid residues (preferably between about 9-30 residues).

Examples include the flu HA tag polypeptide polypeptide and its 12CA5 antibodies (Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)); c-myc marker and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereof (Evan et al., Mol. Cell. Biol. 5 (12): 3610-3616 (1985)); and the herpes Simplex D (gD) glycoprotein marker and its antibodies (Paborsky et al., Protein Engineering 3 (6): 547-553 (1990)). In certain applications, the labeled epitope is a rescue receptor binding epitope, which is an Fc region epitope of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the half-life. serum concentration of the IgG molecule. A cytotoxic agent refers to a substance that inhibits or impedes cell function and / or causes cell destruction. Cytotoxic agents may include radioactive isotopes (eg, 131I, 125I, 90Y and 186Re), chemotherapeutic agents and toxins such as enzymatically active bacterial, fungal, plant or animal toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or impede cell function and / or cause cell destruction. A non-cytotoxic agent may include an agent that can be activated to become cytotoxic. Non-cytotoxic agents may include a globule, liposome, matrix or particle (see for example: U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Such agents may be conjugated, aggregated, linked or associated with an anti-integrin α2β1 antibody as described herein. A chemotherapeutic agent refers to a chemical compound used to treat cancer. Examples of chemotherapeutic agents include but are not limited to Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxine, Taxol, Methotrexate, Cisplatin, Melfal, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Speramycin (see US Pat. No. 4,675,187) and other related mardalin.

A prodrug refers to a precursor or derivative form of a pharmacologically active substance that is less cytotoxic to tumor cells compared to the primary compound and which may be enzymatically activated or converted to a more active primary form (see eg 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 not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing drugs, modified D-amino acid prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, prodrug those containing 5-fluorocytosine and 5-fluorouridine that can be converted to a more active non-cytotoxic drug. Examples of cytotoxic drugs that may be derivatized into a prodrug form may be the chemotherapeutic agents described above.

A label refers to a detectable compound or composition that is conjugated or aggregated, directly or indirectly, to an antibody. The label may itself be detectable (eg radioisotope markers or fluorescent markers) or, in the case of an enzyme label, may catalyze the chemical alteration of a detectable composition or substrate compound.

The solid phase refers to a non-aqueous matrix to which the antibody object of the present invention may adhere. Examples of solid phases discussed herein include those formed partially or wholly of glass (eg, controlled pore glass), polysaccharides (eg agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain applications, depending on the context, the solid phase may be composed of a well of a test plate; in others by a purification column (eg affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.

A liposome refers to a small vesicle composed of various types of lipids, phospholipids and / or surfactants that are useful in administering a medicament (such as antibodies of the invention and optionally a chemotherapeutic agent) to a mammal. Liposome components are commonly organized into a two-layer formation, similar to lipid organization in biological membranes.

An isolated nucleic acid molecule refers to a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule, with which it is normally associated in the natural source of antibody nucleic acid. An isolated nucleic acid molecule is different in shape or configuration from that found in nature. Therefore, nucleic acid molecules are different from nucleic acid molecules found in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally express the antibody where, for example, the nucleic acid molecule is located at a different chromosomal site than it is in natural cells.

A viral vector refers to a vehicle for the transfer of a nucleic acid (eg DNA or RNA) to cells through viral infection or transduction. Examples of viral vectors include retrovirus, adenovirus, pox virus, and baculovirus.

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

Expression control sequences refer to the DNA sequences required for expression of a coded and operably linked sequence in a specific host organism. Control sequences that are suitable for prokaryotic (or prokaryotic) cells, for example, include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers.

Nucleic acid is operably linked when it is placed in a functional relationship with another sequence of nucleic acids. For example, DNA from a leader sequence or secretory sequence is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in 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. Operably linked DNA sequences are generally contiguous and, in the case of the secretory leader sequence, contiguous and in the reading phase. However, enhancers need not be contiguous. Binding is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional conduct.

Another aspect of the present invention is the treatment of α2β1 integrin-associated disorders by administering to a subject a nucleic acid molecule encoding an anti-integrin α2 antibody of the invention. Suitable methods of administration include gene therapy methods (see below). A nucleic acid of the invention may be administered to cells in vivo using methods such as direct DNA injection, receptor-mediated DNA uptake, virus-mediated transfection or non-virus-mediated transfection, and lipid-based transfection, all of which may be used. These methods may involve the use of gene therapy vectors. Direct injection has been used to introduce single (naked) DNA into cells in vivo (see eg Acsadi et al (1991) Nature 332: 815-818; Wolff et al. (1990) Science 247: 1465-1468). An administration device may be used (eg, a "gene gun") to inject DNA into cells in vivo. This device may be commercially available (eg from BioRad). Simple DNA can also be introduced into cells by complexing DNA into a cation, such as polylysine, which is attached to a cell surface receptor ligand (see, for example, Wu, G. and Wu, CH (1988)). J. Biol Chem 263: 14621 Wilson et al (1992) J. Biol Chem 267 963 967 and US Pat. No. 5,166,320). Binding of a DNA-ligand complex to a receptor may facilitate receptor uptake by receptor-mediated endocytosis. An adenovirus capsid-linked DNA ligand complex that disrupts endosomes, thereby releasing material into the cytoplasm, can be used to prevent complex degradation by intracellular lysosomes (see, for example, Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2122-2126).

Defective retroviruses are well characterized for use as vectors in gene therapy (for review, see Miller, A. D. (1990) Blood 76: 271). Protocols for the production of recombinant retroviruses and for in vitro or in vivo cell infection with these viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are well known to experienced practitioners. Examples of suitable packaging virus strains include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce a wide variety of genes into many different cell types, such as epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo (see, for example, Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc Natl. Acad Sci USA 87: 6141-6145; Huber et al (1991) Proc Natl Acad Sci USA 88: 8039- Ferry et al (1991) Proc Natl Acad Sci USA 88: 8377-8381 Chowdhury et al 1991 Science 254: 1802-1805 van Beusechem et al (1992) Proc Natl. Acad. Sci. USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641- 647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; et al (1993) J. Immunol 150: 4104-4115; US Pat. No. 4,868,116; US Pat. No. 4,980,286; PCT Application WO 89/07136; P CT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

For use as a vector in gene therapy, an adenovirus genome can be engineered to encode and express a nucleic acid compound of the invention, but be inactivated in terms of its ability to replicate in a normal lytic viral life cycle. . See for example, Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; and Rosenfeld et al. (1992) Cell 68: 143-155. Suitable adenovirus vectors derived from the dl324 Ad type 5 adenovirus strain or other adenovirus strains (eg, Ad2, Ad3, Ad7 etc.) are well known to experienced practitioners. The use of recombinant adenoviruses is advantageous because there is no need to divide cells to function effectively as gene releasing vehicles and can be used to infect a wide variety of cell types, including airway epithelial cells (Rosenfeld et al. ( 1992), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90 : 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2581-2584).

Adeno-associated viruses (AAVs) can be used as a vector for DNA release for gene therapy purposes. VAA is a naturally occurring defective virus and needs another virus, such as an adenovirus or herpes virus, as an auxiliary virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro, and Immunol (1992) 158: 97-129). VAA can be used to integrate DNA into non-dividing cells (see, for example, Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7: 349-356; Samulski et al. (1989) J. Virol 63: 3822-3828 and McLaughlin et al (1989) J. Virol 62: 1963-1973). A VAA vector as described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 can be used to introduce DNA into cells (see, for example, Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol Cell Biol 4: 2072-2081 Wondisford et al (1988) Mol Endocrinol 2: 32-39 Tratschin et al (1984) J. Virol 51: 611-619 and Flotte et al. (1993) J. Biol. Chem. 268: 3781-3790). Vectors of lentivirus gene therapy may also be adapted for use in the invention.

The general methods of gene therapy are known to qualified professionals. See for example, U.S. Pat. No. 5,399,346 to Anderson et al. A biocompatible capsule for releasing genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer to hematopoietic cells have also been previously reported (see Clapp, DW, et al., Blood 78: 1132-1139 (1991); Anderson, Science 288: 627-9 (2000); and Cavazzana-Calvo et al. ., Science 288: 669-72 (2000)).

Cells, cell lines and cell culture are often used interchangeably and all of these designations include progeny. Transforming and transformed cells (e.g., obtained by transfection, transformation or transduction of nucleic acids, vectors, viruses, etc.) include the subject's primary cell and cultures derived from them without regard to the number of transfers. It is also known that not all progeny can be precisely identical in DNA content due to deliberate or inadvertent mutations.

Mutant progenies that have the same biological function or activity as evaluated in the originally transformed cell were included. When it is intended to use different designations, they will be clarified by the context.

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

Antibodies of the present invention were constructed using heavy chain variable region and light chain variable region CDRs from a BHA2.1 murine monoclonal antibody clone (Hangan et al., Cancer Res. 56: 3142-3149 (1996)). Preferred materials for initiating antibody generation are anti-integrin α2 antibodies such as those excreted by the hybridoma BHA2.1 (eg, TMC-2206) which are blocking antibodies directed to human integrin and whose binding and activity depend on the presence of an intact domain I within the target Î ± 2 integrin. There is a preference for antibodies with TMC-2206 (or BHA2.1) epitope specificity, including antibodies that bind to the inactive conformation of the integrin molecule (x2, and / or do not act as linker mimetics.) There is a preference for antibodies with specificity for the TMC-2206 (or BHA2.1) epitope which, while interacting with α2β1 integrin present in both leukocytes and platelets, does not cause platelet activation, compromises collagen-active platelet aggregation, has minimal or no effect on bleeding and / or are not associated with bleeding complications at administered concentrations, including in vivo therapeutic doses.

Antibodies can be constructed in which the human acceptor molecule for the light chain variable region is selected based on considerations of homology between the potential acceptor molecule variable regions and the murine antibody light chain variable region. Germline candidate human acceptor molecules are preferred to reduce potential antigenicity. The germline databases are made up of antibody sequences that extend to the end of the heavy chain FW3 region and partially into the CDR3 sequence. For the selection of the FW4 region, priority should be given to searching databases of mature antibody sequences derived from the selected germline molecule and also to selecting a reasonably homologous FW4 region for use in the recombinant antibody molecule. Human acceptor molecules are preferably selected from the same light chain class of the murine donor molecule and the same canonical structural class as the variable region of the murine donor molecule. Secondary considerations for selection of the human acceptor molecule for the light chain variable region include CDR length homology between the murine donor molecule and the human acceptor molecule.

Human acceptor antibody molecules are preferably selected by homological searches in the V-BASE database; other databases like Kabat's and the NCBI public databases can also be used. For humanized anti-integrin α2 antibodies having the same or similar epitope specificity and / or functional properties as TMC-2206, the preferred human light chain acceptor molecule is SEQ ID NO: 37 with germline A14 antibody sequence for region FW 1-3 and the sequence FGQGTKVEIK for FW4 (SEQ ID NO: 38) representing a common mature kappa 1 light chain FW-4 (eg light chain sequence AAB24132 (NCBI entry gi / 259596 / gb / AAB24132).

Antibodies can be constructed in which the human acceptor molecule for the heavy chain variable region is selected based on considerations of homology between the potential acceptor molecule variable regions and the trinurin antibody heavy chain variable region. Germline candidate human acceptor molecules are preferred to reduce potential antigenicity. Germline databases are made up of antibody sequences that extend to the end of the heavy chain FW3 region and partially into the CDR3 sequence. For the selection of the FW4 region, priority should be given to searching databases of mature antibody sequences derived from the selected germline molecule and also to selecting a reasonably homologous FW4 region for use in the recombinant antibody molecule. Human acceptor molecules are preferably selected from the same heavy chain class of the murine donor molecule and the same canonical structural class as the variable region of the murine donor molecule. Secondary considerations for the selection of the human acceptor molecule for the heavy chain variable region include CDR length homology between the murine donor molecule and the human acceptor molecule. Human acceptor antibody molecules are preferably selected by homology search in the V-BASE database; although other databases like Kabat's and the public NCBI database can also be used. For anti-Î ± 2 integrin antibodies with the same or similar epitope specificity and / or functional properties as TMC-2206, the preferred heavy chain acceptor molecule is SEQ ID NO: 39 with germline antibody sequence 4-59 to FW 1-3 region (SEQ ID NO: 12) and antibody, CAA48104.1 (input NCBI, gi / 33583 / emb / CAA48104.1) a mature antibody derived from germline sequence 4-59 to FW 4 region (SEQ ID NO: 13) (http://www.ncbi.nlm.nih.gov).

Methods for humanizing a non-human α2 integrin antibody are described herein, including the examples below. To humanize an anti-integrin α.2 antibody, non-human starting material is obtained by preparing immunization or purchasing commercially available antibodies. Examples of techniques for producing antibodies are described herein. The α2β1 integrin antigen to be used for antibody production may be, for example, a soluble form of α2βΐ integrin or another α2β1 integrin fragment (eg, a α2β1 integrin fragment containing a human α2 integrin domain I (SEQ See also, eg, SEQ ID NO: 107). Other ways of using α.2 integrin for antibody production will be apparent to experienced practitioners based on the a2 integrin sequence (eg, a human a2 integrin as in SEQ ID NO: 8).

Polyclonal antibodies are preferably produced in animals by administering various subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the corresponding antigen with or without an adjuvant. It may be useful to conjugate antigen corresponding 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, eg maleimidobenzol sulfosuccinamide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via residues) of lysine), glutaraldehyde, succinic anhydride, SOCl2, or R1N = C = NR, where R and R1 are different alkyl groups.

Animals may be immunized against the antigen, immunogenic conjugates or derivatives by combining the antigen or conjugate (eg 100 μg for rabbits or 5 æg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution by the route. intradermal at various sites. One month later the animals receive another injection of antigen or conjugate (eg, 1/5 to 1/10 of the original amount used for immunization) in Freund's complete adjuvant by subcutaneous injections at various sites. Seven to 14 days later the animals are bled and the serum is analyzed for antibody titer. Animals receive booster injections until concentration reaches a plateau. For conjugate immunizations, animals preferably receive injections with the conjugate of the same antigen but conjugated to a different protein and / or via a cross-linking reagent. Conjugates can also be produced in recombinant cell culture in the form of protein fusions. Aggregating agents such as alum are also suitable for enhancing the immune response.

Monoclonal antibodies may be made using the hybridoma technique first described by Kohler et al., Nature, 256: 495 (1975), or using recombinant DNA methods (eg, US Patent No. 6,204,023). . Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 and also in U.S. Patent Application Publications no. 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 described above) to evidence lymphocytes that produce or may produce antibodies that will specifically bind to the antigen used in immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused to myeloma cells using a suitable fusion agent such as polyethylene glycol to form the hybridoma cell (see, eg, Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)).

Hybridoma cells thus prepared are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the growth or survival of unfused primary myeloma cells. For example, if the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) does not exist in primary myeloma cells, culture media for hybridomas will usually include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent cell growth. HGPRT disabled.

Preferred myeloma cells are those that fuse efficiently, have stable antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Preferred myeloma cell lines are murine myeloma strains, such as those derived from mouse MOP-21 and M.C.-11 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 myelomatous and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (eg, Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody.Production Techniques and Applications, pp. 51-63 (Mareei Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are cultured is prepared for the production of monoclonal antibodies directed against the antigen. The specificity of monoclonal antibody binding produced by hybridoma cells is preferably determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA).

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

Once it has been determined that hybridoma cells produce antibodies of the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution and growth procedures using standard methods (Goding, Monoclonal Antibodies: Principles and Practice). , pp.59-103 (Academic Press, 1986)). An example of suitable culture medium for this purpose is D-MEM or RPMI-1640 medium. Hybridoma cells can also grow in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by subclones are suitably separated from the culture medium, ascites liquid or serum using conventional immunoglobulin purification procedures such as protein A chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, gel electrophoresis. , dialysis, and / or affinity chromatography.

DNA encoding monoclonal antibodies is readily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells are the preferred source for this DNA. Once isolated, DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovarian (CHO) cells, or myeloma cells, including those from other do not produce the immunoglobulin protein to obtain monoclonal antibody synthesis in recombinant host cells. Production of recombinant antibodies is described in more detail below.

The examples presented herein describe methods for humanizing an exemplary anti-integrin α2 antibody. In certain applications, it may be desirable to produce amino acid sequence variants of the humanized antibody, especially when they enhance binding affinity or other biological properties of the antibody. humanized.

Amino acid sequence variants of the α2β1 humanized anti-integrin antibody are prepared by introducing nucleotide-specific changes into the α2β1 humanized anti-integrin antibody DNA or by peptide synthesis. Such variants include, for example, deletions of, and / or insertions in and / or residue substitutions within the amino acid sequences shown for the anti-integrin α2 antibody TMC-2206 (eg derived from or based on variable region sequences). as shown in SEQ ID NOS: 19 and 21),. Any amino acid deletion, insertion and substitution combinations are made to obtain the final structure as long as the final structure has the desired characteristics. Changes in amino acids may also alter post-translational processes of the humanized anti-integrin α2 antibody, such as changes in the number or position of glycosylation sites.

There are several methods for making antibodies human or human-like (eg "humanization"). The approaches used for antibody humanization have varied over the years. One approach was to produce murine variable regions fused to human constant regions, called murine human Fc chimeras (see, eg, Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); US Patent No, 5,807,715). Another approach explored 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 its canonical structure (Chothia and Lesk, J. Mol. Biol. 196 (4): 901-17 (1987)); Lazakani et al. , J. Mol. Biol.

2 72: 929 (1997) and humanized by grafting only non-human CDR regions (called donor CDRs) into a human framework (called acceptor frameworks) as shown, for example by Jones et al. Nature 321 (6069): 522-5 (1986); (see, e.g., U.S. Patent No. 5,225,539; U.S. Patent No. 6,548,640). The six cycles of the CDR are presented in a cluster, and based on crystallographic analysis, it is possible to readily identify the critical residues of the so-called "Vernier" framework laterally to the CDRs or at the light-heavy chain interface (see, eg. : 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 may reverse mutate to the murine residue to restore the correct relative orientation of the six CDRs (see, eg, 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)). Because variable regions can be classified into families with relatively high homology between mice and humans (reviewed eg Pascual and Capra Adv. Immunol. 49: 1-74 (1991)), these early studies also indicated that the potential for Affinity loss could be minimized in the grafted antibody by selecting the human germline sequence with the highest homology for the murine antibody of interest for use as a human acceptor molecule (see: US Patent No. 5,225,539; Verhoyen et al. ., Science 239 (4847): 1534-6 (1988)).

Family homologies and structural relationships that interfere with the correct presentation of a particular canonical structure of a CDR have been reported (see, eg, Al-Lazakani et al., J. Mol. Biol. 273 (4): 927-48 ( 1997) and related references). Preferably, a more suitable human or germline sequence is selected. Available databases of germline antibody sequences can be used to determine the subtype of the family of a given murine heavy and light chain and to identify the most suitable sequences that can be used as human acceptor frameworks within a human subfamily. Both the linear amino acid homology of the donor and acceptor frameworks and the canonical structure of the CDR are taken into account. Examples of heavy chain residues that may be substituted on a humanized anti-integrin α2 antibody include one or more of the following framework residue numbers: H37, H48, H67, H71, H73, H78 and H91 (Kabat Numbering System) ). At least four such residues are preferably substituted. A particularly preferred set of heavy chain substitutions in humanized anti-integrin α2 antibodies exemplified herein is H37, H71, H73 and H78. Similarly, light chain residues can also be replaced. Examples of replacement light chain residues include one or more of the following residue numbers: L1, L2, L4, L6, L46, L47, L49 and L71. At least three such residues are preferably substituted. A particularly preferred set of light chain substitutions in humanized anti-integrin oc2 antibodies exemplified herein is L2, L46 and L49.

One method that can be used to identify certain residues or regions of a humanized anti-integrin α2 antibody that are preferred sites for mutagenesis is called "alanine scanning mutagenesis" (see, eg, Cunningham and Wells Science, 244: 1081-1085 (1989)). Here, a target residue or group of residues is identified (eg, residues charged as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (preferably alanine or polyalanine) to interfere with amino acid interaction. with α2β1 integrin antigen. Amino acid sites that demonstrate functional sensitivity to substitutions are then refined by the subsequent introduction of other variants at or to the substitution sites. Thus, although 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 mutation performance at a particular site, alanine or random mutagenesis is scanned at the target codon or region and the expressed variants of humanized anti-integrin a2 antibody are separated for desired activity.

Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include a humanized anti-integrin α2 antibody with an N-terminal methionyl residue or antibody fused to a labeled epitope. Other variants of the humanized α2-integrin antibody molecule which may be inserted include the N- or C-terminal fusion of a humanized α2-integrin antibody of an enzyme or polypeptide that increases the serum half-life of the antibody ( see below).

Another type of variant is an amino acid substitution variant. In these embodiments, at least one amino acid residue in a humanized anti-integrin α2 antibody molecule is removed and a different residue inserted in its place. Sites of major interest for substitution mutagenesis include hypervariable cycles, but changes in the framework are also contemplated. Residues from the hypervariable region or framework residues involved in antigen binding are generally relatively conservatively substituted. These conservative substitutions are shown below under the heading "preferred substitutions". If these substitutions result in a change in biological activity, more substantial changes will be introduced called "exemplary substitutions" or as described later with reference to the amino acid class, and products will be scanned.

<table> table see original document page 49 </column> </row> <table>

Considerable modifications in the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect on the maintenance (a) of the polypeptide backbone in the substitution area, for example as a leaf or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, vai, 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 properly maintaining the confirmation of a humanized anti-integrin α2 antibody can also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking from occurring. On the other hand, cysteine bonds may be added to the antibody to improve its stability (especially where the antibody is an antibody fragment such as the Fv fragment).

Another type of antibody amino acid variant alters the original antibody glycosylation pattern. By alteration is meant deletion of one or more carbohydrate moieties found in the antibody and / or addition of one or more glycosylation sites not present in the antibody.

Glycosylation of antibodies is generally N-linked or Co-linked. N-Ligated refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.

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

The addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains or not one of the tripeptide sequences mentioned above (for N-linked glycosylation sites). The alteration may consist of the addition, substitution or deletion of one or more serine or threonine residues to the original antibody sequence (for 0-linked glycosylation sites). Nucleic acid molecules encoding amino acid sequence variants of the humanized anti-integrin α2 antibody are prepared using a variety of methods known to skilled practitioners. Such 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 a previously prepared variant or a non-variant version of the humanized anti-integrin a2 antibody.

Normally, the amino acid sequence variants of the humanized anti-integrin α2 antibody will contain an amino acid sequence with at least 75% amino acid sequence identity with the amino acid sequences of the original humanized heavy chain and light chain antibody (e.g. (variable region sequences as in SEQ ID NO: 21 or SEQ ID NO: 19, respectively), preferably at least 80%, preferably at least 85%, preferably at least 90%, 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 is identical to the humanized α2 integrin residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum possible identity in the sequence percentage, and not considering any conservative substitution (as described above) as part of the sequence identity. None of the N-terminal, C-terminal, or internal extensions, deletions, or insertions in the antibody sequence should be interpreted as interfering with sequence identity or homology. Therefore the sequence identity can be determined using standard methods that are. commonly used to compare similarity in amino acid position of two polypeptides. Using a computer program such as BLAST or FASTA it is possible to align two polypeptides to an optimal combination of their respective amino acids (either along the full length of one or both sequences or over a predetermined portion of one or both). the sequences). The programs provide a standard opening penalty and a standard range penalty and a score matrix such as PAM2 50 (a standard score matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, vol 5, supp. 3 (1978)) can be used in conjunction with the computer program. For example, the identity percentage can be calculated as: the total number of identical combinations multiplied by 100 and divided by the sum of the length of the longest sequence within the range of the combinations and the number of intervals entered in the longest sequences for the purpose of align the two sequences.

Antibodies that exhibit the characteristics identified herein as desirable in a humanized anti-integrin α2 antibody are screened using the methods described herein. For example, methods for screening candidate anti-integrin α2 antibodies for preferred characteristics and functionality are provided. These methods include screening for epitope-binding antibodies into an α2β1 integrin linked by an antibody of interest (eg, those that compete, inhibit or block the binding of the TMC-2206 antibody to α2β1 integrin).

Examples of methods and materials are given in Example 13. Cross-blocking assays can be performed and are described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Epitope mapping may be performed additionally or alternatively, for example, as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), to determine if the antibody binds to an epitope of interest (see, e.g., Example 12 for TMC-2206 epitope mapping studies).

Immobilized α2β1 integrin can be similarly used to determine relative binding potencies by measuring K ± values in competitive assays (see, eg, Example 2). 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. The amount of bound Eu-TMC-2206 is determined after a specific incubation period. Inhibition curves are fitted with the "one-site competition" model using Prisma software (GraphPad, Inc. CA) to obtain IC50 values and to calculate Ki using the Cheng and Prusoff equation (Biochem, Pharmacol 22 (23): 3099-108 (1973)).

It is advisable to prepare, identify and / or select humanized anti-integrin α2 antibodies having beneficial binding properties, for example under the conditions described in Example 2, in which candidate antibodies are tested for the ability to block integrin-mediated cell adhesion. α2β1 compared to TMC-2206 and the chimeric mouse-human antibody derived from TMC-22 06 as described in Example 2. For example, CHO cells expressing human a2 integrin and endogenous βΐ hamster cells (Symington et al., J. Cell Biol. 120 (2): 523-35 (1993)) are prepared and labeled with CFSE (Molecular Probes, OR). Labeled cells are prepared and cell concentration is adjusted; The cells are kept in the dark until used. A collagen coated plate (rat tail Type I collagen; BD Biosciences) is prepared and each serially diluted antibody solution is added to the collagen plate. Then the labeled cells are placed in the wells and the plate is incubated. After washing, the cells are lysed and the fluorescence intensity is checked (excitation 485 nm; emission 535 nm). The inhibitory activity of each antibody is calculated.

Binding constants of candidate antibodies to immobilized α2βΐ integrin ligand can also be calculated as described in Example 2. The wells of a 96-well microtiter plate are coated with platelet α2βΐ integrin (a2pide human platelet-tailored coating). GTI Inc., WI) and then locked. For example, to determine TMC-226 6 affinity for its α2 integrin antigen, fluorescently labeled TMC-2206 or isotype control IgG antibody is used (see Examples below). Fluorescently labeled antibody, including Eu-TMC-2206 or Eu-isotype control IgG, is applied to α2β1-integrin-blocked microtiter plates. After incubation of the sealed plates to allow antibody-antigen interaction to reach equilibrium, samples are transferred to other wells containing the enhancer solution which will allow evaluation of the free (unbound) marker. The intensifying solution is also added to the empty wells for evaluation of the bound marker. Ka values of α2 integrin antibody are calculated by Scatchard analysis. The relative affinity of TMC-2206 derivatives (including humanized antibodies derived or based on TMC-2206) can be determined by determining the Ki value in a competition assay. For example, for the competition assay, I-labeled TMC-2206 is placed in α2β1-coated wells in the presence of unlabeled a2 anti-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 an incubation period to reach equilibrium, the wells are washed and bound labeled antibody levels are measured as the I-marker retained in each well. The Ki value can be derived from the EC50 values using the Kd value obtained for the Eu-TMC-2206 antibody by direct binding studies as described above.

In certain applications, the humanized anti-integrin oc2 antibody is an antibody fragment. Several techniques have been developed for the production of antibody fragments. Traditionally, such fragments were obtained by proteolytic digestion of intact antibodies (see eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985). ). However, such fragments may be produced directly by recombinant host cells, such as bacteria (see eg, Better et al., Science 240 (4855): 1041-1043 (1988); US Patent No. 6,204,023. For example, Fab1-SH fragments can be directly recovered from E. coli and chemically aggregated to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). F (ab ') 2 fragments may be isolated directly from a recombinant host cell culture Other techniques for producing antibody fragments will be apparent to the skilled practitioner In some applications it may be desirable to produce humanized anti-integrin antibodies multispecific α2 (eg, bispecific) with binding specificities for at least two different epitopes Bispecific antibodies (eg, with two different binding arms) may bind to two different epitopes of the proton. α2β1 integrin eine. Alternatively, an a2 anti-integrin arm may be combined with an arm that binds to a trigger molecule on a leukocyte such as the T cell receptor molecule (eg, CD2 or CD3), or Fc receptors for IgG ( FcyR), such as FcyRl (CD64), FcyRII (CD32) and FcyRIII (CD16) to concentrate the cellular defense mechanism in a cell that has α2β1 integrin bound to its surface. Bispecific antibodies can be used for cytotoxic agents located in cells with α2β1 integrin bound to their surface. These antibodies have an α2β1 integrin-binding arm and a cytotoxic agent-binding arm ex. : gelonin, saporin, anti-interferon alfa, vinca alkaloid, ricin A chain, or radioisotope hapten). Bispecific antibodies can be made with whole antibodies or antibody fragments (eg, F (ab ') 2 bispecific antibodies) ·

According to another approach for the production of bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from a recombinant cell culture. The preferred interface is comprised of at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains are replaced by larger side chains (eg tyrosine or tryptophan).

Compensatory cavities of size smaller than or identical to the major side chain (s) are created at the interface of the second antibody by replacing the large amino acid side chains with smaller chains (eg. alanine or threonine). This provides a mechanism for increasing the production of heterodimers rather than other undesirable end products such as homodimers (see, eg, WO96 / 27011).

Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate may be added to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient method of crosslinking. Suitable crosslinking agents are well known to skilled practitioners and are described, for example, in U.S. Patent No. 4,676,980 together with various crosslinking techniques.

Techniques for the production of bispecific antibodies from antibody fragments have also been described in the literature. Bispecific antibodies may be

prepared using chemical bonding. For example, Brennan et al. (Science 229: 81 (1985)) describe a procedure in which intact antibodies are proteolytically cleaved to produce F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol arsenite complexing agent. sodium to stabilize dithiols and prevent the formation of intermolecular disulfide. The Fab1 fragments produced are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab1-TNB derivatives is then converted to Fab'-thiol by mercaptoethylamine reduction and mixed with an equimolar amount of another Fab1-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used for selective immobilization of enzymes.

Fab'-SH fragments recovered from E. coli can be chemically aggregated to form bispecific antibodies. For example, Shalaby et al. (J. Exp. Med. 175: 217-225 (1992)) describe the production of a molecule of a fully humanized bispecific antibody F (ab ') 2- Where the Fab1 fragment was separately secreted of E. coli and subjected to direct chemical aggregation in vitro to form the biospecific antibody. The biospecific antibody thus formed was able to bind to HER2 receptor overexpressing cells and normal human T cells, in addition to triggering the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies were produced using leucine zippers (see, eg, Kostgelny et al., J. Immunol. 148 (5): 1547-1553 (1992)). Fos and Jun protein leucine zipper peptides were linked to Fab1 moieties of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to form monomers and then oxidized again to form antibody heterodimers. This method may also be used for the production of antibody heterodimers. Bivalent diabody homodimer technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)) has provided an alternative mechanism for the production of bispecific antibody fragments. Fragments are composed of a heavy chain variable region (VH) connected to a light chain variable region (VL) by a ligand that is too short to allow pairing between the two domains of the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with complementary VL and VH domains of another fragment, thus forming two antigen binding sites. Another strategy for producing bispecific antibody fragments using single chain Fv (sFv or scFv) dimers has also been reported (see, eg, Gruber et al., J. Immunol. 152: 53 68 (1994). )). Alternatively, the bispecific antibody may be a linear antibody, produced for example as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995).

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

Other modifications of humanized anti-integrin a2 antibodies are discussed. For example, it is possible

modify the effector function of the antibody to increase or decrease its effectiveness, for example for cancer treatment. Cysteine residue (s) may be introduced into the Fc region, thereby allowing disulfide bond formation between the chains in the region. The homodimer antibody thus produced may improve the ability to internalize and / or increase complement-mediated cell death (MCM) and / or antibody-dependent cell cytotoxicity (ADCC) (see: Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992)). Homodimer antibodies with enhanced antitumor activity may also be prepared using heterobifunctional crosslinking (see, e.g., those described in Wolff et al., Cancer Research 53: 2560-2565 (1993)). Alternatively, an antibody with two Fc regions can be engineered and thus have their enhanced CMC and / or ADCC capabilities (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989)).

Immunoconjugates composed of a humanized anti-integrin α2 antibody conjugated to a moiety, e.g., a molecule, composition, complex, or agent, for example, a cytotoxic agent such as a chemotherapeutic agent, toxin (eg, an enzymatically active bacterial toxin) , fungus, plant or animal origin, or fragments thereof), or a radioactive isotype (e.g., a radioconjugate), to direct the agent to a cell, tissue or organ expressing the Î ± 2 anti-integrin. This immunoconjugate can be used in a method of targeting the portion or agent of a specific site of action characterized by the presence of α2 or α2β1 integrin.

Chemotherapeutic agents that can be used in the production of these immunoconjugates have been described above. Toxins and enzymatically active fragments thereof which may be used include diphtheria A chain, unbound diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrina A chain, A modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana (PAPI, PAPII, and PAP-S) proteins, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin or trichothecenes. A wide variety of radionuclides are available for the production of radioconjugated alpha 2 integrin antibodies. Examples include 212 Bi, 131In, 90Y or 186Re.

Antibody and cytotoxic agent conjugates are produced using a wide variety of bifunctional protein aggregating agents such as N-succinimidyl-3- (2-pyridylditiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as gluteraldehyde), bis-azide compounds (such as bis (p-azidobenzol) hexanediamine), bis-diazonium derivatives (such as bis- (p- diazoniobenzol) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), or bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al. , Science 238: 1098 (1987). Carbon-14-1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid-labeled acid (MX-DTPA) is an exemplary chelating agent for radionuclide conjugation with antibody (see, eg, W094 / 11026).

In another application, the antibody may be conjugated to a receptor (such as streptavidin) for use in pre-targeting the oc2 integrin expressing cell, tissue or organ, where the antibody-receptor conjugate is administered to the patient followed by removal of the circulation. of the unbound conjugate with the use of a specific agent and subsequent administration of the binder (eg avidin) which is conjugated to an agent such as a cytotoxic agent (eg a radionuclide).

The anti-integrin α2 antibodies described herein may also be formulated as immunoliposomes. Antibody-containing liposomes are prepared using methods known in the art as described in Epstein et al. , Proc. Natl. Acad. Know. USA 82: 3688 (1985); Hwang et al. , Proc. Natl. Acad. Know. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are described in U.S. Patent No. 5,013,556.

Especially useful liposomes may be produced using the reverse phase evaporation method with a lipid composition formed of phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore sizes to produce liposomes of the desired diameter. Fab1 fragments of an anti-integrin α2 antibody can be conjugated to liposomes as described in Martin et al. , J. Biol. Chem. 257: 286-288 (1982) via a disulfide exchange reaction. The liposome may optionally contain a chemotherapeutic agent (e.g., doxorubicin) (see, e.g., Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989)).

Humanized anti-integrin α2 antibodies can also be used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) by conjugating the antibody to a prodrug activating enzyme that converts a prodrug (eg, a peptidil chemotherapeutic agent, see eg .: WO81 / 01145) in an active drug. (see, e.g., WO88 / 07378 and U.S. Patent No. 4,975,278). The enzyme component of the immunoconjugate useful in ADEPT includes any enzyme acting on a prodrug so that it becomes its most active form. Enzymes that may be used include, but are not limited to, alkaline phosphatase, which is used to convert phosphate-containing prodrugs into free drugs; arylsulfatase, used to convert sulfate-containing prodrugs into free drugs; cytosine deaminase, used to convert non-toxic 5-fluorocytosine into an antineoplastic drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins BeL), which can be used to convert peptide-containing prodrugs into free drugs; D-alanylcarboxipeptidases, used to convert prodrugs containing D-amino acid substitutes; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase used to convert glycosylated prodrugs into free drugs; β-lactamase used to convert β-lactam-derived drugs into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, used to convert drugs derived from their amino nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, enzymatic activity antibodies, 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 may be prepared as described herein, including for presenting the abzyme to a αx2 integrin expressing cell, tissue or organ.

Enzymes may be covalently linked to anti-integrin α2 antibodies using techniques well known in the art, including the use of the heterobifunctional cross-linked reagents discussed above. Alternatively, fusion proteins composed of at least the antigen binding region of an anti-integrin α2 antibody bound to at least a functionally active portion of an enzyme may be produced using recombinant DNA techniques well known in the art ( see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

In certain applications of the invention, it may be recommended to use an antibody fragment rather than an intact antibody to, for example, increase tissue or tumor penetration. It may also be recommended to modify the antibody fragment to increase serum half-life. This may be accomplished by incorporating a rescue receptor-linked epitope into an antibody fragment, for example, by mutating the specific region in the antibody fragment or by incorporating the epitope into the labeled peptide which is then fused to the antibody fragment. antibody at either end or medium, for example by DNA or peptide synthesis (see, eg, WO96 / 32478). Covalent modifications of humanized anti-integrin α2 antibodies may be made, for example, by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent antibody modifications may be introduced into the molecule by reacting defined antibody amino acid residues to an organic derived agent that is capable of reacting with selected side chains or N- or C-terminal residues. Cysteinyl residues, for example, commonly react with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derived by reaction with bromotrifluoroacetone, oc-bromo- / 3- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylamaleimides, 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 obtained by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacil bromide may also be used; The reaction should preferably be carried out in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues, for example, react with succinic acid or other anhydride carboxylic acid. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable agents for the derivatization of α-amino containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroboroidide, trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, and glyoxylate catalyzed transaminase reaction. Arginyl residues, for example, are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. For derivatization of arginine residues, the reaction must be performed under alkaline conditions due to the high pKa of the guanidine functional group. In addition, these reagents may react with the lysine groups in addition to the epsilon-amine arginine group. Tyrosyl residues, for example, are specifically modified with the specific intention of introducing spectral markers into tyrosyl residues by reaction with aromatic diazonium or tetranitromethane compounds. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125 I or 131 I to prepare labeled proteins for use in radioimmunoassay. Carboxyl side groups, for example, aspartyl or glutamyl, are selectively modified by reaction with carbodiimides (RN = C = N-R '), where ReR' represent different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4). -ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are often assigned to the corresponding glutamyl and aspargil residues, respectively. These residues are demined under neutral or basic conditions. The demined form of such residues is within the scope of this invention. Other modifications include proline and lysine hydroxylation, phosphorylation of hydroxyl groups from seryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (E. Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine and the amidation of any C-terminal carboxyl group. Another type of covalent modification involves the chemical or enzymatic aggregation of glycosides to the antibody. The advantage of these procedures is that they do not require antibody production in a N- or O-linked glycosylation-capable host cell. Depending on the aggregation model used, sugar (s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine, or tryptophan, or (f) the glutamine amide group (see eg W087 / 05330; Aplin and Wriston, CRC Crit Rev. Biochem., pp. 259-306 (1981)).

Any portion of carbohydrate present in the antibody may be chemically or enzymatically removed. Chemical deglycosylation requires exposure of the antibody to the trifluoromethanesulfonic acid compound, or an equivalent compound. This treatment results in cleavage of most or all of the sugars except bound sugar (N-acetylglycosamine or N-acetylgalactosamine) while leaving the antibody intact (see, eg, 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 using a wide variety of endo and exoglycosidases, (see, eg, Thotakura et al., Meth. Enzymol. 138: 350 (1987)).

Another type of covalent modification of the antibody is accomplished by binding the antibody to one of several nonproteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes (see, e.g., US Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337).

Described herein are isolated nucleic acid (s) encoding a humanized α2-integrin antibody, vectors and host cells that make up nucleic acid, and recombinant production techniques of the antibody. For recombinant antibody production, the antibody-encoding nucleic acid (s) are isolated and inserted into a replicable vector for later cloning (DNA amplification) or for expression. Antibody-encoding DNA is readily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains). Several vectors are available. 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-integrin α2 antibody can be produced by recombinant technique, including as a fusion of a polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide with a specific N-terminus cleavage site on the protein or mature polypeptide. . The selected heterologous signal sequence is preferably one that is recognized and processed (e.g., cleaved by a signaling peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a eukaryotic signal sequence (eg, an immunoglobulin signal sequence), the signal sequence is replaced by a prokaryotic signal sequence including, for example, pectatoliase (such as pelB), alkaline phosphatase , penicillinase, lpp / or thermostable enterotoxin II leader sequence. For yeast secretion, a fungal signal sequence may be used, including, for example, the yeast invertase leader sequence, a leader factor (including Saccharomyces and Kluyveromyces α leader factors), or acid phosphatase leader, albicans glucoamylase leader sequence, or the signal described in W090 / 13646. In the expression of mammalian cells, mammalian signal sequences and viral secretory leader sequences, for example, the herpes simplex gD signal sequence, are available and can be used. The DNA for this percussion region (eg, signal sequence) is linked to the DNA reading frame encoding an anti-integrin oc2 antibody. Both expression vectors 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 host chromosome DNA and includes replication origins and autonomous replication sequences. These sequences are well known for a wide variety of bacteria, yeast and viruses. For example, the origin of replication of plasmid pBR322 is suitable for most gram-negative bacteria, the origin of plasmid 2 μ is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for vector cloning in mammalian cells. Generally, the origin of the replication component is not required for mammalian expression vectors (eg, the SV40 origin can usually be used only because it contains an early promoter).

Expression and cloning vectors may contain a selection gene, also called a selectable marker. Common selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients unavailable in the complex medium, (e.g., the gene encoding the D-alanine racemase for bacilli).

An example selection scheme utilizes a drug to arrest the growth of a host cell. These cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survives the selection scheme.

Examples of this dominant selection use the drugs methotrexate, neomycin, histidinol, puromycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those which enable the identification of cells capable of assuming α2-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 identified first by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive DHFR antagonist. A suitable host cell for use with wwild-type "DHFR" is the Chinese hamster (CHO) ovarian cell line deficient in DHFR activity.

Alternatively, host cells (primarily endogenous DHFR-containing wild-type hosts) transformed or co-transformed with DNA sequences encoding the anti-integrin cc2 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) may be selected by cell growth in medium containing a selectable marker selection agent, including an aminoglycoside antibiotic such as kanamycin, neomycin or G418 (see US Patent No. 4,965,199). ).

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

Vectors derived from the 1.6 μ pKD1 circular plasmid can also be used for Kluyveromyces yeast transformation. Alternatively, an expression system for large-scale production of recombinant calf chemosine has been reported for K. Iactis by Van den Berg, Bio / Technology, 8: 135 (1990). Stable multi-copy expression vectors for secretion of mature human recombinant serum albumin by Kluyveromyces industrial strains have also been described (see, eg, Fleer et al., Bio / Technology, 9: 968-975 (1991)).

Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the α2-integrin antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the arabinose promoter (e.g., araB), phoA promoter, β-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 will also contain a Shine-Dalgamo (S.D.) sequence operably linked to DNA encoding the α2 integrin antibody.

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

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

Transcription of anti-integrin antibody 2a from vectors in mammalian host cells is controlled, for example, by promoters obtained from virus genomes such as polyoma virus, avian pox virus, adenovirus (such as Adenovirus 2), bovine papillomatosis virus , avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus or Simian Virus 40 (SV40), from mammalian heterologous promoters, for example, the actin promoter or an immunoglobulin promoter, from the heat shock promoters. as long as such promoters are compatible with host cell systems. SV40 early and late promoters are conveniently obtained as a SV40 restriction fragment which also contains the SV40 viral origin of replication. The early human cytomegalovirus early promoter is conveniently obtained as a HindIII E restriction fragment. A system for expression of DNA in mammalian hosts using bovine papillomatosis virus as a vector is described in US Patent No. 4,419,446, and A modification of such a system is described in US Patent No. 4,601,978 (see also Reyes et al., Nature 297: 598-601 (1982) on the expression of human β-interferon cDNA in mouse cells under the control of the thymidine promoter. herpes simplex virus kinase). Alternatively, the long terminal Rous sarcoma virus may be used as the promoter. Transcription of the DNA encoding anti-integrin antibody -a2 by higher eukaryotes often increases with the insertion of an enhancer sequence into the vector. A number of mammalian gene enhancer sequences (globin, elastase, albumin, α-fetoprotein and insulin) are known. However, the use of an enhancer obtained from eukaryotic cell viruses is frequent. Examples include the SV40 enhancer on the late side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers (see also, e.g. : Yaniv, Nature 297: 17-18 (1982) on enhancer elements for activation of eukaryotic promoters). The enhancer may be attached to the vector at the 5 'or 3' position of the α2-integrin antibody coded sequence, but preferably is located at the 5 'site of the promoter. Other well-known gene regulatory systems (eg, inducible systems such as tetracycline and GeneSwitch ™ inducible systems) can be used to control the transcription of DNA encoding the α2 anti-integrin antibody.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) will also contain the sequences necessary for transcription termination to stabilize the mRNA. These sequences are commonly available from 5 'and occasionally 3', untranslated regions of viral DNAs or cDNAs or eukaryotes. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-α2 integrin antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region (see, e.g., W094 / 11026 and the expression vector described therein).

Suitable host cells for cloning or expression of DNA in said vectors are prokaryote, yeast, or more developed eukaryotic cells as described above. Suitable prokaryotes for this purpose include gram-positive or gram-negative eubacteria, 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 as B. subtilis and B. licheniformis, Pseudomonas as P. aeruginosa, and Streptomyces. Suitable hosts for E. coli cloning include E. coli 294 (ATCC 31,446), E. coli Β, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325). In addition to prokaryotes, eukaryotic microbes such as filamentous fungus or yeast are suitable hosts for the cloning or expression of vectors encoding anti-integrin alpha 2 antibody. Saccharomyces cerevisiae, or the common yeast used for cooking, is the most frequently used among less evolved eukaryotic host microorganisms. However, several other genera, species and strains are generally 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 as Schwanniomyces occidentalis; and filamentous fungi including Neurospora, Penicillium, Tolypocladium, or Aspergillus hosts such as A. nidulans or A. niger. Suitable host cells for expression of the glycosylated anti-integrin α2 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Several strains and baculoviral variants have been identified and the corresponding permissive host cells of insects such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),

Drosophila melanogaster (fruit fly) and Bombyx mori. A wide variety of transfecting virus strains are openly available, for example Autographa californica NPV variant LI and Bombyx mori NPV strain Bm-5, and these viruses can be used especially for transfecting Spodoptera cells. frugiperda.

Cotton, corn, potato, soybean, petunia, tomato and tobacco cell cultures can also be used as hosts.

However, the interest has been greater for vertebrate cells and the spread of vertebrate cells, including a wide variety of mammalian cells, has become routine procedure. Examples of mammalian host cells that may be used include:

A SV40-transformed monkey kidney CV1 strain (eg, COS - 7, ATCC CRL 1651); a human embryonic kidney line 293 or 293 subcloned cells for growth in suspension culture (see e.g., Graham et al., J. Gen Virol. 36: 59 (1977)); hamster pup renal cells (eg, BHK, ATCC CCL 10); Chinese hamster ovarian (CHO) cells, including CHO cells without 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 (eg, CV1 ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (eg, HELA, ATCC CCL 2); dog kidney cells (eg, MDCK, ATCC CCL 34); buffalo rat liver cells (eg: BRL 3A, ATCC CRL 1442); human lung cells (eg W138, ATCC CCL 75); human liver cells (eg: Hep G2, HB 8065); mouse mammary tumor (eg, 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 strain (eg, Hep G2).

Host cells are transformed with the expression or cloning vectors described above for the production of anti-integrin α2 antibody and cultured in conventional nutrient medium modified as needed to induce promoters by selecting transformants and / or by amplifying the genes encoding the desired sequences.

Host cell culture for the production of anti-integrin α2 antibody can be done in a wide variety of media. Commercially available culture media such as Ham1S FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle1S Medium ((DMEM), Sigma) are suitable for In addition, any culture media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or US Patent Re. No. 30,985 may be used as a culture medium for host cells.

Any of these culture media may be supplemented as needed 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 GENTAMICINA ™), trace elements (defined as inorganic compounds generally present at final concentrations in the micromolar range), and glucose or an equivalent source of energy. Any other necessary supplements may also be included in appropriate concentrations that are known to qualified professionals. Culture conditions, such as temperature, pH, and others, are selected by skilled practitioners, including those culture conditions previously used with the host cell selected for expression. Anti-integrin α2 cell antibodies can be purified from cells, including microbial or mammalian cells, using, for example, protein A affinity 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 Fc immunoglobulin domain present in the antibody. Protein A can be used to purify antibodies based on the human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmark et al., J. Immunol. Meth. 62: 1- (1983)). G protein can be used for mouse isotypes and human γ3 (see, eg, Guss et al, EMBO J. 5: 1516-1517 (1986)). The matrix to which the binder is often attached is agarose, but there are other matrices available. Mechanically stable arrays such as controlled pore glass or poly (divinyl styrene) benzene enable faster flow rates and shorter processing times than those achieved with agarose. When the antibody is composed of a CH3 domain, Bakerbond ABX ™ (J.T. Baker, Phillipsburg, N.J.) may be used for purification. Protein purification may include one or more of the following techniques: fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSE ™ heparin chromatography, anion exchange resin chromatography or cation (eg, a polyaspartic acid column), chromatoconcentration, SDS-PAGE, ammonium sulfate precipitation and / or hydrophobic interaction chromatography. Following the purification step (s), it may be useful, for example, to subject a mixture of the antibody of interest and contaminants to low pH hydrophobic interaction chromatography with the use of pH-elution buffer between about 2.5-4.5, preferably performed with low salt concentration (eg, about 0-0.25M salt).

Formulations of an α2-integrin antibody, including those for therapeutic administration, are prepared for storage by mixing the antibody of desired purity with physiologically acceptable carriers, diluents, excipients or optional 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 non-toxic to the containers at the doses and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl 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 polypeptides (less than about 10 residues); 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 counterions such as sodium; metal complexes (e.g., protein-Zn complexes); and / or nonionic surfactants such as TWEΕΝ ™, PLURONICS ™ or polyethylene glycol (PEG).

The antibody formulation may also contain more than one active compound for the specific indication of treatment, preferably those with complementary activities that do not adversely affect each other. It may be necessary to add an anti-integrin α2 antibody to one or more agents currently used to treat or prevent the disorder in question. It may also be necessary to use an immunosuppressive agent later. Such molecules are present in the combinations in amounts effective for the desired purpose. The active ingredients may also be packaged in a microcapsule prepared, for example, by using co-preservation or interfacial polymerization techniques, for example hydroxymethylcellulose or gelatin microcapsule and poly (methylmethacylate) microcapsule, respectively, in colloidal release systems. (eg liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules) or in macroemulsions. Such techniques are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

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

Formulations can be prepared for sustained release. Suitable examples of sustained release formulations include semipermeable arrays of solid hydrophobic polymers containing the antibody, arrays in the form of articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (US Patent No. 3,773,919), L-glutamic acid and γ ethyl L-glutamate copolymers , non-degradable ethylene vinyl acetate, degradable lactic acid glycolic acid copolymers such as Lupron Depot ™ (injectable microspheres composed of lactic acid copolymer glycolic acid and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid glycolic acid allow molecules to be released for more than 100 days, certain hydrogels release proteins for shorter periods. When encapsulated antibodies remain in the body for a longer period, their nature may change or aggregation may occur as a result of moisture at 37 ° C, resulting in 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 found to be an intermolecular SS-linked formation through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues by lyophilizing acidic solutions by controlling the moisture content. using appropriate additives and developing specific polymer matrix compositions.

Anti-α2 antibodies may be used as affinity purifying agents. In this process, antibodies are immobilized in the solid phase as a Sephadex resin or paper filter using methods well known in the art. The immobilized antibody is contacted with a sample containing the α2β1 integrin protein (or fragment thereof) to be purified and then the support is washed with a suitable solvent that will largely remove all sample material except the protein. α2β1 integrin, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as a pH 5.0 glycine buffer to loosen the α2β1 integrin protein from the antibody. Anti-integrin α2 antibodies may also be useful in diagnostic assays for α2β1 integrin protein, e.g. : detecting its expression in specific cells and tissues and in serum. For diagnostic applications, the antibody will generally be labeled with a detectable moiety. Several markers are available which can generally be grouped into the following categories of radioisotopes, fluorescent markers and enzyme substrate markers. Radioisotopes such as 35 S, 14 C, 125 I, 3 H and 131 I are useful markers. The antibody may be labeled with the radioisotope, for example, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al. , Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) and radioactivity can be measured, for example, by scintillation counting. Fluorescent markers such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansil, lysamine, phycoerythrin and Texas Red may also be used. Fluorescent labels may be conjugated to the antibody, for example, using the techniques described in Current Protocols in Immunology, cited above. Fluorescence can be quantified, for example, using a fluorimeter. Various enzyme-substrate markers may also be used (see, e.g., U.S. Patent No. 4,275,149 for a review). The enzyme usually catalyzes a chemical change in the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate that will be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence have been described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can emit light that can be measured (eg using a chemiluminometer) or donate energy to a fluorescent acceptor. Examples of enzymatic markers include luciferases (e.g., fire fly luciferase and bacterial luciferase; US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glycoamylase, lysozyme, saccharide oxidases (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and others. Techniques for antibody conjugation of enzymes are described, for example, in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed. J. Langone & H. Van Vunakis), Academic press, N.Y., 73: 147-166 (1981). Examples of enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, in which hydrogen peroxidase oxidizes a contrast precursor (eg orthophenylene diamine (OPD) or 3, Tetramethylbenzidine 3 ', 5,5'-hydrochloride (TMB)); (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and (iii) β-D-galactosidase (/ S-D-Gal) with a chromogenic substrate (eg, p-nitrophenyl- / 3-D-galactosidase) or 4-methylumbeliferyl-β-D-galactosidase fluorogenic substrate.

Various other enzyme-substrate combinations are available to qualified practitioners (see, e.g., U.S. Patent Nos. 4,275,149 and 4,318,980 for a general review).

Sometimes a marker is indirectly conjugated to the antibody. An experienced practitioner will know the various techniques that can be used to achieve this result. For example, the antibody may be conjugated to biotin and any of the three broad categories mentioned above may be conjugated to avidin, or vice versa. Biotin selectively binds to avidin and thus the marker can be conjugated to the antibody in this indirect manner. As an alternative to achieving indirect conjugation of the marker with the antibody, the antibody may be conjugated to a small hapten (eg digoxin) and one of the different types of markers mentioned above may be conjugated to the anti-hapten antibody (eg. anti-digoxin antibody). Indirect conjugation of the marker with the antibody can thus be achieved.

An anti-integrin α2 antibody need not be labeled and its presence can be detected using a labeled antibody that binds to the anti-integrin α2 antibody. Anti-integrin α2 antibodies can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, eg, Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987)). Competitive binding assays depend on the ability of a labeled standard to compete with the sample analyte tested for binding to a limited amount of antibody. For example, the amount of Î ± 2β1 integrin protein in the test sample is inversely proportional to the amount of the pattern that will bind to the antibodies. To facilitate determination of the amount of the standard to bind, antibodies are generally insolubilized before and after competition so that the antibody-binding model and analyte can be conveniently separated from the standard and analyte that remain unbound. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody that has been immobilized on a solid support, and then a second antibody binds to the analyte, thus forming an insoluble three-part complex (see, eg, US). Patent No. 4,376,110). The second antibody may be labeled with a detectable moiety (e.g., direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (e.g., indirect sandwich assay). An example of a sandwich assay is the ELISA assay, in which the detectable moiety is an enzyme.

In immunohistochemistry, a tissue sample, including a tumor sample, may be fresh or frozen or may be fixed in paraffin with a preservative such as formalin.

Anti-integrin α2 antibodies can also be used in in vivo diagnostic assays. In general, the antibody is labeled with a radionuclide (111In, 99Tc, 14C, 131I, 125I, 3H, 32P or 35S) so that tissue, for example a tumor, can be localized using immunoscintigraphy.

For convenience, an anti-integrin α2 antibody may be provided in a kit as a combination of reagents in predetermined amounts with instructions, including for carrying out a diagnostic assay. When the antibody is labeled with an enzyme, the kit will include the substrates and cofactors required by the enzyme (eg, a precursor substrate that provides the detectable chromophore or fluorophore). Other additives may be included in the kit such as stabilizers, buffers (eg a blocking buffer or a lysis buffer) and others. The relative amounts of various reagents provided in the kit may vary widely, for example, to provide to the solution reagent concentrations that substantially optimize the sensitivity of the assay. Reagents may be provided as a generally lyophilized dry powder, including excipients, for example, which upon dissolution will provide a reagent solution of the appropriate concentration.

An anti - integrin a.2 antibody can be used to treat various α2β1 integrin associated disorders as described herein. The anti-integrin α2 antibody may be administered by any suitable route including parenteral, subcutaneous, intraperitoneal, intrapulmonary or intranasal route. If recommended for local immunosuppressive treatment, intralesional administration of the antibody (including by infusion or other contact with the graft with antibody before transplantation). Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. The anti-integrin α 2 antibody may also be administered by pulse infusion, for example with reduced doses of the antibody. Administration should preferably be by intravenous or subcutaneous injections. The form of administration may depend in part on whether it will be performed for a short period or for a prolonged period.

For prevention or treatment of a disease, the appropriate dose of antibody will depend on the type of disease to be treated as defined above, the severity and course of the disease, the purpose, that is, whether the anti-integrin oc2 antibody will be administered for prevention or therapeutic purposes, previous treatment, patient's medical history and antibody response, and at the discretion of the treating physician. The antibody is suitable for single patient administration or a series of treatments.

Depending on the type and severity of the disease, the antibody dose [from about 1 μg / kg to about 15 mg / kg or about 0.05 μg / kg to about 20 mg / kg] is the dose of the antibody. recommended starting dose for administration to the patient in one or more separate administrations or continuous infusion. A typical daily dose may vary [from about 1 μg / kg to about 100 mg / kg] or more, depending on the factors mentioned above. For repeated administrations over several days or longer periods, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. However, other administration schemes may be used. The progress of this therapy can be readily monitored by qualified professionals.

An anti-integrin cc2 antibody composition will be formulated, dosed and administered in accordance with good medical practice. Factors that should be considered in this context include the specific disease being treated, the specific mammal being treated, the clinical condition of each patient, the cause of the disease, the place of administration of the agent, the method of administration, the administration schedule, the results of pharmacological and toxicity studies and other factors known to physicians. A therapeutically effective amount of the antibody to be administered is determined based on these factors and is the minimum amount required to prevent, ameliorate, or treat an α2β1 integrin-associated disorder. This amount is preferably less than the amount toxic to the host or makes the host significantly more susceptible to infections.

The anti-integrin antibody a.2 need not be, but may optionally be formulated, co-administered or used as adjunctive treatment with one or more agents currently used to prevent or treat the disorder in question. For rheumatoid arthritis, for example, the antibody may be administered in conjunction with a glucocorticosteroid, Remicaid® or any approved treatment for rheumatoid arthritis. For multiple sclerosis, the antibody may be administered in conjunction with interferonP, Avonex, Copaxon or other approved therapies to treat the signs and symptoms of multiple sclerosis. For transplants, the antibody may be administered separately or concomitantly with an immunosuppressive agent as described above, such as cyclosporin A, to modulate the immunosuppressive effect. Α2β1 integrin antagonists may be given as an alternative or in combination with treatment of mammals suffering from α2β1 integrin disorders. The effective amount of these other agents depends on the amount of α2-integrin antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. Such agents are generally used at the same doses and with the same administration routes used previously or about 1 to 99% of the previously employed doses. An article of manufacture containing materials, including an anti-integrin α2 antibody, useful for treating the disorders described above is provided. The article of manufacture consists of a container and a label. Containers include, for example, vials, ampoules, syringes and test tubes. Containers can be manufactured from a wide variety of materials such as glass or plastic. The container contains a composition that is effective for treating a condition and may have a sterile access port (for example, the container may be a pouch or vial of solution for intravenous administration with a pierceable lid with a hypodermic injection needle). The active agent in the composition is anti-integrin alpha 2 antibody. The label on or associated with the recipient indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container containing a pharmacologically acceptable buffer such as phosphate buffered saline, Ringer's solution or dextrose solution. The product may further include other commercially or user desired materials, including other tampons, diluents, filters, needles, syringes and instructions for use.

The principles described above have been applied, for example, to the anti-integrin α2 antibody secreted by the hybridoma BHA2.1 (Hangan et al., Cancer Res., 56 (13): 3142-9 (1996)). This antibody binds to rat and human α2β1 integrin, but does not bind to murine α2al integrin. The antibody thus produced by hybridoma BHA2.1 is called TMC-2206 and is commercially available from Chemicon (now part of Milliporef catalog number MAB1998). Chimeric, even humanized variants of TMC-2206 were produced and subjected to in vitro analysis. In vivo studies were also performed using the TMC-2206 antibody or similar antibody, including an antibody capable of recognizing murine α2β1 integrin. The following examples are provided by way of illustration and in no way as a means of limitation. Mentions of all citations in the specification are expressly incorporated herein by reference. EXAMPLE 1

Antibodies specific for α2 β1 integrin were made and prepared. Previously unknown sequences of the variable regions of a murine antibody designated TMC-2206 secreted by the hybridoma BHA2.1 have been determined and described herein. VH and VL cDNAs were cloned from BHA2.1 hybridoma cell mRNA by RT-PCR using a set of primers corresponding to the amino acids in the murine heavy (VH) or light chain (VL) N-terminal amino acid , and a second set of primers corresponding to the constant regions of the respective heavy chains γ1 and light γ. The sequence was determined based on the cDNA that had been synthesized from the isolated mRNA according to standard methods described herein.

Cytoplasmic mRNA was isolated from approximately 1 million (1 χ 106) BHA2.1 hybridoma cells expressing TMC-2206 using standard molecular techniques for experienced practitioners. To isolate poly A mRNA, cells were lysed in 5M guanidine thiocyanate, mixed with oligo (dT) cellulose (Ambion, TX) and incubated at room temperature with gentle shaking for 60 minutes. The cellulose-bound oligo (dT) poly (A) RNA was pelleted, washed and then applied to an nWash spin "column (Ambion, TX). The column was centrifuged at 3000 xg for 1 minute, then RNA It was extracted with 200 µl of 10 mM Tris, ImM EDTA (TE) buffer, pH 8.0 and precipitated with 0.1 volume of 5 M ammonium acetate (NH4Ac) and 2.5 volumes of 100% ethanol at -20 ° C. The RNA was pelleted by centrifugation, dried and dissolved in DEPC treated water.

CDNAs were synthesized from BHA2.1 hybridoma mRNA by reverse transcription initiated with murine heavy (VH) or light chain (VL) N-terminal primer-based primers, and a second set of primers corresponding to constant regions of the respective heavy chains γ1 and light γ. The sequence of the BHA2.1 antibody was unknown, so N-terminal degenerate commercial antibody primers were used from the murine heavy and light chain variable regions (Light primer mix, # 27-1583-01 and Heavy Primer mix, # 27-1586-01, from Amersham Biosciences) as shown in Table 1. These primers have been reported to encompass the N-terminal heterogeneous amino acid composition of the murine light and heavy chains, respectively. The RT-PCR reactions (Qiagen RT kit) were adjusted as follows: 0.5 μg mRNA, 10 μL, 5x RT buffer, 2 μm 10 mM dNTP mix, 5 μL; of each 10 mM and 2 μL · primer solution of the enzyme mixture at 50 μm of the total volume. The reaction was initiated with reverse transcription at 500 ° C for 30 minutes, followed by a PCR activation step at 95 ° C for 15 minutes and terminated with a PCR program suitable for degenerate primer mixtures used to amplify heavy chain variable regions and light: 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C for 1 minute for 28 cycles with a final extension performed for 10 minutes at 72 ° C. Subsequent PCR reactions used the primer pairs listed in Table 2, which were synthesized by Retrogen (San Diego, CA). All primers are listed as 5 'to 3' TABLE 1

<table> table see original document page 89 </column> </row> <table>

TABLE 2

<table> table see original document page 89 </column> </row> <table> <table> table see original document page 90 </column> </row> <table>

PCR products of approximately 350 bp in length were obtained for VH and VL. These PCR products were recovered from a 1% agorose gel, cloned into a pCR2.1-TOPO cloning vector (Invitrogen, CA) and sequenced.

Sequencing was performed on a CEQ DNA sequencer using forward and reverse M13 primers (Invitrogen, CA). Plasmid DNA was produced in 1.5 mL bacterial culture using Qiagen kits according to the manufacturer's instructions. Approximately 300 ng of DNA was used for each PCR sequencing reaction, usually in a volume of 10 μL. The DNA was denatured at 96 ° C for 2 minutes and then mixed with sequencing primer at the final concentration of 0.3 μΜ. Four μ] ^ DTCS Quick Start Master Mix (Beckman Coulter, Fullerton, CA) were added to the mix and sequencing was continued for 30 cycles: 96 ° C for 20 seconds, 50 ° C for 20 seconds and 60 ° C for 2 minutes. . Sequencing reactions were precipitated with ethanol in the presence of sodium acetate (NaAc), EDTA and glycogen. The pellet was washed twice with 70% ethanol, dried free and resuspended at 20 μ; Sample Charging Solution (supplied in the kit). Eight individual VH and VL clones were sequenced using standard techniques, and the amino acid sequences determined for VH (SEQ ID NO: 21) and VL (SEQ ID NO: 19) are shown in Tables 3 and 4, respectively. . The sequences obtained with all eight clones were identical except for the first or two first amino acids. In VL clones, Glu-Asn or Gln-Phe occurred with the same frequency. In VHi clones, Gln or Glu occurred with the same frequency. The sequences were verified against the NCBI BLAST protein database (http://www.ncbi.nih.gov/BLAST/, Ye et al., Nucleic acids Res., Jul 1:34 (Web Server Issue): W6-9). All sequences throughout the search (eg. TMH-2206 VH or VL) were aligned by CLUSTALW (Multiple Sequence Alignment) at http: //clustalw.genome. (Aiyar, Methods Mol Biol., 132: 221-41 (2000)). The inserted clones showed better combination with the heavy chain (IgG1) and the murine light chain (κ), which was the expected isotype. The cloned VH and VL region sequences suggested a likely leader and lateral constant region sequences, which were used to design more accurate primers to clone the entire heavy and light chain variable region of the hybridoma mRNA TMC-22 06. All primers were synthesized by Retrogen (San Diego, CA). The primer pair, VHL-for CCATGGCTGTCTTGGGGCTGCTCTTCT (SEQ ID NO: 14) and HC-rev GGGGCCAGTGGATAGAC (SEQ ID NO: 15; Fcy CHI mouse), were used to clone the heavy chain variable region and primer pair again. , VLL-for CCATGGATTTTCAAGTGCAGATTTTCAG (SEQ ID NO: 16) and LCk-rev GTTGGTGCAGCATCAGC (SEQ ID NO: 17), were used to clone the hybridoma mRNA light chain variable region using the same PCR conditions described above. Product sequencing confirmed that the identity of the first two residues of the TMC-2206 VL is L1-Q and L2-F and that the identity of the first two heavy chain residues is H1-Q and H2-V. The remaining nucleotide sequences were identical to those cloned using degenerate primer mixtures. TABLE 3

<table> table see original document page 92 </column> </row> <table> The cloned VL region was 106 amino acids and the VH region was 119 amino acids in length. As shown in Tables 3 and 4, there are three CDRs (CDR1-3) and four frameworks (FW1-4) in the cloned heavy chain (VH) and light chain (VL) cloned variable regions. nFrameworks "and CDRs were identified based on the Kabat numbering system (Kabat et al., 1983) with the exception of heavy chain CDR1 which was defined by the Oxford Molecular's AbM definition as residues from the 26 to 35 range. The Oxford Molecular's software AbM antibody modeling

(http://people.cryst.bbk.ac.uk/~ubcg07s/, 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 MJE (ed.), Protein Structure Prediction. 172. (1996)) combines the Kabat CDR and Chothia hypervariable region numbering systems to define the CDRs. For numbering consistency, insertions made in the framework regions and CDRs region relative to the patterns are named according to the position of the residue followed by the sequence in alphabetical order (for example, residues 82A, 82B, 82C are inserted between residues 82 and 83 in the heavy chain as shown in Table 3) VH and VL sequences have relatively short CDR3s There is a potential glycosylation site (Asp-Ser-Ser, NSS) within the cloned light chain CDR1. consistent with the observation that the TMC-2206 light chain has a molecular weight of 29 kD according to the SDS-PAGE technique that can be changed with endoglycosidase treatment to 25 kD (typical antibody light chain molecular weight).

To confirm that the cloned sequences represented the bioactive VH and VL of the TMC-2206 antibody, the antibody purified from the hybridoma medium was subjected to N-terminal peptide sequencing by Edman degradation. The amino acid sequence obtained from the VH and VL clones indicated the probable presence of an N-terminal glutamine in each one, which raised the hypothesis of N-terminal blockade by cyclization of the N-terminal glutamine residue to produce pyroglutamate (pGlu). Therefore, to remove any potentially cyclized terminal glutamine, the protein was subjected to pyroglutamate aminopeptidase digestion using a heat-tolerant Pyrococcus furiosus enzyme before subjecting the light and heavy chains to N-terminal peptide sequencing. Pyrococcus furiosus (0.01 U) purified pyroglutamate aminopeptidase (Sigma, St. Louis, MO) was reconstituted in 50 μL Digestion Buffer (50 mM sodium phosphate, pH 7.0, 1 mM EDTA and 10 mM dithiothreitol (DTT)). ). The TMC-2206 preparation was digested using a 1: 100 molar ratio of pyroglutamate aminopeptidase: protein at 950C for 1 hour. Digested proteins were resolved using a standard 10% SDS-PAGE gel (Tris-glycine, BioRad

Laboratories, Hercules, CA) with sodium mercaptoacetate (0.1 g in 150 mL of Current Buffer) in the upper reservoir. Then the gel was transferred to Immobilon P PVDF membrane (Millipore, Billerica, MA) in Transfer Buffer (10 mM CAPS, pH 10.5, 0.5 g / L DTT and 25% methanol) at 250 mAmp for 1 hour. Blot staining was performed using 0.1% fresh Ponceau S solution in 1% acetic acid for 1 minute followed by discoloration in 1% acetic acid. The blot was subjected to peptide sequencing. in which it was observed that 20 of the first 21 light chain N-terminal amino acids were successfully sequenced and showed exact identification with the peptide obtained in the sequence obtained by cloning. This confirmed that the identity of the first amino acid of cloned VL is Glu. Pyroglutamate aminopeptidase digested VH did not provide any peptide sequencing data. EXAMPLE 2 Chimeric antibodies with α2β1 integrin specificity were designed and prepared, including mouse-human chimeric antibodies. The cloned TMC-2206 VH and VL regions as described in Example 1 were used to construct and prepare heavy and light chimeric chains, respectively, using standard molecular cloning techniques (see: Molecular Biology Manual by Sambrook and Russell. , 2001). Heavy and light chains were cloned with the introduction of restriction sites as shown below. The primers, TMC-2206-r5 'CCCGAATTCACAGGTGCAGTTGAAGGAGTCA SEQ ID NO: 22) and TMC-22 06-r3' CGGGATCCTTAGGATCATTTACCAGGAGAG TGGGA (SEQ ID NO: 23), were used to clone the TMC-22 06 heavy chain by RT from the hybridoma mRNA BHA2.1 and the primers TMC-2206-k5 'CCCGAATTCACAATTTGTTCTCACCCAGTCT (SEQ ID NO: 24) and TMC-2206 -k3' CGGGATCCTTATCTCTAACACTCATTCCTGTTGAA (SEQ ID NO to 25) were used 2206. These primers introduced EcoRI and BanHI sites at the 5 'and 3' ends, respectively, to allow cloning of cloned heavy and light chains into mammalian pIRES2-GFP and pIRES2-Ds Red expression vectors (Clontech, Catalog Nos. 632306 and 632420), respectively. Both vectors were manipulated to carry an IgK METDTLLLWVLLLWVPGGSTGD leader sequence (SEQ ID NO: 26).

To isolate mRNA, approximately 1 million hybridoma cells expressing TMC-2206 were pelleted at low speed (10 minutes at 800 rpm), washed with PBS, and lysed with 1 ml Trizol (Invitrogen, CA). After vigorous vortexing, the cell suspension was extracted with 0.2 mL chloroform and after centrifugation (14,000 rpm for 5 minutes at 4 ° C), the supernatant was transferred to a new tube in which RNA was precipitated by mixing with 0 ° C. 0.5 mL isopropanol followed by centrifugation (14,000 rpm for 10 minutes at 4 ° C). The RNA pellet was washed with 1 mL of 75% ethanol and dissolved in 50 µl DEPC-treated H2O.

The RT-PCR reaction (Qiagen RT Kit) was performed as described above using 0.5 μg RNA, 10 μL 5x RT buffer, 2 μL 10 mM dNTP mixture, 5 μL each of primer at 10 mM and 2 μL enzyme mix in a total volume of 50 μL. The PCR products were digested with restriction enzymes EcoRI and BamHI and the purified 1% agarose gel fragments were then ligated to the EcoRI / BamHI sites of the pIRES2-GFP (heavy chain) and pIRES2-Ds Red (light chain) vectors. Subsequent sequencing of the variable regions confirmed that no mutations were introduced by RT-PCR.

PCI-neo (Promega, catalog no. E1841) was chosen as the expression vector for cloning chimeric, including humanized, antibody molecules based or derived from TMC-2206 as described above. To reduce the possibility of mutations being introduced into the constant regions by PCR, cloning cassettes were prepared for VH and VL. First, DNA encoding an IgK leader (SEQ ID NO: 26) was cloned into pCI-neo's XhoI and EcoRI cloning sites using the IgK-S (SEQ ID NO: 27) and IgK-AS ( SEQ ID NO: 28) listed in Table 2, which were annealed to each other and then ligated directly into the XhoI-EcoRI digested pCI-neo using T4 ligase. This provided a parental vector for all subsequent cloning steps. Based on this vector two expression cassettes were produced: one for cloning in VH regions adjacent to a human IgG1 Fc (hFc) and the second for cloning in VL regions above the human kappa chain constant region (hKc). No EeoRI, XbaI, HindIII or SalI sites were found in the human IgG1 Fc (hFc) sequences or the kappa chain constant region (hKc), so any of these restriction sites could be introduced at the 5 'end of the constant regions to facilitate cloning. The SalI cloning site was selected for reducing the number of amino acid changes at the variable-constant junction. In the heavy chain chimera, the introduction of a Sal I site at the human VH-Fc mouse junction was accomplished without causing any change in amino acid sequence. First, a

The EcoRI-SalI VH fragment was produced by PCR using the TMC-2206-r5 '(SEQ ID NO: 22) and TMC2206VH-hIgG1 / 4Fc-SalI primer pair (SEQ ID NO: 29) shown in Table 2 for introduce a SalI restriction site at the 31 end of the murine VH sequence using a heavy chain cloned into the pIRES-GFP vector as a template. Human IgG1 Fc was obtained by amplification of DNA IMAGE clone 20688 (Invitrogen, Catalog No. 4764519) using the primers shown in Table 2, hIgG1 / 4Fc-SalI-F (SEQ ID NO: 30) and hIgG1. / 4Fc-NotI-R (SEQ ID NO: 31). The two PCR products were digested with EcoRI / SalI and SalI / Notl, respectively, purified and ligated with the EcoRI / NotI digested pCI-neo-IgK vector. The resulting vector was named pCI-neo-IgK-TMCVH-hFc.

For the light chain chimera, it was not possible to create a SalI site without modifying two amino acids at the VL-kC junction, E105D and L106I. This was achieved by generating a PCR product using the primers shown in Table 2, TMC-2206-k5 '(SEQ ID NO: 24) and TMC22 06VL-hKc-SalI (SEQ ID NO: 32) to amplify the 2206VL region from the plasmid, pIRES-DsRed2-TMC-2206LC above. The PCR product was digested with EcoRI / SalI separated on 1% agarose gel, purified with a Gel Extraction Kit (Qiagen) and ligated with the amplified human IgK light chain constant region from clone IMAGE # 4704496 ( ATCC) using the hKc-SalI-F (SEQ ID NO: 33) and hKc-Notl-R primers (SEQ ID NO: 34) and the vector described above, pCL-neo- IgK. The resulting plasmid was named pCI-neo-IgK-TMC2206VL-hKc.

To assess whether modification of the two amino acids at the VL-kC junction would impact antibody activity, a second light chain chimera encoding the light chain chimera plasmid of the primary amino acid sequence was constructed. First, the human VL and kappa constant regions were amplified with TMC-2206VLwt-hKc-R and TMC-22 06-k5 1 (SEQ ID NOS: 36 and 24) primer pair and the TMC- 2 2 0 6VLwt-hKc-F and hKc-Notl-R (SEQ ID NOS: 35 and 34) respectively, using the pIRES2-DsRed2-Igk-TMC22 06LC vector mentioned above as a template. Second, by dividing the overlapping length of the PCR primers (Horton et al., Gene 77 (1): 61-8 (1989)) TMC-2206-k5 '(SEQ ID NO: 24) and hKc-2 PhotI-R (SEQ ID NO: 34) to bind the two products, and the final PCR product was digested and cloned into pCI-neo-Ig.

To confirm that the cloned mouse-human chimeric antibody had the same specificity as the original monoclonal antibody TMC-2206 secreted by hybridoma BHA2.1, the mouse-human chimeric antibody was expressed in 293F cells using the transient transfection methodology using of a mixture composed of equal parts DNA / OptiMEM and 293fectin / OptiMEM (Invitrogen). Each solution was made with OptiMEM preheated to room temperature. The DNA / OptiMEM mixture contained 20 µg Heavy Chain Expression Plasmid (HC), 20 µg Light Chain Expression Plasmid (LC) and OptiMEM to a total volume of 1.3 mL. 293fectin OptiMEM Mix contained 53 µl 293fectin and OptiMEM to a total volume of 1.3 mL. The 293fectin mixture was added to the DNA mixture, mixed and incubated for 20 minutes at room temperature. The 2.6 mL transfection mixture was added to a tube containing a 40 mL 293F cell culture at 10 6 cells / mL. This tube was incubated at 37 ° C, 8% CO 2 with shaking at 120 rpm. After 3 days, the cell suspension was centrifuged and immediately subjected to protein affinity chromatography to purify the antibody. The final product was concentrated, analyzed using the SDS-PAGE technique and protein concentration was determined by the Lowry method.

To confirm that the mouse-chimeric antibody had the same binding activity as the primary antibody TMC-2206, the purified mouse-chimeric antibody was tested for its ability to block α2β1 integrin-mediated cell adhesion. CHO cells expressing human a2 integrin (SEQ ID NO: 8) and an endogenous hamster β1 cell (Symington et al., J Cell Biol. 120 (2): 523-35. (1993)) were separated from the culture, by incubation in PBS without Ca ++ / Mg ++ containing 5 mM EDTA. The cells were centrifuged (1200 rpm for 8 minutes in a Beckman GH 38 rotor) and the pellet was resuspended in 10 ml RPMI-1640. 30 µl of 17 mM CFSE (molecular probes, OR) was added to the cell suspension and the mixture was incubated at 37 ° C for 15 minutes. Labeled cells were pelleted at low speed, resuspended in 10 ml RPMI-1640 with 0.1% BSA and counted. The cell concentration was adjusted to 8 x 105 cells / mL and kept in the dark until use. A collagen coated plate (rat tail Type I collagen; BD Biosciences) was blocked with 100 μL / well 0.1% BSA in PBS and incubated at room temperature for 30 minutes. Protein samples were serially diluted in serum free medium and 50μL of each serially diluted antibody solution was added to the collagen plate. 50 μL / well of labeled cells were added to the well and the plate was incubated for 1.5 hours at 37 ° C. After washing, cells were lysed with 0.1% Triton X-100 and fluorescence intensity (excitation, 485 nm; emission, 535 nm) was analyzed using a multi-label Victor2 1420 counter (Perkin- Elmer). The cloned TMC-2206 chimera was a potent inhibitor of α2β1-mediated cell adhesion to Type I collagen and had a potency equivalent to that of TMC-2206 with an EC50 value of 1.8 nM compared to 1.2 nM, respectively. In these experiments, the use of the Ig control did not produce binding inhibition, while the use of murine TMC-2206 or the chimeric antibody produced binding inhibition when tested over a molar concentration range of 10 "to 10" 6.

The affinity of the chimeric mouse-human antibody to immobilized α2β1 integrin was also compared with the primary antibody TMC-22 06 for its ability to compete with binding of Eu-labeled TMC-22 06 to α2β1 coated plates, eg by determination of Ki values. First, the affinity of the primary antibody TMC-2206 for immobilized α2β1 integrin was determined by equilibrium binding. The wells of a 96-well microtiter plate were coated with α2β1 platelet integrin (coated on request with human platelet α2β1 by GTI Inc., WI) and blocked with skim milk. For binding and competition assays, the fluorescently labeled TMC-2206 antibody or the IgG control isotype was used. To label the antibodies with the Eu-NI-ITC reagent, approximately 2 mg of the TMC-22 06 or control isotype, MOPC-21 (Invitrogen) was suspended and dialyzed in phosphate buffered saline (PBS; 1, 47 mM KH 2 PO 4, 8.1 mM Na 2 HPO 4 (pH 7.4, 138 mM NaCl and 2.67 mM KCl). After concentration in pre-washed MicroSep concentrators (30-kDa cutoff; Pall Life Sciences at 9500 rpm (7000 χ g) in a JA-20 rotor (Beckman Instruments, Inc.) for 20 minutes at 4 ° C), the antibodies were adjusted to 4.0 mg / mL with PBS containing the final concentration of 100 mM NaHCO3, pH 9.3. The mAb / bicarbonate solution (0.200 mL) was carefully mixed in a vial containing 0.2 mg tetraacetic acid N1- (p-isothiocyanatobenzyl) diet Ilenetriamine - N1, Nz, N3, N3 chelated with Eu3 + (Eu (IT-1; Perkin Elmer Life Sciences) and allowed to react overnight at 40 ° C without stirring. Each mixture with labeled antibody was applied to a separate PD-10 column (GE Biosciences, Piscataway, NJ) pre-equilibrated with Buffer (50 mM Tris, pH 7.4 and 138 mM NaCl). Fractions (0.5 mL) were collected and analyzed to assess total protein (Bradford reagent; Bio-Rad Laboratories, Hercules, CA) using the SpectraMax 384 absorbance plate reader and europium after 1: 10,000 in a time-synchronized fluorescence (TRF) DELFIA (Perkin-Elmer) intensifier solution using a Victor2 multi-label plate reader (Perkin Elmer). Positive fractions for both protein marker and Eu were pooled and applied to new PD-IO columns and samples were collected and analyzed for determination of protein and europium content by calibrated TRF compared to a standard europium solution ( Perkin-Elmer) to calculate fluorine: protein ratio. The fluorescently labeled antibody, either the control IgG Eu-TMC-2206 or Eu-isotype, was then applied to the α2β1 integrin-blocked microtiter plates at a volume of 10 aL / well. After incubation of the sealed plates for 1 h at 37 ° C to allow binding to reach equilibrium, two 2 μL samples were transferred from their wells to a new well containing the DELFIA intensifier solution (100 μL / well; Perkin-Elmer) to evaluation of free (unbound) marker. The intensifying solution (100 μL / well) was added to the empty wells for evaluation of the bound marker. The plate was shaken (The titration shaker plate was set at speed 5 for 5 minutes at room temperature) and TRF (time-resolved fluorescent) intensities were evaluated using a Victor2 multi-label plate reader (Perkin- Elmer Wallac, Boston, MA). The Kd value calculated by Scatchard analysis was 0.374 nM for TMC-2 2 0 6. The relative binding potencies of immobilized α2β1 integrin were analyzed by evaluating Ki values in a 100 pM competition assay. Fluorescently labeled Eu-TMC-2206 in the presence of varying concentrations of unlabeled TMC-2206 antibody or chimeric antibody as competitors. A test system similar to that described above was used. These antibody combinations were then applied to α2β1 integrin coated wells, tested in the concentration range 10 -11 to 10 -7 M, and the amount of Eu-TMC-2206 was determined after the specified time period. Inhibition curves were fitted with a "one-site competition" model, using Prisma software (GraphPad, Inc. CA) to obtain IC50 values and, using the Ki calculation, the Cheng and Prusoff equation (1973). ) and the value above 0.374 nM for Kd. The primary TMC-2206 antibody had a Ki of 0.22 ± 0.04 nM (n = 10) compared to 0.27 ± 0.07 nM (n = 5) for wild type (wt) chimera. The activity of the wt chimera was comparable to that of the chimeric form in which two LC mutations were introduced by manipulation of the SalI site (tambémi also 0.27 nM), confirming that these mutations do not affect activity. In these experiments, BSA-coated control wells tested with either an IgG control or TMC-2206 showed no antibody binding.

EXAMPLE 3

Humanized antibodies with specificity for α2β1 integrin were designed and prepared. Residues of the cloned TMC-2206 antibody that is composed of the heavy and light chain CDR regions were determined and humanized variants were prepared as follows. Three hypervariability regions were found within the less variable framework regions, both in the heavy and light chain variable regions. In most cases, these hypervariability regions correspond to CDRs, but may extend beyond them. The amino acid sequences of the TMC-2206 heavy and light chain variable regions were respectively specified in Tables 3 and 4 above. The CDR and framework regions were generally elucidated according to the Kabat system by aligning with other VH and VL regions using general homology search in the BLAST NCBI protein database (http: // www. .cbi.nih.gov / BLAST /, Ye et al., Nucleic acids Res., Jul 1:34 (Web Server Issue): W6-9)), except for HCDR1. HCDRI was defined based on the AbM definition as residues in the range 26 to 35. The Oxford Software. Molecular's AbM antibody modeling (http://people.cryst.bbk.ac.uk/~ubcg07s/, Martin et al., Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al. al., Methods Enzymol., 203, 121-153 (1991); Pedersen et al., Immunomethods, 1,126 (1992); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 (1996)) combines the Kabat and Chothia numbering systems to define CDRs. Thus, the heavy chain CDR regions were defined as follows:

HCDR1 aa26- aa35

HCDR2 aa50- aa65

HCDR3 aa95- aal02

Similarly, the light chain CDR regions were defined as follows:

LCDR1 aa24- aa34

LCDR2 aa50- aa56

LCDR3 aa89- aa97

Maintaining the binding affinity of the murine antibody to the corresponding humanized antibody is recommended. It may be necessary to select a human acceptor molecule that shares homology with the murine antibody. Preferred human acceptors are human genetic frameworks, due to the absence of somatic mutations that may reduce the degree of immunogenicity. However, individual mature antibody frameworks can also be used as acceptor molecules. The V-BASE database (http://base.mrc-cpe.cam.ac.uk) provides a comprehensive listing of human heavy and light chain germline sequences and was used as a source for comparison with VH and TMC-2206 VL; Kabat's database was also used (http://kabatdatabase.com/, Johnson, G. and Wu, T.T. (2001), Nucleic Acids Res., 29, 205-206).

The TMC-2206 VH aligned well with three of the 51 human gene sequences from the V-BASE database, 4-59, 4-61, and 4-30.4, with no sequence fitting well in the framework 3 region. CDR Hl and H2 lengths in 4-59 were identical to those of TMC-2206 VH, and 4-59 (SEQ ID NO: 39) was selected as the acceptor framework. It had the same class 1 canonical structure for CDR H1 and CDR H2 as that of the TMC-2206 CDR H1 and CDR H2. VH CDR3 and FW4 regions are not included in VBASE gene sequences because CDR3 and FW4 regions are derived from a different noncontiguous gene that varies during mutation of each antibody. The antibody sequence, CAA48104 (NCBI: gi / 3 3 583 / emb / CAA4 8104; http://www.ncbi.nlm.nih.gov/BLAST) was used to provide CDR3 and FW 4 sequences for alignment, and a FW4 acceptor molecule sequence. A comparison of TMC-2206 VH with 4-59 and the CDR3 and FW4 regions of the CAA48104 antibody sequence is presented in Table 5. Table 5 The germline sequence, A14 (SEQ ID NO: 37), was one of 38 human VL antibody sequences from the V-BASE database and was selected as the VL acceptor framework. A14 is in the VK VI family and its LCDR1 and LCDR2 fall into canonical classes 2 and 1 respectively. The TMC-2206's LCDR2 is also included in class 1, although the TMC-2206's LCDR1 is similar to, but not identical to, the class 1 canonical structure. The VL gene sequences extend to the CDR-L3, so it has been selected. an additional sequence for human VL FW4. The selected sequence represents a gene in framework 4, commonly used for kappa light chains in mature human antibodies (eg AAB24132, NCBI: gi / 2 59596 / gb / AAB24132; http://www.ncbi.nlm.nih.gov/ BLAST). Although the introduction of a SalI site caused the two amino acid sequence modification during the construction of the light chain chimera (E105D and L106I, which had no impact on antibody binding, see above), the FW-4 chain acceptor Human light already has an isoleucine at position 106, so this modification caused a single conservative amino acid mutation (E105D) in humanized variants. A comparison of the TMC-2206 VL with A14 and the FW4 region of the AAB24132 antibody sequence is shown in Table 6. TABLE 6

<table> table see original document page 107 </column> </row> <table> Humanized variants of the TMC-2206 were prepared using CDR sequences from the TMC-2206 VH and VL sequences and selected human frameworks as per described above. To maintain proper presentation of the CDR, some canonical residues of acceptor frameworks (see, eg., Chothia et al, 1985, 1992; Queen et al., 1989; Foote and Winter, 1992;

http://people.cryst.bbk.ac.uk/-ubcg07s) can be

exchanged for the corresponding canonical murine donor residues, a process called reverse mutation. Tables 7 and 8 present lists of residues that may affect CDR conformation and inter-chain packaging, respectively, and show the differences between the TMC-2206 donor VH and VL residues and the corresponding human acceptor framework residues (highlighted in bold and italics). The asterisk-labeled L46 residue in Table 8 may play a role in presenting the canonical structure of the CDR and inter-chain packaging.

As shown in Tables 7 and 8, the eleven framework residues that affect the canonical presentation of CDR and two residues that affect cross-chain marshaling differ between donor TMC-22 06 and the acceptor gene sequences A14 and 4-59. with the L46 residue present in both categories. Specifically, these differences are located at positions H37, H48, H67, H71, H73, H78, and H91 for the heavy chain and L2, L4, L46, L47, L49, and L71 in the light chain variable framework regions. These residues were identified and selected as reverse mutation candidates. TABLE 7

<table> table see original document page 109 </column> </row> <table>

TABLE 8

<table> table see original document page 109 </column> </row> <table>

* Mutation that also affects CDR conformation The 13 candidates identified for reverse mutations, being

11 with canonical structure presentation and 2 with inter-chain packaging, were included in the first humanized variant of TMC-2206. A Q-terminal amino was also retained in the humanized VL. The position was retained with the murine identity as it is adjacent to Phe in L2, which is an unusual amino acid at this position.

These humanized heavy and light chain variants were named TMC-2206VH1.0 and TMC-2206VL1.0. Other humanized variants were prepared with fewer reverse mutations, changing the murine residues back to human framework residues.

In this way, framework residues sensitive to reversion to the human residue (in terms of maintenance of antibody potency) were identified. In parallel, a computer modeling was performed to assist in the selection of reversal candidate residues for the human counterpart.

The light and heavy chain chimera pCI-neo expression vectors described in Example 1 were used to express all humanized variants. Humanized TMC-2206 VH version 1.0 (hVH1.O, SEQ ID NO: 40) and humanized VL version 1.0 (hVL1.O, SEQ ID NO: 41) incorporating the 14 reverse mutations defined above, were translated into a nucleotide sequence optimized for mammalian cell expression using Vector NTI software. These sequences were synthesized tandem tailored into a single plasmid constructed by Retrogen (San Diego, CA) and cloned into the EcoRI and SalI sites of the primary TMC-2206 LC and HC expression vectors replacing the mouse VH and VL regions . Specifically, EcoRI-SalI digestion of the plasmid DNA resulted in two fragments of different sizes, the largest being hVH1.0 and the smallest hVL1.0. These two fragments were cloned into the EcoRI and SalI sites of the primary pCI-TMC-2206 chimeric LC and HC expression vectors, replacing the mouse VH and VL regions, respectively, followed by EeoRI and SalI digestion and gel purification in the larger fragment of CI-neoIgk-TMC22 06VG-hFc and pCI-neoIgK-TMC-22 06VLh * c, respectively. This strategy was used for the preparation of subsequent variants. The resulting plasmids contained: leader IgK, the Kozak optimized translation initiation sequence, variable region and human constant region.

The activity of the humanized TMC-2206 VH version 1.0 variant (SEQ ID NO: 40) and the humanized VL version 1.0 (SEQ ID NO: 41) as described above was tested in the adhesion assay. α2β1 integrin-mediated cell phone and in the immobilized α2β1 integrin binding competition assay together with the chimera and the original TMC-2206 antibody as described in Example 2. The Ki value for the humanized prototype was 0.32 nM which was comparable measured Ki of primary antibody TMC-2206 (0.21 nM) and also chimera (0.27 nM), indicating that this first humanized version maintained binding affinity. Similarly, the first humanized prototype showed inhibition activity comparable to that of the primary antibody TMC-2206 in blocking α2β1-mediated cell adhesion to collagen (eg, 1.5 nM EC50 for both).

Using data produced by version 1.0 of the variant, a series of reverse mutations for human VH or VL framework residues were performed using the PCR methodology and minimum numbers of reverse mutations (murine residues) were determined to avoid impairment of specificity and affinity of the original TMC-2206 mAb. Recommended humanized variants are those that preserve the biological activity of the primary murine antibody and also contain less murine residues in order to decrease the immunogenicity potential.

The individual primer sequences were synthesized by Sigma-Genosys and their sequences are shown in Table 9. The primer pairs and models used for the variants produced are shown in Tables 10 and 11. PCR reactions were produced under the following conditions: Primer 1 and 2 (final concentration 0.6 μΜ), dNTP (final concentration 1 mM), DNA model (1 to 10 ng), and 1 unit of Pfx DNA polymerase (Invitrogen, CA) usually in a final volume of 50 μl; The initial PCR program consisted of initial denaturation at 950 ° C for 2 minutes, followed by 30 cycles, each cycle at 95 ° C for 30 seconds, 56 ° C for 45 seconds and at 68 ° C for one and a half minutes. The final step was performed at 68 ° C for 10 minutes. TABLE 9

<table> table see original document page 112 </column> </row> <table> <table> table see original document page 113 </column> </row> <table> <table> table see original document page 114 < / column> </row> <table>

TABLE 10

<table> table see original document page 114 </column> </row> <table> <table> table see original document page 115 </column> </row> <table>

TABLE 11

<table> table see original document page 115 </column> </row> <table> <table> table see original document page 116 </column> </row> <table>

Table 12 lists VH variants and Table 13 lists selected variants and compares selected human acceptor frameworks with the initial (1.0) VH and VL variants. VH variants listed in Table 12 include: hVH1.0 (SEQ ID NO: 21); hVH2.0 (SEQ ID NO: 70); hVH3.0 (SEQ ID NO: 71); hVH4.0 (SEQ ID NO: 72); hVH5.0 (SEQ ID NO: 73); hVH6.0 (SEQ ID NO: 74); hVH7.0 (SEQ ID NO: 75); hVH8.0 (SEQ ID NO: 76); hVH9.0 (SEQ ID NO: 77); hVHIO.0 (SEQ ID NO: 78); hVH11 O (SEQ ID NO: 79); hVH12. 0 (SEQ ID NO: 109); hVH13. 0 (SEQ ID NO: 110); hVH14.0 (SEQ ID NO: 11). VL variants listed in Table 13 include: hVL10 (SEQ ID NO: 41); hVL2.0 (SEQ ID NO: 80); hVL3.0 (SEQ ID NO: 81); hVL4.0 (SEQ ID NO: 82); hVL5.0 (SEQ ID NO: 83); hVL6.0 (SEQ ID NO: 84); hVL7.0 (SEQ ID NO: 85); hVL8.0 (SEQ ID NO: 86); hVL9.0 (SEQ ID NO: 87); hVL10 (SEQ ID NO: 88); hVL11.O (SEQ ID NO: 89); hVL12.0 (SEQ ID NO: 108). Retained murine residues are shown in bold. Also presented are each of the additional built variants (see below). Each of the variants shown in Table 12 below VH1.0 has the same sequence as VH1.0 (shown with a dash [-]) unless a specific amino acid substitution is shown that changes the retained murine residue to the corresponding human framework. Similarly, each of the VL variants shown in Table 13 have the same sequence as the VL1.0 variant, except for the specific amino acid substitutions indicated. Table 12

<table> table see original document page 117 </column> </row> <table> Table 12 (continued)

<table> table see original document page 118 </column> </row> <table> TABLE 13

<table> table see original document page 119 </column> </row> <table> Table 13 (continued)

<table> table see original document page 120 </column> </row> <table> Aligning the TMC-2206 amino acid sequence with the germline sequence showed a clustering of residues in the framework that mutated back to the murine equivalent in the humanized initial variant of TMC-2206 (TMC-2356)]. As shown by the alignment, two clusters were found between FW2 and FW3 in the heavy chain, and two other clusters, one located in FW1 and one in FW2, in the light chain. Two hVH and hVL variants containing reverse mutations for human residues at the sites of interest were variants 3.0 and 4.0, designed to clone mutations to help define the regions in which residues of interest may reside (Tables 12 and 13). In addition to the differences in residues highlighted in Tables 7 and 8 for the VL regions, the L1 position was changed for human Asp in VL4.0, as this is a common residue for human κ light chains. The heavy chains hVH3.0 and hVH4. O were co-transfected with the hVL3.0 and hVL4.0 light chains in various VH / VL combinations and the resulting antibodies were compared with the hVH1.0 / hVL1.0 antibody described above to verify affinity for the ligand by head-to-head comparisons with unlabelled monoclonal antibody TMC-2206. In Table 14, residues in version 1.0 of humanized VH or VL that have been reverted to human waste are shown in bold and italics. The numbers in parentheses in Table 14 represent the change in power compared to the hVH1.O, hVL1.0 variant.

TABLE 14

<table> table see original document page 121 </column> </row> <table> Looking at the Ki values, it is evident that the changes in variant VH 3.0 (human residues inserted in H67, H71, H73 and H78, designated cluster FW-3) induced a large reduction in the potency of hVH3.0-containing antibodies; similarly, a reduction was also observed for hVL4.0 variants (human residues inserted into LI, L2 and L4, called cluster FW-

1). With the exception of the antibodies hVH1.0 / hVL3.0 and hVH4.0 / hVL1.0, which showed a 1.9 and 1.3 fold change in potency compared to the antibody hVH1.0 / hVL1.0, respectively, all remaining combinations showed a more than 4-fold reduction in potency (Table 14). These data showed that the reverse mutations of H37 (V to I) and H48 (L to I), both conservative amino acid changes, were well tolerated. Changes in L46 (K to L) and L47 (W to L) of the human-reverted murine residues were reasonably well tolerated in association with hVH1.0, but showed marked synergistic adverse effect on antibody affinity when in combination. with variant hVH3.0. Examination of differences in residues between human and murine VH and VL frameworks showed that some of these changes were conservative. Additionally, three-dimensional computer models of the murine TMC-2206 VH and VL, human acceptor molecules, and hVH 1.0 and hVL 1.0 structures were made. To guide computer modeling, a BLAST search was performed to identify appropriate database structures for the TMC-2206 VL and VH. The ISY6.pdb structure (resolution 2.0 Â) was chosen for the TMC-2206 VL and the IGIG.pdb structure (resolution 2.3Â) was chosen for the TMC-2206 VH. The ICE1.pdb structure (resolution 1.9A) was chosen for the human light chain acceptor molecule A14, while the IDNO structure (resolution 2.3A) was chosen for the human heavy chain acceptor molecule, 4-59. The modeling predicted the probability of contact of preserved murine residues in humanization of VL 1.0 with the antigen, except for two (LI and L4). The models also indicated that there was no contact of human genetic framework residues with CDRs, while preserved murine framework residues were generally clustered around CDRs.

Three areas of differences were identified between the modeled murine and human VH regions in the heavy chain variable regions. The first area was residues H27-H33, for which probable contact with antigens and CDR Hl was considered. These residues can also affect the angle of the VL / VH interface and have additional indirect effects on antigen binding. The second area was the first CDR H2 loop that may require an H71 residue on the FW. The third area was CDR H3.

Three areas of differences were also observed between the murine and human modeled VL structures in the light chain variable regions. The first structure was CDR1 which had one more residue in murine TMC-2206 VL. Murine Y at L71 (F at A14) was useful to accommodate this difference. The second area was the L40 to L43 murine loop that was pushed deeper into the solvent compared to the human, which indicated that the human L40-43 may be problematic, although the activity of the first humanized prototype showed that these reverse mutations were well tolerated in hVLl.0. The third area was the human framework residues L55 to L59, which were shifted relative to the murine structure. The framework residue L73 (L in murine, F in human) was considered responsible for this difference, although the reverse mutation was well tolerated in hVL1.0.

Using in silico analysis, the results were predicted for the specific heavy chain residues of interest and are summarized in Table 15 below. Similar results for the light chain residues of interest are presented in Table 16.

TABLE 15

<table> table see original document page 124 </column> </row> <table>

TABLE 16

<table> table see original document page 124 </column> </row> <table>

Using in silico analysis, positions were evaluated and ranked to reflect the likelihood that a human substitution would have an effect on antibody performance: H48, H67 <H37, H91 <H73 <H78, H71. In this classification, the H48 position reverse mutation was considered to be the most likely to be tolerated, while the H78 or H71 position reverse mutations were considered to be the least tolerated. Similarly, the following order was predicted for positions of interest within the light chain region: L1 <L4 <L71 <L2 <L47 <L46 <L49. In general, these ratings were in accordance with the Ki values obtained with the hVH3.0, hVH4.0, and hVL4.0 variants. However, a difference was detected between the predicted effects of substitution by computer modeling and the effects observed with the variants produced in the hVL3.0 variant. For example, the antibody variant composed of the combination of VH1.0 / VL3.0 showed reasonable activity, although computer data predicted that the activity of the hVL3.0 variant would be greatly reduced. The impact of the preserved murine framework residues was further assessed by producing other humanized variants, including eight VH variants and six V variants, each carrying a single mutation of the preserved murine residue to the corresponding human. The relative contributions of these changes to the activity were evaluated. Table 12 presents the VH variants and Table 13 shows the VL variants.

The K ± values obtained for these variants indicated that residues in mouse H71, H78, L2 and L46 were preferably retained to maximize activity, whereas H37, H48, H67, H91, LI, L4 and L71 could be altered. to their human counterparts without causing significant loss in activity. Using computer modeling, it was predicted that alteration of mouse residue L47 (Tryptophan, a rare residue at this position in human antibodies) and H73 would impact antigen binding. However, the change to Val-L47 did not significantly affect antigen binding and the change to Thr-H73 caused only a slight deviation (reduction of 1.6) as measured by Ki. It was predicted that L49 (Tyrosine) would bind antigen by in silico modeling and that alliteration to a human lysine would cause major change in potency. However, the shift of human lysine to this position caused only a slight 3.3-fold reduction in potency, as assessed by Ki.

A significant change was observed in VH for the change in H78 from murine valine to phenylalanine, which caused a 70-fold reduction in the potency of hVH11.O, hVL1.0 compared to hVH1.0, hVL1.O, as assessed by Ki. Modeling indicated that this residue has a function in the canonical structure of HCDR1. These results suggest that HCDR1 plays an important role in antigen binding. To maximize activity, the H71 was also maintained. Changing this residue from Lys to Val resulted in a 6.4-fold reduction observed in the hVH9.0 antibody variant, hVL1.0. In addition, among canonical light chain residues, phenylalanine in L2 showed sensitivity to change as evidenced by the marked loss of binding affinity observed with the hVL4.0 variant compared to the hVL5.0, hVL6.0 and hVL7 variants. 0 Computer modeling has indicated that this Phe-L2 can make extensive contact with the LCDR3. Searches in murine and human antibody databases also indicated that this

Phe at position L2 is rare, suggesting that this may represent a somatic mutation that impacts antigen binding.

These residues selected after analysis, as defined above as being tolerant to reverse mutation, were combined with hVH variants from 12.0 to 14.0 and VL variants VL10.0 to 12, and the activity of these variants was compared with the original monoclonal antibody TMC-2206 and the chimeric mouse human antibody TMC-2206. Results indicated that the number of murine residues in hVL could be reduced to three (eg L2 [Phe], L46 [Lys] and L49 [Tyr]) without causing any loss of activity of the variants. Similarly, the number of murine residues in hVH could be reduced from seven to three (eg. H71 [Lys], H73 [Asn] and H78 [Vai]) without causing statistically significant changes in affinity and potency. These 5 results are summarized in Table 17.

TABLE 17

<table> table see original document page 127 </column> </row> <table>

[ANOVA analysis with Dunnett's multiple comparisons test showed no statistically significant differences with TMC-2206 or chimera].

In parallel, homologues of these variants were constructed in which the consensus glycosylation sequence within the LCDRI was altered. Elimination of the glycosylation site (NSS to QSS) may be useful for the downstream manufacturing process and the development process. The change of N26Q in variants hVL10, hVL10.O and hVL12.0 (denoted hVL10.0 [SEQ ID NO: 90], hVL10.0Q [SEQ ID NO: 91] and hVL12.0Q [SEQ ID NO: 92 ]) was introduced in the VL variant using the primer pairs shown in Table 18, whose sequences are shown in Table 9. The change in N2 6Q had no statistically significant effect on the activity of any of the resulting antibodies, as shown in Table 17. Although this glycosylation site occurs at the light chain CDR 1 of the wild-type antibody TMC-2206, these data indicate that it does not appear to play any role in the affinity of the TMC-2206 antibody function blocking activity.

TABLE 18

<table> table see original document page 128 </column> </row> <table>

EXAMPLE 4

Human γ1 antibodies perform effector functions associated with complement and Fe receptor-mediated functions. Experienced practitioners prefer to avoid antibody-dependent cellular cytotoxicity (ADCC) and complement responses to an γ chain without its functionality, such as the region. human γ4 constant. To produce a γ4 version of antibodies VH12.0, VL10.0Q and VH14.0, VL10.0Q, a constant region γ1 sequence was replaced by a constant region γ4 sequence in the VH12.0 and VH14.0 heavy chains, as shown. described below. The constant region γ4 sequence was obtained from the Genbank sequence K01316. Both the IMAGE clone 20688-derived γ1 Fc sequence used to produce intact IgG1 antibody heavy chains with hVH and hVL regions, as described herein, and the γ4 Fc derived from the KO1316 sequence, contain a naturally occurring Apal restriction site. of the junction of the variable of the constant regions. This site was used to clone a constant region γ4 to replace a constant region γ1. The BamH1 and Not1 sites were placed at the 31st end of the sequence to facilitate subcloning into the pCI-neo expression vector. The γ4 sequence (SEQ ID NOS: 105 and 106) was then synthesized as a de novo synthetic gene by Blue Heron Biotechnology (Bothell, WA). The Blue Heron Biotechnology plasmid containing the newly synthesized IgG4 constant region was digested with ApaI and NotI, the constant region fragment Ikb -γ4 was gel purified and ligated to plasmids pCI-VH12.0 and pCI-VH14.0. of the digested ApaI / NotI to produce plasmids encoding VH12.0-y4 and VH14.0-y4. These were combined individually with plasmid pCI-VL10.0Q and transfected into CHO cells. Four days after transfection, culture supernatants were harvested and IgG4 isotypes of VH12.0, VL10.0Q antibodies and VH14.0, VL10.0Q antibodies were purified using protein A affinity chromatography. Transient transfections of γ1 constructs of these variants were performed in parallel.

After acid elution and neutralization, size exclusion analytical chromatography by HPLC indicated the highest presence of order oligomeric forms in the purified protein A IgG4 formulations. Therefore, a second purification step was performed by 26/60 exclusion chromatography using Sefacril S-300 to obtain a monomer fraction. For this, Sefacril S-300 column 26/60 was pre-equilibrated in 660 ml SEC of Buffer Buffer (40 mM HEPES, pH 6.5, 20 mM L-histidine, 100 mM NaCl and 0.02% Tween-80 ). Pooled fractions containing protein extracted from the protein A column were loaded (12.5 ml sample injection) via a Superloop (Amersham Biosciences). SEC fractions (5 ml each) were collected at a flow rate of 2.0 ml / min. Fractions corresponding to the monomeric form (extraction peak at 168.4 ml) were pooled, and protein content was determined by the method. from Lowry. Exemplary IgG1 antibodies have either an hVH 14.0 γ heavy chain (SEQ ID NO: 181) or an hVH12.0 γ1 heavy chain (SEQ ID NO: 182) and an hVL10.0Q light chain (SEQ ID NO: 178). Exemplary IgG4 antibodies have an hVH14.0 γ4 heavy chain (SEQ ID NO: 174) or an hVH12.0 γ4 heavy chain (SEQ ID NO: 176) and an hVL 10.0Q light chain (SEQ ID NO: 178). Purified antibodies were tested in the competition assay to compare potency using K ± values as well as in the collagen cell adhesion assay, where potency is measured based on EC50 values. No significant difference was observed between the isotypes of the different variants, nor did they differ significantly in the original TMC-2206 mAb in any of the assays, as shown in Table 19.

TABLE 19

<table> table see original document page 130 </column> </row> <table>

EXAMPLE 5

The effect of anti-a2 on neutrophil extravasation was studied in a mouse and rat inflammatory peritonitis model. Intraperitoneal administration of certain antigens such as carrageenan or thioglycolate induces a rapid mast cell response that initiates an acute peritoneal reaction (Edelson et al., Blood 103 (6): 2214-2220 (2004)). This peritonitis is characterized by rapid neutrophil infiltration (in hours), followed by slower infiltration and macrophage proliferation (3-5 days). Therefore, this model was used to evaluate for the first time the use of anti-integrin α2 antibodies in the prevention or functional reduction of neutrophil response.

The acute peritonitis model was performed in rats and mice. The TMC-2206 antibody recognizes mouse α2β1 integrin, but not its murine counterpart. However, several in vivo models of inflammatory models are performed in mice and a replacement anti-a2 antibody, Ha 1/29 (Pharmingen, Becton Dickenson, CA, catalog No. 559987), was used in the murine acute peritonitis model.

Animals received either IV or IP injections with the anti-integrin a2 antibody or control isotype at doses ranging from 0.1 to 10 mg / kg 15 minutes before challenge. An IP injection of 1 mL of 9% casein (mouse) or carrageenan (rats) was administered and the animals were taken back to their cages for specific periods of time: 3 hours (mouse) or 5 hours (rats) (n = 4 per group). Animals were sacrificed with halothane and peritoneal cavity was washed with 5 mL (mouse) or 10 mL (rats) of PBS containing 5 mM EDTA. The cells were collected by low speed centrifugation and resuspended in 5 mL PBS / EDTA and an aliquot of 100μL was analyzed with a microscope. Most of these cells were found to have a neutrophil-compatible polymorphonuclear morphology. Remaining cells from the suspension were subjected to low speed centrifugation to obtain a washed cell-free pellet.

Neutrophil content was quantified by analyzing the level of myeloperoxidase (MP0) activity (eg, Speyer et al., Am J Pathol. 163 (6): 2319-28 (2003)). Cell pellet was recovered from the wash liquid and resuspended in 500 µL of 50 mM KH2PO4 (pH 6.0) buffer containing 0.5% hexadecyl trimethyl ammonium bromide (HTAB; Sigma-Aldrich, MI). Samples were sonicated for 60-90 seconds and centrifuged at 14,000 rpm for 5 minutes at 4 ° C. Serial 2: 1 dilution of the clean supernatant was performed by transferring 50 μL to 50 μL HTAB buffer in the well of a microtiter plate. In the next step, 50 μL of this well solution was transferred to another 50 μL buffer, and so on in the dilution series. 200 μΟ L of substrate buffer (50 mM KH2PO4 buffer (pH 6.0) containing 0.168 mg / mL o-dianisidine and 0.0005% H2O2) was added to initiate the colorimetric reaction which was monitored with a plate reader set. Molecular Devices at a wavelength of 460 nm. The number of Units of enzyme activity in the original cell suspension was then calculated (500 μl washed cell suspension for each individual animal). A calibration curve, set to titrate neutrophils / mL (assessed by direct neutrophil count) against MPO activity, indicated a strong linear correlation between U / mL and the number of neutrophils / mL within the measured range. As shown in Table 20, both the Hal / 29 murine anti-a2pi integrin antibody and TMC-2206 had a marked effect on neutrophil infiltration into the peritoneal cavity following administration of 9% casein in mice or 1% carrageenan. rats as measured by the total MP 0 activity recovered from the peritoneal lavage fluid. The ED50 value obtained in Hal / 29 mouse was approximately 0.07 mg / kg, while the ED50 for TMC-2206 in rats was approximately 5 mg / kg. This difference is partly correlated with the relative fineness of human α2β1 integrin by mouse α2β1 integrin compared to the affinity of Hal / 29 antibody for mouse α2β1 integrin and partly to differences in antigen used in rat and mouse models. mouse (carrageenan and casein, respectively). TABLE 20

<table> table see original document page 133 </column> </row> <table>

EXAMPLE 6

The effect of anti-integrin a2 antibodies was studied in a mouse model (dextran sulfate-induced colitis) of inflammatory bowel disease. In this model, colitis is induced in mice by administration of a 5% sodium dextran sulfate (DSS) solution in drinking water (Elson et al., Gastroenterology 109 (4): 1344-67 (1995); Egger B., et al., Digestion 62 (4): 240-8 (2000)) The effect of treatment with murine α2β1 integrin antibody on the development of clinical signs and symptoms of colitis, as well as the effect on infiltration of proinflammatory leukocytes in the colon.

Balb / C mice (Harlan, IN) weighing 16-21 grams were separated in pairs. Animals received distilled water or water containing 5% sodium dextran sulfate (DSS); (ICN, Irvine, CA) ad libitum for 7 days. At this stage the mice had diarrhea and remarkable weight loss. The study design consisted of four groups of six mice each; one group was used as a control without treatment; one group was used as a DSS control and two groups were assigned to treat intraperitoneal injections with doses of 2 or 5 mg / kg of anti-integrin a2 PS / 2 antibody on Days 0, 2, 4 and 6. Mice were sacrificed on Day 7 (168 hours after commencement of DSS administration). The animals were weighed to observe any changes since the beginning of the study, the colon length was measured and then the animals were rated using a scale of 0 to 2, with 2 being the most severe classification for diarrhea, bleeding. colon and rectal bleeding as follows: rectal bleeding: score 0 = no visible blood; 2 = visible blood stool consistency: score 0 = normal; 1 = loose stools; 2 = watery stools colon bleeding: score 0 = no visible blood; 2 = visible blood

The colon was processed for immunohistochemistry. The colon from caecum to rectum was carefully removed and fixed for 2 hours at 40 ° C in 4% paraformaldehyde (PFA), left overnight in 20% sucrose and frozen quickly in OCT compound for freezing (Tissue Tek ). Thin sections (10 μΜ thick) were made in series using a Leica cryostat, air-dried, blocked for 2 hours in 3% goat serum in PBS and incubated overnight in primary antibody at room temperature. environment. Primary antibodies used included murine mouse anti-CD11b / mac-1 (a marker for activated macrophages and neutrophils, Clone M1 / 70, BD-Pharmingen), hamster-murine anti-CD3 (a T cell marker, BD-Pharmingen), and an F4 / 80 clone (a macrophage marker, Research Diagnostics, Inc). The slides were then washed, incubated for 2 hours in the corresponding Alexa 488- or TRITC-labeled secondary antibody (Molecular Probes, 0R) and washed three times for 5 minutes in PBS and mounted in Dect-containing Vectashield medium (Vector Labs, CA). The sections were analyzed with a Leica epifluorescence microscope connected to a Spot RT (Research Diagnostics) camera. The fluorescence intensity and the number of fluorescent cells within the selected region of interest (ROI) that delineated the region between the lamina propria and the ends of the villus, but eliminated the serous surface (hyperautofluorescence) and the enteric lumen from a total of Five fields of view in five separate sections were quantified for each animal using ImagePro software (Media Cybernectics, MD).

As shown in Table 21, a statistically significant dose-related effect of murine anti-integrin a2 treatment on reversal of weight loss and stool consistency associated with DSS feeding was observed. Both treatment groups (2 mg / kg and 5 mg / kg) had significant effects on rectal bleeding and colon bleeding, but had no significant effect on colon shortening associated with the development of colitis. Treatment was also correlated with a marked reduction in the number of infiltrating leukocytes (data not shown).

TABLE 21

<table> table see original document page 135 </column> </row> <table> * p <0.05 ** p <0.01

In another study, the effects of α4 anti-integrin (clone PS / 2; Southern Biotech, AL), α2 anti-integrin (Hal / 2 9 clone, BD Pharmingen, CA) and α1 anti-integrin ( clone Ha8 / 31; Invitrogen, CA) compared to mice treated with DSS alone (n = 8 per group).

Doses of antibody treatment were 5 mg / kg. These function blocking anti-integrin antibodies have been reported to modulate experimental colitis (Kriegelstein et al., J Clin Invest. 110 (12): 1773-82 (2002); Watanabe et al., Am J Physiol Gastrointestinal Liver Physiol.283 ( 6): G1379-87 (2002)). As shown in Table 22, the three anti-integrin antibodies were associated with a reversal in colon shortening, but only anti-α2 treatment was associated with significant improvement in stool consistency (diarrhea). Both anti-α2 and anti-a4 treatment resulted in significant improvement in colon bleeding. In this study, none of the antibody treatments induced a significant effect on weight loss. When resident T-cell, macrophage and neutrophil numbers were assessed by indirect immunofluorescence using anti-CD3, F4 / 80 and anti-Macl (as described above), the three anti-integrin-treated groups showed significant reduction compared to the saline control group, as shown in Table 23. These data support the conclusion that α2 antagonizing function has a profound effect on the steady-state levels of these immune effector cells that accumulate in the inflamed colon in response. DSS and that these changes are simultaneously related to an improvement in clinical evaluations associated with colitis.

TABLE 22

<table> table see original document page 136 </column> </row> <table>

* p <0.05 ** p <0.01 TABLE 23

<table> table see original document page 137 </column> </row> <table>

* p <0.05 ** p <0.01

EXAMPLE 7

The effects of anti-integrin a2 antibody were studied based on clinical signs and symptoms observed in a multiple sclerosis experimental allergic encephalomyelitis (EAE) model. The synthetic encephalogenic peptide injection-induced multiple sclerosis EAE model together with Freund's adjuvant in SJL mouse is considered a predictive model of relapsing-remission of multiple sclerosis (Encinas et al., J Neurosci Res.45 (6): 655-69 (1996)).

In a study of 4 groups of mice (8 / group; see Figure 1), two groups received 5mg / kg dose on Days 10, 11, 12, 14, 15, 18, and 20 with the control isotype or antibody. murine anti - integrin cx2, Hal / 29, from onset of disease symptoms to Day 20, which encompasses the first acute exacerbation. This was an acute scheme of administration. Day 0 was defined as the beginning of preparation with Freund's most adjuvanted synthetic peptide PLP139-151. Disease onset was defined as the second consecutive day of weight loss. The other two groups were assigned to a late administration schedule in which they received 5 mg / kg of murine anti-integrin a2 antibody or saline three times a week from Day 18 to Day 36, which should coincide with the second / first exacerbation. relapse. The mice were classified according to clinical signs and symptoms as follows: score 0.5 = 2 consecutive days of weight loss

score 1 = unstable tail score 2 = ataxia score 3 = hind limb paralysis score 4 = dying score 5 = death

As shown in Table 24, treatment with 5 mg / kg anti-a2 antibody since the onset of the first exacerbation (eg, acute treatment) reduced the extent of the first exacerbation and also limited the extent of the second exacerbation. When administration was initiated after the end of the first exacerbation on Day 18 (eg, late treatment; see Figure 1), mice treated with 5 mg / kg murine anti-integrin antibody 2 also had lower clinical scores during first relapse by Day 32. At this point, both groups had similar scores for the disease as shown in Table 24. When mice were treated only in the induction phase as shown in Figure 2, anti-integrin mAb ot2 had little or no effect on the first attack or subsequent phases of the EAE model and mice developed essentially the disease equivalent to that of animals treated with the control isotype. These results contrast with the results previously obtained with the use of the anti-a4 antibody PS / 2 in the EAE model (Yednock et al., Nature 356 (6364): 63-6 (1992); Theien et al., J Clin Invest Invest 107 (8): 995-1006 (2001)), used to support the treatment of multiple sclerosis relapse episodes with the anti-a4 natalizumab antibody (Miller et al., N. Engl. J. Med. 348 (1): 15-23 (2003)). These results show that unlike the function that integrin a4

unstable tail ataxia

dying hind limb paralysis

10

death plays in neuroinflammatory disorders, where antagonism of this receptor may delay the onset of exacerbation relapses associated with multiple sclerosis, a2 integrin antagonism is a useful treatment modality to reduce and treat the occurrence of exacerbations when they occur.

TABLE 24

<table> table see original document page 139 </column> </row> <table>

Histological examinations were performed on the brains and vertebral columns obtained from mice in

dying (stage 4) or at the end of the study. The mice were sacrificed with halothane in the dying state (clinical score 4), or after an observation period of 55-60 days. The animals were perfused with PBS, followed by a 4% paraformaldehyde solution. The brains were divided into five coronal sections, the vertebral columns into twelve transverse sections and the tissues were fixed in paraffin and the 4 μ thick sections were stained with Luxol blue for myelination visualization. The degrees of tissue myelination, infiltration (meningitis) and perivascular infiltrate were classified as blinded. For the classification of the spinal sections, each section was divided into quadrants: the anterior, posterior and each lateral funicular. Any quadrant containing meningitis, perivascular infiltrate or demyelination was classified with score 1 of the respective pathological class. The total number of positive quadrants in each class was determined, then divided by the total number of quadrants present in the slide and multiplied by 100 to determine the percentage of involvement for each pathological class. An overall classification for each pathological class was also determined by assigning a positive score if quadrant lesions were present.

Demyelinating inflammatory lesions were observed in the spine, most often in the posterior fascicular graciles fascicle and the emergence of the ventral root in the anterolateral funicular. Only mild perivascular infiltrate was observed. A strong correlation was observed between meningitis and demyelination with clinical signs (r = 0.84 and 0.79, respectively) and significantly lower scores were also observed in the anti-a2 group (P <0.01 between groups). anti-a2 and IgG control group for both parameters). These data indicate that anti-integrin α2 antibody treatment inhibits meningitis and demyelination associated with an exacerbation and / or facilitates remyelination and regeneration. The overall result is an improvement in clinical outcome.

Another study (n = 17 to 20 per group) was performed to compare a2 anti-integrin with a4 anti-integrin antibody PS / 2 (obtained from Southern Biotech), treatment during the first acute exacerbation until the onset of remission. (Days 10 to 20). Two additional groups were treated with IgG control or anti - integrin a2 from the beginning of remission (Day 18) to the first relapse (Day 36) and at the onset of the chronic phase of the disease. Again, treatment with anti-integrin cc.2 had a marked effect on the incidence of neurological sequelae (paralysis) and a statistically significant reduction in the mean maximum clinical score during the first EAE exacerbation and also in the subsequent phase of relapse ( chronic phase of EAE; Table 24). There was a small effect of late treatment improvement with anti-a2 on clinical scores during the chronic phase of the disease. The incidence of the disease during the first attack (anti-a2, 61%; IgG control 85%) and during the chronic phase (anti-oc2, 77%; IgG control 10%) was lower in the anti-a2 treated group. -a2 compared to the control group. In the case of the group treated with anti-a4, the incidence of the disease during the first attack (anti-a4 treatment, 94%; control 82%) and relapse (anti-a4 treatment, 89%; control 92%). ) was similar between the anti-a4 antibody treated group versus the control group. However, the extent of subsequent relapses has been markedly reduced. These anti-a4 antibody data are comparable to previous reports on the effect of anti-a4 antibody treatment on clinical outcome in EAE (Theien et al., J. Clin. Invest. 107 (8): 995-1006 ( 2001)).

EXAMPLE 8

The effects of platelet α2β1 integrin binding (α2βΐ is expressed on the platelet surface), including the effects on platelet function, were studied. Different sets of trials were performed to study these effects.

The first studies evaluated whether TMC-2206 binding causes platelet activation as measured by P-selectin over-regulation or platelet allba3 integrin activation that was measured using an allba3-specific activating antibody such as PAC-1. Using a 21-gauge needle, human venous blood was collected from the cubital vein from healthy donors who had not taken any medication for at least 10 days in a 1/10 volume of acidified citrate dextrose (ACD) buffer. : 85 mM sodium citrate, 111 mM dextrose, and 71 mM citric acid, without pH adjustment, containing 500 ng / ml prostaglandin I2 (PGI2, Sigma-Aldrich) if used to make washed platelets. Whole blood was centrifuged at 160xg for 20 minutes at room temperature and platelet rich plasma (PRP) was removed without breaking the buffer. Washed platelets were prepared by diluting 2.5 times PRP with glucose citrate saline buffer (CGS; 13 mM trisodium citrate, 120 mM sodium chloride and 30 mM dextrose, pH 7.0) buffer and PGI2 (500 ng / ml) and centrifugation at 160xgr for 20 minutes at room temperature to remove any contaminating leukocytes. The supernatant was collected and centrifuged at 100 xgr for 10 minutes and the resulting platelet pellet was lightly resuspended in CGS buffer, washed and resuspended in normal Tyrodes-Hepes buffer (12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl 10 mM HEPES, 1 mM CaCl 2, 1 mM MgCl 2, pH 7.4). Platelets recovered for up to 30 minutes at 37 ° C. Washed platelets were then counted prior to the addition of 1 mM CaCl2 and MgCl2. Washed rat platelets were prepared similarly, however blood was collected from the vena cava to minimize platelet activation during blood collection.

50 μΟ L of freshly prepared PRP were incubated with μg / mL of TMC-2206 or IgG control antibody for 30 minutes, washed and incubated with Alexa-594-labeled goat anti-mouse in the presence or absence of P-labeled selectin antibody. Alexa 488 (BD Pharmingen, # 555523), Alexa-488-labeled PAC-I antibody (BD Pharmingen, No. 340507) or 150 ng of Alexa-488-labeled fibrinogen (Molecular Probes) for 40 minutes at room temperature. P-selectin and PAC-1 are markers of platelet activation and activated platelets are capable of binding to fibrinogen. At the end of the incubation period, platelets were fixed in 1/10 volume of 4% paraformaldehyde and analyzed using the FACScalibur ™ flow cytometer. The results of these experiments unexpectedly showed that although platelets clearly bound TMC-2206, as indicated by the Iog shift in the increase in fluorescence intensity observed in platelets treated with TMC-2206, but not with platelets treated with the IgG control; There was no concomitant increase in P-selectin or PACl staining, indicating that TMC-2206 binding did not activate platelets.

The following studies evaluated whether TMC-2206 binding causes platelet activation as measured by the effects on collagen-induced platelet aggregation. Soluble collagen is a potent platelet aggregation agonist and is routinely used to assess platelet responsiveness (see eg, Hemostasis and Thrombosis; (2001), ed. Colman et al). Studies with a2 deletion in mice have suggested that platelets of these mice exhibit mild impairment of collagen response (Holtkotter et al, J. Biol. Chem. 277 (13): 10789-94 (2002) E. pub Jan, 11, Chen et al., Am. J. Pathol. 161 (1): 337-344 (2002)). To assess whether TMC-2206 would have any adverse effects on platelet response to collagen, human and rat platelet aggregation assays were performed using conventional optical aggregometry using the Bio-data PAR4 aggregometer. PRP was prepared as described above and the platelet count was adjusted to 3x10 8 / ml. 450 / µL PRP or washed platelets were mixed with a magnetic bead for 1 minute at 37 ° C in the presence of 5 µg / mL TMC-2206 prior to the addition of calf skin Type I collagen (Biodata Corp) to initiate the aggregation. The final volume of 500 μL was composed of Tyrodes buffer for washed platelet aggregation and platelet poor plasma (PPP) to platelet rich plasma (PRP) assays. 500 µL of Tyrodes or PPP buffer were used as blanks for the assays. PPP was prepared by pelleting the platelets in PRP with centrifugation at 3000 rpm for 5 minutes in a microfuge. The rate and extent of platelet aggregation after the addition of soluble collagen was comparable in the presence or absence of TMC-2206. The results of these experiments demonstrated that the binding of TMC-2206 to platelets unexpectedly had no effect on platelet aggregation. collagen in in vitro tests at the concentrations tested.

The following studies evaluated whether TMC-22 06 binding causes thrombocytopenia, a potential consequence of antibody binding to platelets in vivo. (Hansen and Balthasar, J. Pharmacol Exp Ther. 298 (1): 165-71 (2001)). To assess whether thrombocytopenia would occur with TMC-2206 administration, the rats received a 10 mg / kg dose of TMC-2206 or murine IgG control by IP injection. Prior to injection, tail blood was used for baseline cell counts. Blood samples were collected at specific timepoints following IP administration of the drug (eg, 10, 30, 60 minutes, and 4, 24, and 72 hours) from unaesthetized rats by retroorbital puncture using a Unopipet capillary. Then approximately 40 μL. of blood were transferred to a tube containing 5 μL. ACD and immediately analyzed on a Hemavet blood cell counter (Drew Scientific). The results of this study unexpectedly showed no significant change from baseline platelet counts at doses of 5 mg / kg or 10 mg / kg TMC-2206. In contrast, 0.1 mg / kg injection of an antibody to another platelet receptor (αIIb, anti-CD41 antibody (BD Pharmingen, CA)) induced thrombocytopenia, with a platelet count decline of almost 80% within 15 minutes. administration of the antibody.

EXAMPLE 9

The effects of anti-integrin a2 antibodies on platelet adhesion to collagen, including adhesion to various collagen subtypes, were studied. Α2β1 integrin is the only collagen-binding integrin, although it is not the only collagen receptor expressed by platelets. However, as discussed above, there are other mechanisms, especially through platelet activation, which facilitate firm adhesion to the collagen matrix. In this example, the ability of the TMC-2206 antibody to block platelet adhesion to Type I, II, III, IV and VI collagen was evaluated for both inactive and activated platelets with the moderate ADP platelet agonist.

Collagen Type I, II, III, VI (Rockland Immunochemical) and IV (Sigma, St. Louis, MO) collagen coated platelets, which were solubilized without foam in 5 mM acetic acid to a final concentration of 1 mg / ml.

The wells were washed twice using Ca ++ or BSA modified Tyrode-HEPES buffer but with 2 mM / Mg ++ and blocked with 100 µL / well Tyrode-HEPES buffer containing 2 mM / Mg ++ and 0.35% BSA, but without Ca ++ '

Human venous blood was used for the preparation of platelets, including PRP, as described above in Example 8. Platelet poor plasma (PPP) was produced by centrifuging PRP at 1100 χ g for 10 minutes at room temperature. The resulting platelet pellet was carefully resuspended for labeling in 1.0 mL CGS (13 mM trisodium citrate, 120 mM sodium chloride and 30 mM dextrose pH 7.0, transferred to a 5 mL round bottom tube and 3 μL CFSE stock (final concentration 53.7 μΜ) were added with gentle stirring for exactly 20 minutes.The labeled platelets were diluted in CGS buffer and washed.The platelet pellet was resuspended in 1 mL CMFTH buffer (5 mM HEPES, pH 7 , 12 mM sodium bicarbonate, 137 mM NaCl, 3 mM KCl, 0.3 mM NaH 2 PO 4, 5 mM dextrose and 0.35% BSA) and kept in the dark for as long as possible. similarly, although blood was collected from the vena cava in a syringe containing 500 ng / mL PGE1 in ACD to minimize platelet activation during blood collection CFSE-labeled platelets were diluted to 2. OxlO5 / xL using Tyrode-HEPES buffer containing 0.35% BS A. The labeled platelets (1. Ox107 wells) were applied to wells containing 20 xM ADP in Tyrode-HEPES buffer with 0.35% BSA and varying concentrations of the inhibitor under test. Microtiter plates containing platelet mixtures were centrifuged at 550 x g for 10 minutes at room temperature and then incubated in the dark for an additional 10 minutes. The wells were washed with Tyrode-HEPES buffer. Fluorescence was assessed using a Victor2 fluorescent plate reader. To determine the relationship of fluorescence intensity versus platelet number, labeled platelets were diluted to varying degrees in Tyrode-HEPES buffer containing 0.35% BSA (without Ca ++ or ADP) applied to Type I or Type collagen-coated wells. IV, centrifuged, and CFSE fluorescence measurements were performed. As shown in Table 25, the TMC-2206 blocked collagen binding under these static conditions, with an EC50 of 1.7 nM. Similar studies were performed with rat platelets using TMC-2206. EC50 values for inhibition of rat platelets in Type I rat collagen were 6.3 nM indicating an approximately 5-fold shift in rat α2β1 affinity compared to humans on platelets. TABLE 25

<table> table see original document page 147 </column> </row> <table>

As shown in Table 25, TMC-2206 was a potent inhibitor of platelet adhesion to fibrillar collagen, but was less potent for Type IV non-fibrillar collagen (10 nM compared to 1-2 nM). In the presence of ADP, a 10 to 20-fold reduction in the potency of fibrillar collagen binding inhibition was unexpectedly observed and the antibody was no longer effective in preventing adherence to Type IV collagen. These unexpected observations suggest that the. TMC-2206 and antibodies with epitope with the binding specificity of TMC-2206 are less active in inhibiting fibrillar collagen-activated platelet interactions and would have little or no effect [on the therapeutic dose range] on Type IV collagen binding. , the predominant collagen subtype in the endothelial vessel wall.

EXAMPLE 10

The effects of anti-integrin a2 antibodies on bleeding time were studied. Qualified practitioners expect that administration of an antibody against platelet integrin may cause bleeding complications and lead to increased clotting time in a patient treated with this antibody following an acute injury. To assess whether antibodies directed against α2 integrin would increase the propensity for bleeding in vivo, the effect of TMC-2206 on bleeding time in rats was determined. Rats received an IP or IR injection of TMC-2206 15 minutes before the bleeding time test. Unanesthetized rats were immobilized in a restraining apparatus and 0.8 cm from the tail tip was cut rapidly to initiate bleeding. The tail was immediately placed in a beaker containing 30 mL of PBS and kept at 37 ° C. The time required for tail bleeding to stop was recorded as bleeding time. As shown in Table 26, data showed that administration of TMC-2206 doses up to 10 mg / kg had no significant effect on bleeding time.

TABLE 26

<table> table see original document page 148 </column> </row> <table>

EXAMPLE 11

The effects of anti-integrin a2 antibodies were studied in an arterial thrombosis model. Another potential manifestation of a bleeding disorder caused by the administration of platelet-reactive α2β1 antibodies could be an increase in time to thrombotic occlusion that occurs following acute arterial injury due to undesirable effects of platelet function. Thus, anti-integrin a.2 antibodies such as TMC-22 06 were tested in a model of ferric chloride-induced rat arterial thrombosis. This is a standard model that has been used for the development of antithrombotic agents and the activity is manifested as a delay in occlusion time after exposure of the blood vessel endothelial bed to a FeCl3 solution (Kurz et al., Thromb Res 60 (4): 269-80 (1990); Hoekstra et al., J Med Chem. 42 (25): 5254-65 (1999)).

TMC-2206 antibody was administered to rats by injection into the tail vein approximately 30 minutes before induction of arterial injury at doses ranging from 1 mg / kg to 15 mg / kg. For IV injections, most antibodies were concentrated at 4-5 mg / mL to reduce injection volumes to the highest doses. Treatment groups were 1.0, 2.5, 5.0, 10.0 and 15 mg / kg TMC-2206, 5.0 mg / kg murine control IgGI (k) (MOPC21 clone) 5.0 mg / kg polyclonal rabbit anti-vWF (DAKO) or saline solution; Each treatment group consisted of 3-4 animals.

Sprague-Dawley (Harlan) rats weighing 220-270 grams were anesthetized with 60 mg / kg sodium pentobarbital. Once sufficient anesthesia was achieved their carotid artery was exposed and placed on a piece of filter paper (4 mm χ 5 mm) that was bent on the 4 mm side to accommodate the carotid artery and provide a surface for the chloride ferric (35%) involve the carotid artery. Twelve μL 35% FeCl3 was applied for 5 minutes and then the paper filter was removed and the Transonic Systems Inc. flow probe (Ithaca, NY) was placed around the carotid artery. Flow was measured for up to 45 minutes.

Mean values and SBM for flow rates of various animals per group were recorded at specific timepoints after administration of ferric chloride. No significant differences in time to occlusion were observed with any of the TMC-2206 doses tested, even at the high dose of 15 mg / kg compared to saline control. This indicates that there appear to be no adverse effects on thrombosis due to TMC-2206 administration. Although initial flow values may vary substantially between animals, time to occlusion consistently occurred between 10 and 16 minutes after administration of ferric chloride in the TMC-2206 treated groups. This time was very similar to that found in the saline-treated and control IgG groups, in which the average occlusion times were 12 and 14 minutes, respectively. The only treatment tested that was associated with occlusion prevention was positive control, a polyclonal anti-vWf antibody, which resulted in no reduction in flow parameters for up to 45 minutes after FeCl3 addition. EXAMPLE 12

The binding properties of anti-α2 integrin antibodies were studied, including epitope mapping study, to characterize the nature of the TMC-2206 binding site in the α2 integrin subunit. An α2 integrin antibody that binds directly to the target binding site and serves as a direct competitor for the binding ligand can be expected to cause platelet activation with α2α1 integrin binding. On the other hand, an anti-integrin cx2 antibody that binds to the inactive state α2β1 integrin and does not promote integrin activation may have a platelet non-activation profile similar to that unexpectedly observed for TMC-2206. Antibodies with the same or similar binding epitope as TMC-2206 would inhibit leukocyte cell adhesion to collagen and thus have significant therapeutic utility, but would not be associated with bleeding complications that an antibody that has bound and activated an α2β1 integrin may have. Studies have been conducted to investigate whether the epitope recognized by TMC-2206 is in domain I of α2 integrin subunit ligand binding or whether it simply depends on the presence of an intact domain I (Hangan et al., Cancer Res. 56: 3142- 3149 (1996)). For these studies, a GST-a.2 I domain I fusion protein was produced using a modified version of the protocol described by Tuckwell et al., J. Cell Sci. 108 (Pt 4): 1629-37 (1995). The human α2 domain I was cloned from the isolated raRNA of approximately 106 CHO cells expressing human α2 integrin (Symington et al., J Cell Biol. 120 (2): 523-35. (1993). lysates in Trizol reagent (Gibco) and chloroform was added for extraction of the aqueous phase prior to the addition of 0.2 volumes of isopropanol to precipitate RNA which was collected by centrifugation and resuspended in water without RNAse.

Human a.2 domain I side primers were synthesized by Sigma-Genosys. Primers were engineered with BamHI and EcoRI sites at the 5 'and 3' ends, respectively, for cloning into the pGEX-2TK vector (GE Biosciences). The halphal primers F

(5'GGGGATCCAGTCCTGATTTTCAGCTCTCAG; SEQ ID NO: 117) and halphal R (5'GGGAATTCAACAGTACCTTCAATGCTG; SEQ ID NO: 118) (see Table 27) were used for a one-step RT-PCR reaction using a standard kit Qiagen to amplify amino acids 123 to 346 of the mature α2 integrin subunit and incorporate an amino terminal BamHI site (which adds a GS at the 5 end of domain I residue 124) and an additional hexapeptide EFIVTD as part of the KcoRI cloning site via of the final codon. A single band was detected by agarose gel electrophoresis. The PCR reaction was performed using the Qiagen PCR Quic kit, the restriction enzyme digested product and cloned into the pGEX-2TK vector (Amersham, GE) using standard molecular biology techniques. Transformed bacteria were analyzed for insertion detection and several sequenced clones using a Beckman-Coulter CEQ system. The deduced amino acid sequence as cloned was identical to the sequence available from a human a2 domain I (SEQ ID NO: 11, shown in Table 28). A single clone containing the correct DNA insertion was amplified in DH5a cells (Invitrogen) and transformed back into BL21 (Invitrogen) electrocompetent bacteria. TABLE 2 7

<table> table see original document page 152 </column> </row> <table> <table> table see original document page 153 </column> </row> <table> <table> table see original document page 154 < / column> </row> <table>

The human o 1 domain GST fusion protein was expressed in BL21 log growth bacteria using IPTG as an inducing agent. Approximately 4 hours after induction, the bacteria were harvested and pelleted at 3000 RPM in 50 mL conical tubes. The pellet was resuspended in PBS containing 1% Triton X-100 and protease inhibitors. The homogenate was sonicated for 1 minute and centrifuged at 3000 RPM to remove cell debris from the lysate. GST fusion protein was purified from bacterial lysates using glutathione-Sepharose resin (GE-Amersham) according to the manufacturer's instructions and eluted in TBS (pH 8.0) containing 20 mM free glutathione. Purified GST-α.2 I domain I bound to collagen with the same specificity as previously reported (Tuckwell et al., J Cell Sci. 108 (Pt 4): 1629-37 (1995)), ie, a higher affinity for Type I collagen compared to Type IV collagen. It also bound to immobilized TMC-22 06 with an apparent Kd assessed by the 0.31 nM ELISA, which was comparable to the observed affinity of binding TMC-2206 to the intact 0.37 nM α2β1 integrin obtained from the studies of Direct binding described in Example 2. The stability of the soluble GST-α2 domain I fusion protein to compete with Eu-labeled TMC-2206 for binding to α2β1 coated plates was then evaluated as described in Example 2. The Ki value for domain I of soluble GST-a2 was found to be similar (0.18 nM compared to 0.28 nM) to that obtained with unlabeled TMC-2 2 6, indicating that the binding site for TMC-2206 accommodates within a2 domain I and did not require the presence of a βΐ subunit Studies have been conducted to investigate the binding cation dependence on TMC-2206. Cation dependence indicates that the ligand moiety targets the divalent binding site Integrating cation (MIDAS) cation and therefore acts as a mimetic ligand. Α2-binding collagen is Mg ++ dependent under normal physiological conditions whereas binding does not occur when Mg ++ is replaced by Ca ++ (Staatz et al., Cell Biol. 108 (5): 1917-24 (1989); Emsley et al., Cell 101 (1): 47-56 (2000)). For these studies, the GST-a2 I domain fusion protein was immobilized on glutathione-coated Reacti-Bind microtiter plates (Pierce Biotechnology, Inc. Rockford, IL) and the ability of the labeled I-TMC-22 06 was determined. bind under different cation conditions (Ca- and without Mg, Ca ++ or Mg ++ at concentrations ranging from 0.1 μΜ to 3 mM). The plates were coated by incubating 100 µL / well of GST-a2I fusion protein (2.0 µg / ml divalent cation-free binding buffer: 50 mM HEPES, pH 7.4, 150 mM NaCl and 0.5% Tween-20 ) for 1 hour at room temperature, and the wells were washed four times in divalent cation-free wash buffer. The wells were blocked using 100 µL / well blocker buffer (wash buffer containing 3.0 mg / ml IgG-free BSA [Jackson ImmunoResearch Laboratories, Inc., West Grove, PA]) for 1 hour at room temperature, washed four times in cation-free divalent wash buffer and dipped in cation-free divalent wash buffer (300 μL / well) for 45 minutes at room temperature. The wells were equilibrated in wash buffer (300 µL / well) containing the desired level of divalent cations for 30 minutes and then incubated for 1 hour at 37 ° C in the presence of 41 pM, 199 pM 345 pM or 1 nM TMC-. 22 06 labeled with Eu or control antibody. Murine antibody TMC-2206 bound in a concentration-dependent manner with similar potency under all conditions, indicating that its binding to the α2 I domain is cation-independent and therefore did not involve the MIDAS site. Eu-labeled IgG control did not bind to α2βΐ integrin-coated wells confirming that binding was specific.

Additional studies were conducted to investigate the TMC-2206 binding site. Integrin ligands usually have a key acid that forms the final chelating bond for the divalent metal ion (Haas and Plow, Curr. Opin. Cell. Bio. 1994; Lee et al., Structure 1955), a feature shared by many antagonists. integrin, including the anti-integrin al integrin mAb, AQC2 (Karpusas et al., J. Mol. Biol. 2,003) in which the acid is provided by the residue D101 in CDR-H3. By analogy, the TMC-2206 CDR-H3's D00 could provide this kind of interaction with a2 MIDAS. Therefore two murine VH-containing variant antibodies were produced, one with a DOOA mutation and one with a DLOOR mutation. The ability to compete for the Eu-TMC-2206 binding was then assessed in the Ki assay compared to the mouse-human chimeric antibody TMC-2206. The D100A mutant was completely inactive at concentrations up to 0.9 μΜ, which represented a relative power shift greater than 1600 times compared to the chimeric mouse-human antibody TMC-2206. In contrast, the reverse charge D100R mutant was nearly as potent as the mouse-human chimeric antibody TMC-2206, as evidenced by similar Ki values (0.41 nM compared to 0.52 nM). This fact provides evidence against any function of the TMC-2206 D100 residue to couple to the metal chelation complex that forms the MIDAS binding site. Additional studies were conducted to investigate the binding specificity of TMC-2206, including epitope mapping studies, with this murine monoclonal antibody that is directed against human α2β1 integrin domain I. TMC-22 06 cross reacts with mouse α2β1 integrin, but does not cross react with mouse α2β1 integrin. As α2β1 integrin proteins share high homology between species, residues within α2β1 that are important for antibody binding were identified using methods capable of identifying the differences between those species that cross-reacted with the antibody compared. with those who do not (eg, Champe et al., J. Biol. Chem. 270 (3): 1388-94 (1995); Karpusas et al., J. Mol. Biol. 327 (5): 1031- 41 (2003); Bonnefoy et al., Blood 101 (4): 1375-83). The crystal structure I of the Î ± 2 integrin domain was analyzed alone and when complexed with its target ligand, collagen (Emsley et al., J. Biol. Chem. 272 (45): 28512-7 (1997); Emsley et al., Cell 101 (1): 47-56 (2000)). The sequence comparison of human a2 I domains (SEQ ID NO: 11), rat α2 I domains (SEQ ID NO: 93), and mouse ct2 I (SEQ ID NO: 94), obtained from Genbank are shown in Table 28. This analysis reveals that mouse domain I contains 14 residues that differ from rat and human a2 domains I (shown in bold and underlined in Table 28). These residues were used to further study the TMC-2206 binding epitope. TABLE 2 8

<table> table see original document page 158 </column> </row> <table>

GST-a2 I domain I from mice and rats were cloned as GST fusion proteins to confirm that the appropriate cross reactivity was maintained by the respective I domains and the PCR methodology described in Example 3 was used. murine a2 was cloned from isolated mouse kidney Balb / C mRNA by RT-PCR using the malphal F (SEQ ID NO: 121) and malphal R (SEQ ID NO: 122) primers and the I domain of a2 of the Sprague Dawley rat kidney by RT-PCR using the ralphal F (SEQ ID NO: 123) and ralphal R (SEQ ID NO: 124) primers. In addition, α2 domains from two non-human primates were cloned from leukocyte pellets obtained by low speed centrifugation of fresh blood collected from rhesus and cynomolgus monkeys. The leukocytes were then frozen in liquid nitrogen. In total, 5 χ106 (rhesus) and 2 χ106 (ciynomolgus) cells were lysed in 1 mL Trizol (Invitrogen, Cat # 15596-026) and total RNA was prepared as described above. 0 RNA pellet

The final solution was resuspended in 50 μΐ H2O-treated with DEPC. This preparation served as a model for the first step of reverse transcriptase (RT). The RT reaction consisted of 8 μl (2.24 μg for Rhesus mRNA, 1.44 μg for cynomolgus mRNA) of cellular RNA, 1 μΐ (10 mM) of DNTPs and 1 μl (2 μΜ) of primer forward. human domain I (GGGGATCCAGTCCTGATTT; SEQ ID NO: 119). This mixture was incubated at 650 ° C for 5 min, cooled on ice. Five μΐ of this cDNA was then used as a template for PCR amplification using forward and reverse primers (Forward: GGGGATCCAGTCCTGATTT, SEQ ID NO: 119; Reverse: GGAATTCAACAGTACCTT, SEQ ID NO: 120). Cycle times were: 1 cycle at 94 ° C for 30 s, 94 ° C for 30 s., 55 ° C for 30 s, 40 cycles at 68 ° C for 1 min. and 1 cycle at 68 ° C for 5 min. The PCR products were separated by 1% agarose gel electrophoresis and the band (of the expected size) was purified directly from the BamliI and EcoRI digested agarose gel and cloned into the same sites in the pGEX-2TK vector and transformed into bacteria. BL21.

Single colonies were isolated and the inserts sequenced using the CEQ 8000 DNA analyzer to verify the identity of murine and rat a2 domain I and to determine the sequence homology of two monkey species with human. The cloned murine sequence showed exact identity to the domain I region of the deposited sequence, NM_008396.1. Similarly, the cloned rat sequence was identical to the Genbank deposited rat integrin, XM_34156.1, except that the cloned sequence contained 6 additional residues compared to the deposited sequence, which allowed the region between the 16 and 16 residues. and 21 (intact domain Î ± 2 integrin residues 139 to 144) of the rat domain be accurately translated. The amino acid sequence, ACPSLV, was identical to mouse residues at these positions.

At the nucleotide level, the two primate sequences showed very high homology to the human a2 domain I sequences. The a2 domain I of rhesus monkeys in the nucleotide sequence (SEQ ID NO: 104) showed only a difference in the human nucleotide sequence at codon 50, a change from CTT to CTG, but as both encode a leucine, the sequences of deduced proteins were identical to human ones. The α2 domain I of cynomolgus monkeys in the nucleotide sequence (SEQ ID NO: 103) was identical to human, except for codon 40, in which a change from AAG to human GAC was observed. This results in the change of a lysine residue to aspartic acid at this position. However, other studies have shown that this change in nucleotide occurs due to a polymorphism and that this does not occur in all animals, as other cynomolgus monkeys exhibited 100% homology to human a2 domain I (see Example 18). .

The fusion proteins were then expressed and purified according to the above description referring to the human GST-a2 domain I fusion protein. Analysis of the material extracted from the glutathione-Sepharose column indicated that rodent fusion proteins contained aggregate forms. Therefore, these together with primate fusion proteins were further purified by size exclusion chromatography on a sefadex 75 10/30 (GE-Amersham) (primate) column by FPLC on an Akta-Basic FPLC system (GE-Amersham ) to obtain a monomer fraction. Next, the ability of GST fusion proteins to bind to immobilized TMC-2206 and to compete with Eu- labeled TMC-2206 for binding to a human immobilized α2βΐ integrin was evaluated. K ± assays were performed as described in Example 2. To assess direct binding to TMC-2206, Immulon 4 plates were coated with 50 mL of a bicarbonate solution (pH 9.0) containing 5 Mg / mL TMC-2206. 2206. The plates were sealed and coating occurred overnight at 4 ° C. The next morning, the plates were washed twice with TBS solution and blocked using 200 μL of blocking solution as described above for 1 hour at room temperature with shaking. After blocking, the blocking solution was removed, but the alveoli were not washed. Instead, serial dilution of the GST fusion protein was added to the wells and incubated for 2 hours at room temperature with shaking. Then, the wells were aspirated and a TBS wash buffer was applied for 5 minutes at room temperature. The wash step was repeated twice more before secondary antibodies were applied. The secondary antibody application step consisted of an Amersham rabbit anti-GST HRP-conjugated antibody diluted in 1: 2000 blocking buffer. One hundred μL. Secondary antibodies were added to each well and incubated at room temperature for 1.5 hours with shaking. The wells were again aspirated and washed three times with wash buffer before adding the substrate mixture for reaction. One hundred μL of the reaction substrate mixture (1: 1 dilution of the TMB kit) was then added to each well for 6 minutes. The reaction was stopped with the addition of 100 mL of 0.1 M H2SO4. Then, the reactions in the alveoli were evaluated and quantified by spectrophotometric absorption using the molecular dynamics reader and associated software Softmax, respectively. Kd values were then calculated based on EC50 values using Prisma software (Graphpad, CA).

Kd 3-fold shift in rat α2 binding to TMC-2206 compared to human α2 was observed, whereas murine α2-I-GST fusion protein showed only slightly specific binding at the highest concentration, representing a shift in greater than 1500 times in affinity (see Table 29). GST-α2 I of rhesus monkeys showed comparable affinity to humans, while GST-α2 I of cynomolgus monkeys unexpectedly showed detectable affinity for TMC-2206 at concentrations up to 1 μΜa. These relative classifications were also observed in the Ki assay. The absence of cross-reactivity of domain I of the GST fusion protein of cynomolgus monkeys by TMC-2206 indicates that the K40 residue may be an epitope determinant. The difference in affinity of cloned rat GST-α2 I (Kd 0.54 nM and Ki value 3.8 nM) compared to GST-human fusion protein α2 I (Kd 0.18 and 0.33 nM Ki) is compatible with deviations in EC50 values found in assays performed to assess the ability of TMC-2206 to antagonize adhesion of fresh rat platelets compared to human platelets to Type I collagen as described above. similarly the absence of cross-reactivity of mouse α2 I GST fusion protein by TMC-2206 is consistent with the lack of cross-reactivity of the antibody with intact α2β1 mouse integrin.

TABLE 29

<table> table see original document page 162 </column> </row> <table>

* ND means not detectable at concentrations up to approximately 1 μΜ

In other studies, the 14 residues corresponding to the unique differences in murine α2 I domain I compared to human and rat α2 domains I were mutated individually in the a2 domain I fusion protein cloned by PCR with the use of standard molecular biology methods (primer sequences are shown in Table 30). Individual bacterial clones were sequenced to verify that the correct mutation had been incorporated into domain I. A tested variant, the mutant G101R did not produce a correct clone and was no longer studied. Primers designed to create the Y93H mutation resulted in a set of clones with the Y93D mutation instead of the previous one. Both variants Y93 were evaluated. The others were correct in the sequence. The fusion proteins were then expressed and purified according to the above description referring to the human GST-a2 domain I fusion protein. Their activity was then tested in three ways: first, their relative ability to bind to different collagens to ensure that mutations did not induce conformation disturbances that could interfere with ligand binding; secondly, the apparent affinity for the TMC-2206 (direct binding by the immobilized TMC-2206 evaluated by the ELISA assay) and thirdly, its ability to act as competitive ligands in the Ki assay. Data regarding Ki values and apparent Kd values are also summarized in Table 30.

TABLE 30

<table> table see original document page 163 </column> </row> <table> <table> table see original document page 164 </column> </row> <table>

* ND = not detectable at concentrations up to approximately 1 μΜ

Of the 13 residues evaluated, 12 shifted to the murine counterpart with little effect on affinity, but changes in Y93 caused marked loss of affinity as shown in Table 31. The Y93D mutation abolished antigen binding capacity even at concentrations 3. -Iogs above the κ d value for the I wt domain GST fusion protein. The change in murine histidine (Y93H) caused a 23-fold reduction in apparent affinity for the TMC-2206 antigen. Both mutations abolished the ability of the domain I GST to antagonize the eu-labeled antibody. The change in murine H93 to a Y conferred the ability of the murine a2 domain I to bind to TMC-226, albeit with a reduced 200 times the relative potency for the human w2 domain I wt, as shown in Table 31.

TABLE 31

<table> table see original document page 164 </column> </row> <table> <table> table see original document page 165 </column> </row> <table>

Comparison of the crystal structures for the ligand-bound (NCBI PDB inlet 1A0X) and open, ligand-bound (PDB inlet 1DZI) conformation ct2 I domain reveals that Y93 is located on the face of the domain behind the helix A1, which shows a large downward movement when ligand binding occurs (Emsley et al., J. Biol. Chem. 272: 28512 (1997) and Cell 100: 47 (2000).) Although not previously identified as a change In conformation associated with ligand binding, examination of crystal structures indicates that in closed conformation, the aromatic ring Y93 extends off the surface of the protein, moves laterally and downward to align along the face of domain I in the open conformation of the bound ligand To investigate whether the binding of TMC-2206 to α2βΐ integrin depends on a particular conformation, mutations were introduced in domain I in favor of the open conformation of domain I. Fo The E195W mutation (E318 in intact cx2 integrin) has been reported to lock human a2 domain I into open conformation (AquiIina et al., Eur. J. Biochem. 269 (4): 1136-44 (2002)), therefore, its use allows to observe whether the antibody recognizes activation-dependent confirmation or not. Crystallographic studies have also shown that E195 forms a salt bridge with residue R165 located in aC Ioopl that serves to hold aC Ioop in a conformation that protects the ligand binding site (Emsley et al., Cell 100: 47 ( 2000)). Ioop BC assumes an extended conformation in the open position and both residue R165 and adjacent residue R166 have been postulated to contribute to collagen binding (Emsley et al., J Biol Chem 272: 28512 (1997) and Cell 100: 47 (2000); Kâpylà et al., J Biol Chem 275: 3348 (2000)). Therefore, four mutations were constructed, the E195W; an R165D mutation to reverse the charge and thereby break the salt bridge forming the E195W in the closed conformation, and an N166D mutation also to reverse the charge on the aC helix. The change in E195W caused a 45-fold reduction in Ki values as shown in Table 31 indicating that the TMC-2206 antibody has higher affinity for closed conformation. Changes in R165D and N166D abolished the domain's ability to bind to the TMC-22 06 epitope, even at concentrations up to 1 μΜ, again suggesting that the TMC-2206 antibody recognizes closed conformation. Based on mutagenesis and conformation studies, Y93 in closed conformation appears to play a role in the binding of TMC-2206 and may provide a determinant for species specific binding. Unexpected results obtained with cynomolgus polymorphic domain I indicated that residue K40 may also play a role in antigen - TMC-2206 interaction. Computer models of the TMC-2206 antibody indicated that the CDRs form a relatively flat binding site, suggesting that the antibody makes multiple contacts with the antigen. Since the residues in the CDRs are charged, the charged residues surrounding Y93 in the closed position which also exhibit marked position changes in the open conformation were identified from the PDB structures in both open and closed conformations as K40, R69, Ν73 and Q89 The charges from three of these residues were reversed in that generation of the following K40D, R69D and N73D mutants and modified in the fourth by generation of a Q8 9H variant, as shown in Table 31. In addition, a third variant of residue 93 was obtained, a change from tyrosine to phenylalanine, to determine whether the aromatic character of tyrosine was the important structural characteristic, or whether the activity was dependent on the aromatic hydroxyl character, which is the characteristic of tyrosine. In the case of this set of mutations, all were subjected to HPLC purification to enrich for the monomeric fraction of protein preparations obtained outside the glutathione-seraphose affinity column. Each variant was first tested for functionality by assessing collagen binding. All but the R69D variant bound to collagen with an EC50 value similar to the human α2 I domain wt. Consequently, R69D has not been further studied. Of the remaining mutants, the introduction of the K40D variant abolished the ability to compete for TMC-2206 epitope binding. This was consistent with the results obtained with the cloned cynomolgus domain I which showed polymorphism in this residue (lysine change to aspartic acid). Similarly, the Y93F mutation also abolished the ability to compete for binding to EU-TMC-2206. N73D and Q89H showed a reduction of 7.8 and 15.9 times the Kif values respectively (Table 30). Taken together, the mutation data indicate that residues K40, Y93, R165 and N166 may be determinant for the binding of TMC-2206 to its epitope, and that N73 and Q89 also contribute energy to the binding.

These data indicate that the TMC-2206 antibody, its derivatives and antibodies such as TMC-2206 (ex.AK7) which recognize the same or similar epitope as TMC-2206, (see, eg, Example 13) are antagonists. non-mimetic atypical collagen-a2pi interactions ligand. This conclusion is based on: i) its ability to block α2β1 integrin-mediated collagen adhesion in a divalently cation-independent manner, ii) its inhibition does not involve the interaction of a critical group, such as DOO within H-CDR3, with MIDAS, iii) the antibody binds to the surface of domain I which is distal to the direct ligand binding site, iv) the TMC-2206 binding site favors closed conformation of the receptor and encompasses amino acid residues K40, N73, Q89, Y93, R165 and N166. Therefore, binding of TMC-2206 and antibodies such as TMC-2206 (eg, which recognize the same or similar epitope as TMC-2206) will not carry the integrin-mediated outside-in signaling that would normally occur with use. cognate ligand, and it is this mode of binding that can contribute to the non-bleeding profile of this antibody and antibodies such as TMC-2206. EXAMPLE 13

Studies were performed to compare the binding of other function-blocking anti-integrin cc2 antibodies to TMC-2206. The results of the mapping studies as described in Example 12 indicate that the TMC-2206 antibody appeared to bind to a closed conformation of the Î ± 2 integrin domain I and / or did not act as a ligand mimetic. These expected results, together with the unexpected results from the platelet-related studies described in Examples 8, 9, 10 and 11, demonstrated that the TMC-2206 epitope is particularly advantageous and that antibodies with functional properties similar to those of TMC-2206 are particularly useful. . Screening methods have been developed for the identification of these similar antibodies and antibodies have been identified by these methods.

To determine which function blocking anti-integrin α2 antibodies bound similarly to TMC-2206, a series of cross-competition studies were performed. For commercially available human anti-integrin α2 antibody studies, the human fusion protein GST-α2 I was immobilized on microtiter plates as described above. The antibodies tested were 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)). ) commercially distributed by Chemicon, (Temecula, CA; catalog number, CBL477 (AK7); MAB1950 (P1E6); MAB1988 (IOG11) and Upstate (Waltham, MA; A2-IIE10, catalog number 05-227) , respectively). Antibodies were tested together with the same batch of α2α1 coated microtiter plates used in epitope mapping studies to assess its ability to antagonize the binding of Eu-labeled TMC-2206. In another set of studies, it was evaluated. the ability of antibodies to antagonize the binding of isolated fresh inactive platelets to Type I collagen. Thus, the ability of different antibodies directed against human α2 integrin to antagonize the binding of eu-labeled TMC-2206 antibody to α2β1 coated microtiter plates Platelet counts were evaluated with Ki values and the ability to antagonize adhesion of inactive platelets to Type I collagen under static conditions were evaluated based on EC50 values. The results, shown in Table 32, demonstrate that AK7 antibody is an effective competitor of TMC-2206. Clone 10G11 showed clear biphasic competition from TMC-2206, suggesting that it did not act as a single competitive agonist. A2-IIE10 showed a 10-fold deviation from TMC-2206 in platelet adhesion blockade, but approximately 350-fold deviation in its ability to compete with Eu-labeled TMC-22 06 again indicating no agreement between the two antibodies. Ρ1Ε6 had no effect on any of the tests, indicating that it recognizes an activated conformation.

TABLE 32

<table> table see original document page 170 </column> </row> <table>

* Not detected under the test conditions described.

These data demonstrate for the first time that not all function blocking antibodies bind to α2 integrin in the same manner and also show methods for identifying a new antibody subgroup with similarity to the TMC-2206 epitope with blocking activities. similar functions. These data also demonstrate that this new anti-a2 antibody subgroup, which includes TMC-2206 and antibodies with similar epitope specificity as TMC-2206, are characterized by unexpected in vivo absence of bleeding complications and / or absence of activation. of platelet α2βΐ integrin. Epitope specificity, function blocking activities and advantages (eg, non-platelet activation) are not characteristic of all function blocking anti-a2pi antibodies, but in fact a new antibody subgroup including TMC-2206 and similar antibodies, including derivatives and / or variants of TMC-2206, which may be identified and / or selected as described herein.

Based on the fact that not all function-blocking antibodies that bind to domain a2 domain also bind to the same or similar epitope as TMC-2206 (eg, overlap), studies have been performed to determine if a Substitute antibody used in murine efficacy studies had properties similar to that of TMC-2206. Because Hal / 29 antibody cross-reacts with mouse and mouse integrin α2 integrin, and TMC-2206 antibody binds to mouse and human α2 integrin, the GST fusion protein was used to determine whether two antibodies bind to overlapping sites (eg shared epitope specificity). To this end, a a.2 domain I GST fusion protein was immobilized on glutathione-coated Reacti-Bind microtiter plates (Pierce Biotechnology, Inc. Rockford, IL). First, the Eu-TMC-2206 Kd bound to a 370C-immobilized human and rat α2 domain I GST protein was determined as described in Example 2. Scatchard analysis on the bound Eu-TMC-2206 versus The free sample showed Kd values of 0.2 nM for human a2 domain I and 1.3 nM (a 6-fold reduction) for rat a.2 domain I. Next, the ability of Eu-labeled TMC-2206 to bind to rat α2 domain I in the presence of different concentrations of competition antibody was assessed as described in Example 2 using the KcJ value. 1.3 nM to derive a Ki value based on the observed EC50 values. Hal / 29 antibody (Mendrick and Kelly, Lab Invest. 69 (6): 690-702 (1993)), but not HMa2 antibody (Miyake et al., Eur. J. Immunol. 24: 2000-2005 (1994 )) was an effective eu-TMC-2206 binding antagonist, indicating that the Hal / 29 antibody bound to similar sites (e.g., overlap) to the TMC-2206 binding site.

EXAMPLE 14

Another study was performed with exemplary IgG4 antibodies containing an hVH14.0 γ4 heavy chain (SEQ ID NO: 174) or an hVH12.0 γ4 heavy chain (SEQ ID NO: 176) and an hVL 10.OQ light chain (SEQ ID NO: 178). In this study, it was evaluated whether the binding of these IgG4 antibodies causes platelet activation, assessed based on the effects on collagen-induced platelet aggregation. Blood samples were collected by venipuncture of the antecubital vein in vacuum collection tubes containing 3.8% sodium citrate after discarding the first 3.0 ml of the collected blood. All antibodies were diluted in saline to final concentrations of 140 Mg / ml. Each disposable cuvette (containing a disposable electrode) was divided into 0.5 ml aliquots of citrated whole blood and 0.5 ml saline or antibody solution. Each cuvette was preheated to 37 ° C for 5 minutes in the aggregometer heating socket (Model 591A, Chrono-Log, Havertown, PA), and then placed in the socket for reaction; baseline was determined and 20 μl of saline or collagen (1 mg / ml; equine type I, Chrono-Log) were added to initiate the aggregation reaction. During aggregation, platelet buildup occurred on exposed electrode surfaces, resulting in increased impedance. Data acquisition proceeded for 6 minutes and the change in impedance (ΔΩ, ohms) was recorded by a graph recorder (Model 707, Chrono-Log).

The data (Table 33) were analyzed using the Kruskal-Wallis test, which tested the hypotheses that the means used in the populations (saline or collagen) of each of the four groups were equivalent, and the hypothesis would be rejected if (95% confidence) P values were less than or equal to 0.05. In the saline group (P value = 0.148) neither isotype control nor the two humanized antibodies induced platelet aggregation compared to the negative saline control group. In the collagen group (P value = 0.201) neither isotype control nor both humanized antibodies inhibited collagen-induced platelet aggregation compared to the negative saline control group. The results of this study and Example 8 show that binding of TMC-2206 and the two humanized IgG4 antibody variants have no effect on collagen-induced platelet aggregation in in vitro tests at all concentrations tested.

TABLE 33

<table> table see original document page 173 </column> </row> <table>

EXAMPLE 15

The ability of humanized TMC-2206 (hIgG4 / kVH12.0 / VL10.0Q) to block type I collagen-binding integrin α2β1-mediated cell adhesion was tested using CHO-α2, HT1080 (human fibrosarcoma) cells and platelets Following the procedures described in Example 2. Humanized OTMC-2206 was a potent inhibitor of collagen cell binding with EC50 values comparable to those of TMC-2206 (Table 34).

<table> table see original document page 173 </column> </row> <table> EXAMPLE 16

The ability of the humanized TMC-2206 to bind to immobilized human αΐβΐ was assessed using the ELISA assay. Human α1 / 31 integrin (Chemicon International) was diluted in coating buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2) to a final concentration of 0.5 Mg / ml. 96-well immunoplates were coated with α1β1 at 50ng / well and incubated overnight at 40 ° C. The plates were washed three times with wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.5% Tween-20) and blocked with 5% skim milk w / v / in wash buffer by one hour at room temperature. Humanized TMC-2206 antibodies, human IgG4 / κ (control isotype) and mouse human anti-al (FB-12, Chemicon International) were serially diluted in binding buffer (0.1 mg / ml BSA, IgG "). Fifty microliters / well of the diluted antibody solutions were added to the αΐβ revest coated plates, which were incubated for one hour at room temperature and washed three times. goat (secondary antibody; Jackson ImmunoResearch Laboratories, West Grove, PA) was added to wells containing the control isotype and humanized TMC-2206; goat mouse alkaline phosphatase conjugate (Sigma) was added to wells After one hour incubation at room temperature, the plates were washed three times, incubated in substrate solution (1 mg / ml 4-nitrophenyl phosphate, 0.1 M diethanolamine, 5 mM MgCl2, pH 9.8) for 2 0 minutes and quenched with NaOH. Absorbance (405 nm) was analyzed using a Spectramax Plus reader plate and Softmax Pro software. Similar to TMC-2206, humanized TMC-2206 and 35 IgG4 / κ antibodies did not bind to α1β1. The anti-al / 31 control antibody (FB-12) bound to α1β1 with an EC50 of 0.79 ± 0.15 nM. Example 17

K0 and Ki values for TMC-2206 and TMC-2206 MAbs bound to immobilized α2β1 were determined by a competitive binding assay. The wells of a 96-well microtiter plate were coated with platelet integrin α2β1 (coated to human platelet α2β1 by GTI Inc., WI) and blocked with skim milk. The humanized antibody TMC-2206 was labeled with the I-NI-ITC regent, approximately 2 mg was suspended and dialyzed in phosphate buffer saline (PBS; 1.4'7 mM KH2PO4, 8.1 mM Na2HPO4; pH 7 (138 mM NaCl and 2.67 mM KCI). Following concentration in pre-washed MicroSep concentrators (30-kDa cutoff; Pall Life Sciences at 9500 rpm (7000 g) in a JA-20 rotor (Beckman Instruments, Inc.) for 20 minutes at 4 ° C), the antibody It was adjusted to 4 ~ 6 mg / ml with PBS, 100 mM NaHCO 3, pH 9.3. The MAb / bicarbonate solution (0.250 mL) was carefully mixed in a vial containing 0.2 mg tetraacetic acid N1- (p-isothiocyanatobenzyl) diethylenetriamine-JV1, N21N31N3 chelated with Eu3 + (Eu-NI-ITC; Perkin Elmer Life Sciences) and incubated overnight at 4 ° C without stirring. The labeled antibody mixture was applied to a separate PD-10 column (GE Biosciences, Piscataway, NJ) pre-equilibrated with Current buffer (50 mM Tris, pH 7.4 and 138 mM NaCl). Fractions (0.5 mL) were collected and analyzed to assess total protein (Bradford reagent; Bio-Rad Laboratories, Hercules, CA) using SpectraMax 384 plate and europium after diluting 1: 10,000 in a DELFIA enhancer solution. (Perkin-Elmer) by time-synchronized fluorescence (TRF) using a Victor2 multi-label plate reader (Perkin Elmer). Positive fractions for both protein marker and europium were pooled and applied to a new PD-10 column and samples were collected and analyzed for protein TR and europium content determination by calibrated TRF compared to a standard europium solution. (Perkin-Elmer) to calculate fluorine: protein ratio. Eu-labeled antibody TMC-2206 was then applied to α2β1 integrin-blocked microtiter plates at a volume of 10 µl / well. After incubation of the sealed plates for 1 h at 37 ° C to allow binding to reach equilibrium, 2 μΐ; The sample was transferred from each well to a new well containing the DELFIA intensifier solution (100 μL / well; Perkin-Elmer) for evaluation of the free (unbound) marker. The intensifying solution (100 μL / alveol) was added to the emptied alveoli for evaluation of the bound marker. The plate was shaken (Titration Shaker Plate, speed setting 5 ° C for 5 minutes at room temperature) and TRF intensities were evaluated using a Victor2 TPerkin-Elmer WaXlac plate reader, Boston, MA). KD values were calculated by Scatchard analysis. The relative binding potencies of immobilized α2β1 integrin were analyzed by evaluating K ± values in a competition assay using 100 pM Eu-TMC-2206 in the presence of varying concentrations of unlabelled TMC-2206 antibody or antibody. chimeric as competitors. A test system similar to that described above in this example was used. Antibody combinations were then applied to α2β1 integrin coated wells, tested at the concentration range of 10 "11 to 10" 10 M, and the amount of humanized Eu-TMC-2206 determined after the specified time period. . Inhibition curves were fitted with a "one-site competition" model with the aid of Prisma software (GraphPad, Inc.) to obtain IC50 values and to calculate K ± t using the Cheng and Prusoff equation (1973). ) and the respective Kdi values given above. The humanized Kd and Ki values for the humanized TMC-2206 and TMC-2206 were between 2 times each other (Table 35). Therefore, the binding affinities of TMC-2206 and TMC-2206 to α2β1 were similar.

TABLE 35

<table> table see original document page 177 </column> </row> <table>

Humanized TMC-2206 and TMC-2206 were subjected to surface plasma resonance (SPR) analysis to determine kinetic dissociation and associated constants, ka and ka (also known as k0ff and kon), respectively in the a.2 I domain. SPR, a method used to characterize macromolecular interactions, is an optical technique that uses the transient wave phenomenon to accurately measure small changes in refractive index, very close to the surface sensor. The binding between an antigen in solution (eg fusion protein) and its MAb receptor (immobilized on the surface of a chip sensor) results in alteration of the refractive index. Interaction is monitored in real time and the amount of bound antigen as well as association and dissociation rate constants can be measured with great accuracy. The equilibrium dissociation constant can be easily calculated based on: Kd = kd / ka = koff / kon. Cloning of human α2 domain I and purification of α2 domain I expressed human GST-fusion protein were described in Example 12. Analyzes were performed at 20 ° C using a Biacore 2000 optical sensor with a CM5 research grade chip (Biacore Life Sciences, Uppsala, Sweden) and equilibrated with standard buffer (50 mM HEPES, 150 mM NaCl, 0.25 mM MgCl2, 0.25 mM CaCl2, 0.5% Tween-20.0, 1 mg / ml BSA, pH 7.4). For TMC-2206 capture on the chip sensor, two of the chip flow cell surfaces were coated with mouse anti-IgGs; the other two flow cells were coated with Protein A to capture humanized TMC-2206. Each cycle of the antigen (cx2 domain I GST-human fusion protein) bound to the surface attached to the mouse anti-IgGs involved three steps. In the first step, TMC-2206 was captured on an anti-mouse surface and then humanized TMC-2206 was captured on a protein A surface. [The other two surfaces (one anti-mouse and one protein A ) were used with analytical references.] In the second step, the α2 domain GST-human fusion protein was injected into the four surfaces. Responses obtained from reference surfaces (due to inconsistent refractive index between antigen and current buffer) were subtracted from responses obtained from reaction surfaces. In the third step, the outer layer of antigen / antibody complexes was removed from the surfaces so that these surfaces could still be used for another binding cycle. The highest concentration of oI2 domain I GST-human fusion protein was 41 nM. The antigen solution was applied to the surfaces for 2 minutes at 50 μΐ / min and surface antigen dissociation was monitored for six minutes. The rate constants for binding of α2 domain I GST-human fusion protein to TMC-2206 and humanized TMC-2206 were determined and found to be similar (Table 37).

Competitive binding assays and SPR analysis confirmed that the humanization process did not affect the binding affinity of the humanized TMC-2206 to the human oI domain I.

EXAMPLE 18

Cross-species reactivity to humanized TMC-2206 was evaluated by biochemical analytical techniques. In the first study, the binding affinities (Kj values) of the humanized TMC-2206, humanized TMC-2206, and GST-o; 2-domain I fusion proteins derived from different species were determined (Ki values) by competitive binding with the Humanized and europium-labeled TMC-2206 to α.2β1-coated plates (Example 17). Cloning of α2 domains I from humans, rhesus monkeys, rats and mice was described in Example 12. α2 domains I from cynomolgus and other rhesus monkeys were cloned from total RNA-derived cDNA extracted from skin tissues (MediCorp, Inc ., Montreal, QC). There was a 9-fold decrease in Kj for rat α2 I binding to humanized TMC-2206 compared to human o? 2 domain I, whereas murine GST o? 2 I fusion protein showed only discrete specific binding at the highest concentration (4 μΜ; Table 36). [Negative control of GST fusion protein and negative control of IgG4 / k isotype did not show any competitive binding effect at 0.4 μ concentrations.] A2 I GST fusion proteins from rhesus, cynomolgus and human monkeys showed comparable link. Therefore, all species demonstrated cross-reactivity with humanized TMC-2206.

TABLE 3 6

<table> table see original document page 179 </column> </row> <table>

In a second study, the frequency constants and binding equilibrium of the humanized TMC-2206 and TMC-22 06 and selected oi2 I-GST fusion proteins were evaluated by SPR analysis (Table 37). All kinetic and equilibrium constants derived for primary and humanized TMC-2206 for human and rat α2 I domains were similar. In addition, the frequency constants of the humanized TMC-2206 for the human a2 I and cynomolgus domains were similar. Humanized TMC-2206 did not bind to mouse a.2 I domain at concentrations of 4.0 μ concentrações of mouse a.2 I-GST fusion protein. Comparable binding of GST-cynomolgus-oi2-domain I fusion protein to TMC-2206 was not consistent with the result of Example 12 - in which no competitive binding was observed at concentrations up to 1 μΜ (Table 29). However, DNA sequence analyzes performed on monkey-extracted mRNA-derived cDNA populations (Medicorp Inc.) revealed a single amino acid polymorphism (position 40) compared to the human a2-I domain. This polymorphism was not conserved between animals, so a cynomolgus monkey and a rhesus monkey exhibited heteromorphism, while other animals exhibited 100% homology to the human cx2 I domain. The cynomolgus a2 domain I GST studied in this example by competitive binding and SPR analysis encoded a sequence identical to the human a2 I domain. These biochemical studies demonstrated that humanized TMC-2206 cross-reacted with human, rhesus, cynomolgus, and rat a2 I domains, but not with mouse a2 I domains. In vitro cross-reactivity studies (Example 20) were performed to verify whether humanized TMC-2206 cross-reacted with blood cells of different species.

TABLE 37

<table> table see original document page 180 </column> </row> <table> <table> table see original document page 181 </column> </row> <table>

Example 19

Cross-species reactivity was also assessed by the binding of TMC-2206 to blood cells of different species by flow cytometry. In the first study, the cross-reactivity of humanized TMC-2206 with platelets of different species was evaluated. Blood was obtained by venipuncture from human donors, rhesus / cynomolgus monkeys. Human blood was collected in 3.8% sodium citrate; blood from rhesus and cynomolgus monkeys was collected in 10 mM EDTA; and rat blood was collected in heparin. Whole blood of primates (inhumans, rhesus, cyroomo 1 gus) was fused with humanized THC-225 to a final concentration of 140 Mg / ml for 10 minutes at room temperature, followed by incubation for 10 minutes of IgG4-conjugated Mab. Mouse anti-human FITC (Clone HP6023; Southern Biotech), followed by incubation with species-specific platelet marker antibodies conjugated with fluorescent molecules. Human platelets were identified with CD42b mouse-anti-human conjugate PE (BD Biosciences) and rhesus / cynomolgus platelets were identified with CD41a mouse-anti-human conjugate PE (BD Biosciences). Rat whole blood was incubated with 500 mg / ml of Alexa- 488-conjugated humanized TMC-2206 (Alexa Fluor 488 Protein Labeling kit, A10235, Molecular Probes) for 10 minutes at room temperature, followed by incubation with PE-hamster-conjugate -anti-CD61 (rat platelet marker; BD Biosciences). All samples were washed once, suspended in phosphate-buffered saline and then subjected to flow cytometric analysis. [Frontal and lateral spreading channels were used to adjust the logarithmic scale to better discriminate platelets from larger red blood cells and leukocytes.] Humanized TMC-2206 bound to platelets of the four species (Table 38). In the second study, the cross-reactivity of humanized TMC-2206 with leukocytes of different species was evaluated. Blood was obtained from the same four species and human blood was collected in 10 mM EDTA. Humanized TMC-2206 conjugated with Alexa 488 was added to whole blood (final concentrations between 225-400 μg / mL) for 10 minutes, followed by incubation for 30 minutes at room temperature with marker antibodies. Anti-CD45 antibodies were used to stain leukocytes [for PE-Cy5-mouse-conjugated anti-human human leukocytes (clone H130, BD Biosciences); for rhesus and cynomolgus leukocytes PE-Cy5-conjugate-mouse-anti-human (clone TuT16, BD Biosciences); and for PE-Cy5 mouse-conjugated mouse anti-rat leukocytes (BD Biosciences). Labeling antibodies were used to stain platelets: for human PE-Cy5-conjugate-mouse-anti-human-CD42b platelets (BD Biosciences); for rhesus and cynomolgus R-PE-mouse-anti-human-CD41a conjugated platelets (BD Biosciences); and for R-PE-conjugated-hamster-anti-mouse-CD61 rat platelets (BD Biosciences] .A milliliter of water was added to the reaction (approximately 250 μl), incubated for 5 minutes at room temperature to lyse red blood cells, followed by by adding 2 ml of PBS (to tonicity levels that prevent leukocyte lysis), and centrifuged.The cell pellet was resuspended in 0.5 mls PBS and subjected to flow cytometric analysis. [The lateral dispersion channel was adjusted to the linear scale and the CD45 channel was adjusted to the logarithmic scale to discriminate granulocytes, monocytes, and lymphocytes.] How varying levels of endogenous platelet activation lead to the formation of platelet-leukocyte microaggregates is essential to identify. non-platelet-bound leukocytes (which constitutively express α2β1 integrin), so only cells that were CD45 + / CD41a "CD45 + / CD42b", or CD45 + / CD61 "were evaluated for binding to humanized TMC-2206. Humanized TMC-2206 bound to lymphocytes, monocytes and granulocytes of the four species (Table 38).

These results are consistent with the results of Example 19, that is, humanized TMC-2206 cross-reacts with human GST-CX2-domain I fusion proteins from rhesus monkeys, cynomolgus and rats (by Kj and SPR analysis) . A relatively low percentage of rat blood cells bound humanized TMC-2206 compared to primate cells. In three initial studies (Examples 9, 19, and 12), the binding affinities of humanized primary antibodies and TMC-2206 to the rat α2 integrin subunit were shown to be of an order of magnitude lower than the human α2 subunit binding affinities. In the first study, Example 9, the EC50 values for inhibition of TMC-2206 binding of rat platelets and human platelets to rat type I collagen was 6.3 nM and 1.7 nM, respectively. In the second study, Example 19 (Table 38), Kj values for inhibition of humanized TMC-2206 binding to immobilized α2β1 by competing fusion proteins GST-human-α2-domain I and GST-rat-a2-domain I were 0.57 nM and 5.23 nM, respectively. Similarly, in the third study, Example 12 (Table 29), Kj values for inhibition of binding of TMC-2206 to α2β1 by fusion proteins

GST-human-a2-domain I and GST-rat-a; 2-domain I were 0.33 nM and 3.8 nM, respectively. In addition, in platelet and leukocyte studies, all cell samples were washed before being subjected to flow cytometric analysis, and more humanized TMC-2206 was washed from low affinity rat subunit a.2 than from a.2 subunits of primates. Combined with previous results, this led to a relatively lower percentage of rat blood cells being counted as "positive" when compared to primate blood cells (taking into account similar α2β1 receptor densities). In summary, platelets, lymphocytes, monocytes and granulocytes for the four species tested (humans, rhesus macaque, cynomolgus macaque, and rat) bound to humanized TMS-2206.

TABLE 38

<table> table see original document page 184 </column> </row> <table>

EXAMPLE 20

Another study evaluated whether TMC-2206 binding to α2β led to platelet activation as measured by flow cytometry. Platelet activation was measured as both P-Seletin up-regulation and GPIIbIIIa (αIIbβ / 33) integrin activation. Blood samples were collected by venipuncture of the antecubital vein in vacuum tubes containing 3.8% sodium citrate after discarding the first 3.0 ml of standard blood. Whole blood was diluted 1:10 in TBS (pH 7.4) and then incubated for 10 minutes at room temperature with saline, isotype control IgG4 / k (final concentration 132 æg / ml), or humanized TMC-2206 ( final concentration del44 μg / ml). For platelet activation, thrombin receptor activation peptide 6 (TRAP-6, 10 μΜ final concentration; AnaSpec Inc., San Jose, CA) or adenosine diphosphate (ADP, 20 μΜ final concentration; Sigma) was added to the samples, followed by an incubation period of 5 minutes at room temperature. Cells were processed for flow cytometry by incubation with marker antibodies: PE-Cy5-mouse-anti-human-CD4 2b conjugate (BD Biosciences) to stain platelets; PE-mouse-anti-human-CD62P conjugate (BD Biosciences) to stain P-selectin; and FITC-PAC-I conjugate (BD Biosciences) to stain activated GPIIbIIIa (PAC-1 binds to active conformation of integrin GPIIbIIIa). The sampling error for each sample was less than 5% (95% confidence interval).

Early experiments evaluated whether binding to humanized TMC-2206 leads to platelet activation as measured by P-selectin over-regulation. Activation was quantified as the percentage of platelets (CD42b +) that were stained by the P-selectin marker (CD62P) (Table 39). By ANOVA analysis (single-tailed, 95% confidence interval), P-selectin expression of platelets incubated with saline, IgG4 / k, and humanized TMC-2206 was not statistically different (P = 0.96). Therefore, humanized TML'-22Ub ligation to platelets did not induce platelet activation. In paired (side-by-side) experiments, TRAP-6 induced a significant increase in P-selectin expression. The addition of humanized TMC-2206 did not statistically affect TRAP-6-induced P-selectin expression compared to saline or control isotype (P - 0.96; single-tailed ANOVA, 95% confidence interval). Therefore, the binding of humanized TMC-2206 did not inhibit TRAP-6 induced platelet activation.

TABLE 39

<table> table see original document page 185 </column> </row> <table>

The following study evaluated P-selectin up-regulation after incubation with / without the ADP agonist (percentage of platelets expressing P-selectin, Table 40). As before, P-selectin expression on platelets incubated with humanized TMC-2206, IgG4 / k and saline were comparable, however, humanized TMC-2206 did not induce platelet activation. ADP induced P-selectin expression comparable to TRAP-6 induction. P-selectin expression apparently increased further with ADP incubated platelets, followed by humanized IgG4 / c or TMC-2206. However, the increase in P-selectin up-regulation was similar for the control isotype and humanized TMC-2206, again indicating that humanized TMC-22 06 binding to platelets does not induce platelet activation. Concomitantly, expression of ADP-induced platelet P-selectin incubated with humanized IgG4 / β or humanized TMC-2206 did not decrease. Therefore, binding of humanized TMC-226 "did not inhibit ADP-induced platelet activation.

TABLE 40

<table> table see original document page 186 </column> </row> <table>

The following study evaluated GPIIbIIIa activation after incubation with / without TRAP-6 or ADP agonists, quantifying the percentage of platelets that bound the PAC-I marker antibody (which binds to the active conformation of GPIIbIIIa; Table 41). Activated GPIIbIIIa expression level on platelets incubated with humanized TMC-2206, IgG4 / k and saline were comparable - humanized TMC-2206 did not induce platelet activation. IgG4 / k and humanized TMC-2206 did not inhibit activation induced by TRAP-6 or ADP. TABLE 41

<table> table see original document page 187 </column> </row> <table>

In summary, humanized TMC-2206 did not induce platelet activation (without increased P-selectin over-regulation or GPIIbIIIa activation), nor did it inhibit agonist-induced platelet activation (TRAP-6, ADP). These data complement the study of platelet aggregation (Example 15, Table 34) which showed that humanized TMC-2206 did not induce platelet aggregation and did not inhibit collagen-induced aggregation.

EXAMPLE 21

The effect of humanized TMC-22 06 on extrinsic and intrinsic coagulation pathways was evaluated based on prothrombin time (PT) and activated partial thromboplastin time (aPTT). A human plasma lyophilized preparation (Citrex I, Bio / Data Corporation, Horsham, PA) was used to evaluate both PT and aPTT. Humanized TMC-2206 was added to plasma to give final concentrations of 179, 214, and 286 Mg / mL (corresponding to C max of a single antibody dose at 12.5, 15.0, and 20.0 mg / kg, respectively) before subjecting the samples to coagulation tests. Standardized procedures were followed for both PT and aPTT with coagulation times measured with a BBL fibrometer (BD, Franklin Lakes, NJ). Table 42 summarizes the data from a series of six experiments (3 PT and 3 aPTT). A saline control was made for each experiment. Statistical analysis with Student-t test of each pair (humanized TMC-2206 and saline solution) showed in each experiment that there were no statistically significant differences between the average coagulation time for humanized TMC-2 2 O 6 compared to the solution. saline. (The hypothesis that the clotting time was different would be rejected at 95% confidence intervals if the calculated individual P values were less than 0.05.) Therefore, humanized TMC-22 06 had no effect on coagulation according to measurement by PT and aPPT.

TABLE 42

<table> table see original document page 188 </column> </row> <table>

EXAMPLE 22

The effects of TMC-2206 on coagulation times in rats were evaluated. Sprague-Dawley rats (190 - 200g) received intravenous injection (tail vein) of saline, heparin (0.6 mg / kg, positive control), or humanized TMC-2206 at doses of 5 and 15 mg / kg, a one hour before the standard transection of the tail tip (0.5 mm). The rats were not anesthetized and were conscious while observing bleeding time. The severed tail tip of each rat was immediately immersed 2 cm deep in a test tube containing saline at 37 ° C. The time required for the onset of a 15 second period to stop bleeding was quantified as bleeding time. A maximum cut-off time of 20 minutes was used. Blood loss was quantified by the amount of hemoglobin released after hemolysis (by spectrophotometry) from the blood collected in the test tube. Humanized TMC-2206 showed no statistically significant effect on bleeding time with both doses tested compared to naiVe and saline controls (Table 43; P = 0.08, single-tailed ANOVA analysis). Humanized TMC-22 06 showed no statistically significant effect on blood loss at both doses tested compared to naive and saline controls (P = 0.22, single-tailed ANOVA analysis). Therefore, humanized TMC-2206 has no in vivo effect on bleeding time or blood loss.

TABLE 43

<table> table see original document page 189 </column> </row> <table>

EXAMPLE 23

A study was conducted to determine the effect of a single dose of humanized TMC-2206 on circulating cytokine levels in rats as a means of determining whether humanized TMC-2206 causes detectable leukocyte activation in vivo. Saline (negative control), human IgGA / κ isotype control (15 mg / kg), humanized TMC-2206 (15 mg / kg) or lipopolysaccharides (LPS, positive inflammation control; 0.75 mg / kg) were administered to rats by via IV. Mice that did not receive the injection were used as a naive control (without previous exposure). At 2, 4, 6, and 8 hours after injection, blood samples were collected from the saphenous vein and processed for plasma. Plasma samples were subjected to multiplex immunoassay (MIA; Linco Diagnostics, St. Charles, MO) to determine IL-10 ;, IL-II, IL-2, IL-4, IL-5, IL-6 levels , IL-12, GM-CSF, IFN-γ, and TNF-α (Table 44; pg / ml, mean ± SEM). MIA involves the simultaneous detection of analytes (up to 100) in the same sample volume (25 μΐ) by combining several individual antigen-antibody binding reactions into microsphere sets of different spectral regions. Depending on the antigen, MIA sensitivity is between 1.5 - 50 pg / ml. Each cytokine dataset was subjected to two-tailed ANOVA analysis with a 95% confidence interval to test the hypothesis that individual cytokine levels were equal for the four timepoints and for the four conditions (naive, vehicle, IgG4 / (humanized TMC-2206). The hypothesis would be rejected if P values were less than 0.05.

There were no statistically significant differences in each of the ten sets (all P values ranged from 018 to 1.0, Table 44) of cytokine levels observed in rats injected with the humanized, IgG4 / κ, TMC-2206 vehicle or received no (naive) injection · - {} - Therefore, intravenous injection of a single dose (15 mg / kg) of humanized TMC-2206 did not induce increased expression of cytokines involved in inflammation.

TABLE 44

<table> table see original document page 190 </column> </row> <table> <table> table see original document page 191 </column> </row> <table>

Although the present invention has been described in this

With reference to specific applications, it is important to understand that such applications are merely illustrative of the various aspects of the invention. Thus, it should be understood that numerous modifications may be made to the illustrative applications and other uses may be envisioned without departing from the principle and scope of the invention. LIST OF SEQUENCES

<110> GLENMARK PHARMACEUTICALS S.A. LAZARIDES, Elias WOODS, Catherine BERNARD, Mark

<120> "HUMANIZED ANTI-INTEGRINE α2 ANTIGOD HUMANIZED ANTIBODY, METHOD FOR DETERMINING IF A SAMPLE CONTAINS INTEGRINE (X2, NUCLEIC ACID KITf, VECTOR, HOST CELL, PROCESS FOR PRODUCING AN ANTIGOD HYDROGENINE, , METHOD FOR TREATMENT OF α2β1 INTEGRINE-ASSOCIATED DISORDERS IN PATIENTS, METHOD FOR INHIBITION OF COLLAGEN LEUKOCYTES, METHOD OF DIRECTING A Molecule, USE OF AN INTEGRUM AND EYTHROPHINE ANTIBODY ANTIBODY, <2> CONNECT ANTI-INTEGRINE ANTIBODY a2 ".

<130> 14575.2

<150> US 60 / 738.303

<151> 2005-11-18

<160> 186

<170> PatentIn version 3.3

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actgttcaag gaggagacaa ctttcagatg gaaatgtcac aagtgggatt cagtgcagat 1200

tactcttctc aaaatgatat tctgatgctg ggtgcagtgg gagcttttgg ctggagtggg 1260

accattgtcc agaagacatc tcatggccat ttgatctttc ctaaacaagc ctttgaccaa 132 0

attctgcagg acagaaatca cagttcatat ttaggttact ctgtggctgc aatttctact 1380

ggagaaagca ctcactttgt tgctggtgct cctcgggcaa attataccgg ccagatagtg 1440

ctatatagtg tgaatgagaa tggcaatatc acggttattc aggctcaccg aggtgaccag 1500

attggctcct attttggtag tgtgctgtgt tcagttgatg tggataaaga caccattaca 1560

gacgtgctct tggtaggtgc accaatgtac atgagtgacc taaagaaaga ggaaggaaga 162 0

gtctacctgt ttactatcaa aaagggcatt ttgggtcagc accaatttct tgaaggcccc 1680

gagggcattg aaaacactcg atttggttca gcaattgcag ctctttcaga catcaacatg 1740

gatggcttta atgatgtgat tgttggttca ccactagaaa atcagaattc tggagctgta 1800

tacatttaca atggtcatca gggcactatc cgcacaaagt attcccagaa aatcttggga 1860

tccgatggag cctttaggag ccatctccag tactttggga ggtccttgga tggctatgga 1920

gatttaaatg gggattccat caccgatgtg tctattggtg cctttggaca agtggttcaa 1980

ctctggtcac aaagtattgc tgatgtagct atagaagctt cattcacacc agaaaaaatc 2040

actttggtca acaagaatgc tcagataatt ctcaaactct gcttcagtgc aaagttcaga 2100

cctactaagc aaaacaatca agtggccatt gtatataaca tcacacttga tgcagatgga 2160

ttttcatcca gagtaacctc cagggggtta tttaaagaaa acaatgaaag gtgcctgcag 222 0

aagaatatgg tagtaaatca agcacagagt tgccccgagc acatcattta tatacaggag 2280

ccctctgatg ttgtcaactc tttggatttg cgtgtggaca tcagtctgga aaaccctggc 2340

actagccctg cccttgaagc ctattctgag actgccaagg tcttcagtat tcctttccac 2400

aaagactgtg gtgaggatgg actttgcatt tctgatctag tcctagatgt ccgacaaata 2460

ccagctgctc aagaacaacc ctttattgtc agcaaccaaa acaaaaggtt aacattttca 2520

gtaacactga aaaataaaag ggaaagtgca tacaacactg gaattgttgt tgatttttca 2580

gaaaacttgt tttttgcatc attctcccta ccggttgatg ggacagaagt aacatgccag 2640

gtggctgcat ctcagaagtc tgttgcctgc gatgtaggct accctgcttt aaagagagaa 2700

caacaggtga cttttactat taactttgac ttcaatcttc aaaaccttca gaatcaggcg 2760

tctctcagtt tccaagcctt aagtgaaagc caagaagaaa acaaggctga taatttggtc 2820

aacctcaaaa ttcctctcct gtatgatgct gaaattcact taacaagatc taccaacata 2880

aatttttatg aaatctcttc ggatgggaat gttccttcaa tcgtgcacag ttttgaagat 2940

gttggtccaa aattcatctt ctccctgaag gtaacaacag gaagtgttcc agtaagcatg 3000

gcaactgtaa tcatccacat ccctcagtat accaaagaaa agaacccact gatgtaccta 3060

actggggtgc aaacagacaa ggctggtgac atcagttgta atgcagatat caatccactg 3120

aaaataggac aaacatcttc ttctgtatct ttcaaaagtg aaaatttcag gcacaccaaa 3180

gaattgaact gcagaactgc ttcctgtagt aatgttacct gctggttgaa agacgttcac 3240

atgaaaggag aatactttgt taatgtgact accagaattt ggaacgggac tttcgcatca 3300

tcaacgttcc agacagtaca gctaacggca gctgcagaaa tcaacaccta taaccctgag 3360

atatatgtga ttgaagataa cactgttacg attcccctga tgataatgaa acctgatgag 3420

aaagccgaag taccaacagg agttataata ggaagtataa ttgctggaat ccttttgctg 3480

ttagctctgg ttgcaatttt atggaagctc ggcttcttca aaagaaaata tgaaaagatg 3540

accaaaaatc cagatgagat tgatgagacc acagagctca gtagctgaac cagcagacct 3600

acctgcagtg ggaaccggca gcatcccagc cagggtttgc tgtttgcgtg catggatttc 3660

tttttaaatc ccatattttt tttatcatgt cgtaggtaaa ctaacctggt attttaagag 3720

aaaactgcag gtcagtttgg atgaagaaat tgtggggggt gggggaggtg cggggggcag 3 78 0

gtagggaaat aatagggaaa atacctattt tatatgatgg gggaaaaaaa gtaatcttta 3840

aactggctgg cccagagttt acattctaat ttgcattgtg tcagaaacat gaaatgcttc 3900

caagcatgac aacttttaaa gaaaaatatg atactctcag attttaaggg ggaaaactgt 3960

tctctttaaa atatttgtct ttaaacagca actacagaag tggaagtgct tgatatgtaa 4020

gtacttccac ttgtgtatat tttaatgaat attgatgtta acaagagggg aaaacaaaac 4080

acaggttttt tcaatttatg ctgctcatcc aaagttgcca cagatgatac ttccaagtga 4140

taattttatt tataaactag gtaaaatttg ttgttggttc cttttatacc acggctgccc 4200

cttccacacc ccatcttgct ctaatgatca aaacatgctt gaataactga gcttagagta 4260

tacctcctat atgtccattt aagttaggag agggggcgat atagagacta aggcacaaaa 4320

ttttgtttaa aactcagaat ataacattta tgtaaaatcc catctgctag aagcccatcc 4380

tgtgccagag gaaggaaaag gaggaaattt cctttctctt ttaggaggca caacagttct 4440

i cttctaggat ttgtttggct gactggcagt aacctagtga atttttgaaa gatgagtaat 4500

ttctttggca accttcctcc tcccttactg aaccactctc ccacctcctg gtggtaccat 4560

tattatagaa gccctctaca gcctgacttt ctctccagcg gtccaaagtt atcccctcct 4620

ttacccctca tccaaagttc ccactccttc aggacagctg ctgtgcatta gatattaggg 4680

gggaaagtca tctgtttaat ttacacactt gcatgaatta ctgtatataa actccttaac 4740

ttcagggagc tattttcatt tagtgctaaa caagtaagaa aaataagcta gagtgaattt 4800

ctaaatgttg gaatgttatg ggatgtaaac aatgtaaagt aaaacactct caggatttca 4860

ccagaagtta cagatgaggc actggaaacc accaccaaat tagcaggtgc accttctgtg 4920

gctgtcttgt ttctgaagta ctttttcttc cacaagagtg aatttgacct aggcaagttt 4980

gttcaaaagg tagatcctga gatgatttgg tcagattggg ataaggccca gcaatctgca 5040

ttttaacaag caccccagtc actaggatgc agatggacca cactttgaga aacaccaccc 5100

atttctactt tttgcacctt attttctctg ttcctgagcc cccacattct ctaggagaaa 5160

cttagattaa aattcacaga cactacatat ctaaagcttt gacaagtcct tgacctctat 5220

aaacttcaga gtcctcatta taaaatggga agactgagct ggagttcagc agtgatgctt 5280

tttagtttta aaagtctatg atctgatctg gacttcctat aatacaaata cacaatcctc 5340

caagaatttg acttggaaaa g 5361

<210> 8

<211> 1181

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<223> Human alpha-2 integrin

<400> 8 Met Gly Pro Glu Arg Thr Gly Ala Pro Wing Leu Pro Leu Leu Leu Val 1 5 10 15 Leu Wing Leu Ser Be Gln Gly Ile Leu Asn Cys Cys Leu Tyr Asn Val 20 25 30 Gly Leu Pro Glu Ala Lys Ile Phe Be Gly Pro To Be Glu Phe 35 40 45 Gly Tyr Wing Val Val Gln Phe Ile Asn Pro Lys Gly Asn Trp Leu Leu 50 55 60 Val Gly Be Pro Trp 80 Tyr Lys Cys Pro Val Asp Read Thr Be Wing Thr Cys Glu Lys Read Asn 85 90 95 Read Gln Thr Be Thr Be Ile Pro Asn Val Thr Glu Met Lys Thr Asn 100 105 110 Met Be Read Gly Leu Ile Leu Thr Arg Asn Met Gly Thr Gly Gly Phe 115 120 125 Read Thr Thr Cys Gly Pro Read Trp Wing Gln Gln Cys Gly Asn Gln Tyr Tyr 130 135 140 Thr Thr Gly Val Cys Ser Asp Ile Be Pro Pro Wing Thr Gln Pro Cys Pro Be Read Ile Asp Val Val 165 170 175 Val Val Cys Asp Glu Be Asn Be Ile Tyr Pro Trp Asp Wing Val Lys 180 185 190 Asn Phe Leu Glu Lys Phe Val Gln Gly

195 200 205

Thr Gln Val Gly Leu Ile Gln Tyr Wing Asn Asn Pro Arg Val Val Phe

210 215 220

Asn Leu Asn Thr Tyr Lys Thr Lys Glu Glu Met Ile Val Wing Thr Ser 225 230 235 240

Gln Thr Be Gln Tyr Gly Gly Asp Read Thr Asn Thr Phe Gly Ala Ile

245 250 255

Gln Tyr Wing Arg Lys Tyr Wing Tyr Being Wing Wing Being Gly Gly Arg Arg

260 265 270

Ser Wing Thr Lys Val Met Val Val Val Val Asp Gly Glu Ser His Asp

275 280 285

Gly Ser Met Leu Lys Wing Val Ile Asp Gln Cys Asn His Asp Asn Ile

290 295 300

Leu Arg Phe Gly Ile Wing Val Leu Gly Tyr Leu Asn Arg Asn Wing Leu 305 310 315 320

Asp Thr Lys Asn Leu Ile Lys Glu Ile Lys Wing Ile Wing Ser Ile Pro

325 330 335

Thr Glu Arg Tyr Phe Phe Asn Val Ser Asp Glu Wing Wing Leu Leu Glu

340 345 350

Lys Wing Gly Thr Read Gly Glu Gln Ile Phe Ser Ile Glu Gly Thr Val

355 360 365

Gln Gly Gly Asp Asn Phe Gln Met Glu Met Ser Gln Val Gly Phe Ser

370 375 380

Asp Wing Tyr Ser Gln Asn Asp Ile Leu Met Leu Gly Val Gly Wing 385 390 395 400

Wing Phe Gly Trp Being Gly Thr Ile Val Gln Lys Thr Being His Gly His

405 410 415

Read Ile Phe Pro Lys Gln Wing Phe Asp Gln Ile Read Gln Asp Arg Asn

420 425 430

His Be Ser Tyr Leu Gly Tyr Ser Val Wing Ile Ser Thr Thr Gly Glu

435 440 445

Be Thr His Phe Val Gly Wing Pro Wing Arg Wing Asn Tyr Thr Gly Gln

450 455 460

Ile Val Leu Tyr Ser Val Asn Glu Asn Gly Asn Ile Thr Val Ile Gln 465 470 475 480

Wing His Arg Gly Asp Gln Ile Gly Ser Tyr Phe Gly Ser Val Leu Cys

485 490 495

Ser Val Asp Val Asp Lys Asp Thr Ile Thr Asp Val Leu Leu Val Gly

500 505 510

Pro Wing Met Tyr Met Ser Asp Leu Lys Lys Glu Glu Gly Arg Val Tyr

515 520 525

Phe Thr Read Ile Lys Lys Gly Ile Read Gly Gln His Gln Phe Leu Glu

530 535 540

Gly Pro Glu Gly Ile Glu Asn Thr Arg Phe Gly Ser Wing Ile Wing Wing 545 550 555 560

Read Ser Asp Ile Asn Met Asp Gly Phe Asn Asp Val Ile Val Gly Ser

565 570 575

Pro Read Glu Asn Gln Asn Ser Gly Wing Val Tyr Ile Tyr Asn Gly His

580 585 590

Gln Gly Thr Ile Arg Thr Lys Tyr Be Gln Lys Ile Read Gly Be Asp 595 600 605 Gly Wing Phe Arg Be His Read

610 615 620

Tyr Gly Asp Read Asn Gly Asp Ser Ile Thr Asp Val Ser Ile Gly Wing 625 630 635 640

Phe Gly Gln Val Val Gln Read Trp Ser Gln Ser Ile Wing Asp Val Wing

645 650 655

Ile Glu Wing Be Phe Thr Pro Glu Lys

660 665 670

Gln Wing Ile Ile Leu Lys Leu Cys Phe Ser Ala Lys Phe Arg Pro Thr

675 680 685

Lys Gln Asn Asn Gln Val Wing Ile Val Tyr Asn Ile Thr Read Asp Wing

690 695 700

Asp Gly Phe Be Being Arg Val Thr Be Arg Gly Read Phe Lys Glu Asn 705 710 715 720

Asn Glu Arg Cys Read Gln Lys Asn Met Val Val Asn Gln Wing Gln Ser

725 730 735

Cys Pro Glu His Ile Ile Tyr Ile Gln Pro Glu Ser Asp Val Val Asn

740 745 750

Be Read Asp Read Le Arg Val Asp Ile Be Read Glu Asn Pro Gly Thr Be

755 760 765

Pro Wing Read Glu Wing Tyr Wing Be Glu Thr Wing Lys Val Phe Ser Ile Pro

770 775 780

Phe His Lys Asp Cys Gly Glu Asp Gly Leu Cys Ile Ser Asp Leu Val 785 790 795 800

Read Asp Val Arg Gln Ile Pro Wing Wing Gln Glu Gln Pro Phe Ile Val

805 810 815

Be Asn Gln Asn Lys Arg Leu Thr Phe Be Val Thr Leu Lys Asn Lys

820 825 830

Glu Arg Be Wing Tyr Asn Thr Gly Ile Val Val Asp Phe Be Glu Asn

835 840 845

Read Phe Phe Ala Be Phe Be Read Pro Val Asp Gly Thr Glu Val Thr

850 855 860

Cys Gln Val Wing Ward Be Gln Lys Be Val Wing Cys Asp Val Gly Tyr 865 870 875 880

Pro Wing Read Lys Arg Glu Gln Gln Val Thr Phe Thr Ile Asn Phe Asp

885 890 895

Phe Asn Leu Gln Asn Leu Gln Asn Gln Wing Be Leu Be Phe Gln Wing

900 905 910

Leu Be Glu Be Gln Glu Glu Asn Lys Wing Asp Asn Leu Val Asn Leu

915 920 925

Lys Ile Pro Read Leu Tyr Asp Wing Glu Ile His Leu Thr Arg Be Thr

930 935 940

Asn Ile Asn Phe Tyr Glu Ile Being Ser Asp Gly Asn Val Pro Being Ile 945 950 955 960

Val His Ser Phe Glu Asp Val Gly Pro Lys Phe Ile Phe Ser Leu Lys

965 970 975

Val Thr Thr Gly Ser Val Pro Val Ser Met Val Thr Val Ile Ile His

980 985 990

Ile Pro Gln Tyr Thr Lys Glu Lys Asn Pro

995 1000 1005

Val Gln Thr Asp Lys Wing Gly Asp Ile Be Cys Asn Wing Asp Ile 1010 1015 1020 Asn Pro Read Lys Ile Gly Gln Thr Be Ser Be Val Ser Phe Lys

1025 1030 1035

Be Glu Asn Phe Arg His Thr Lys Glu Read Asn Cys Arg Thr Wing

1040 1045 1050

Ser Cys Ser Asn Val Thr Cys Trp Read Lys Asp Val His Met Lys

1055 1060 1065

Gly Glu Tyr Phe Val Asn Val Thr Thr Arg Ile Trp Asn Gly Thr

1070 1075 1080

Phe Wing Be Being Thr Phe Gln Thr Val Gln Read Thr Wing Ward Wing

1085 1090 1095

Glu Ile Asn Thr Tyr Asn Pro Glu Ile Tyr Val Ile Glu Asp

1100 1105 1110

Thr Val Thr Ile Pro Read Met Ile Met Lys Pro Asp Glu Lys Wing

1115 1120 1125

Glu Val Pro Thr Gly Val Ile Ile Gly Ser Ile Ile Wing Gly Ile

1130 1135 1140

Leu Leu Leu Leu Wing Leu Val Wing Ile Leu Trp Lys Leu Gly Phe

1145 1150 1155

Phe Lys Arg Lys Tyr Glu Lys Met Thr Lys Asn Pro Asp Glu Ile

1160 1165 1170

Asp Glu Thr Thr Glu

1175 1180

<210> 9

<211> 3700

<212> DNA

<213> Homo Sapiens

<22 0>

<221> misc_feature

<22 3> Human beta-1 integrin DNA <4 0 0> 9

agccgccgcc acccgccgcg cccgacaccc gggaggcccc gccagcccgc gggagaggcc 60

cagcgggagt cgcggaacag caggcccgag cccaccgcgc cgggccccgg acgccgcgcg 12 0

gaaaagatga atttacaacc aattttctgg attggactga tcagttcagt ttgctgtgtg 180

tttgctcaaa cagatgaaaa tagatgttta aaagcaaatg ccaaatcatg tggagaatgt 240

atacaagcag ggccaaattg tgggtggtgc acaaattcaa catttttaca ggaaggaatg 300

cctacttctg cacgatgtga tgatttagaa gccttaaaaa agaagggttg ccctccagat 360

gacatagaaa atcccagagg ctccaaagat ataaagaaaa ataaaaatgt aaccaaccgt 420

agcaaaggaa cagcagagaa gctcaagcca gaggatatta ctcagatcca accacagcag 480

ttggttttgc gattaagatc aggggagcca cagacattta cattaaaatt caagagagct 540

gaagactatc ccattgacct ctactacctt atggacctgt cttactcaat gaaagacgat 600

ttggagaatg taaaaagtct tggaacagat ctgatgaatg aaatgaggag gattacttcg 660

gacttcagaa ttggatttgg ctcatttgtg gaaaagactg tgatgcctta cattagcaca 720

acaccagcta agctcaggaa cccttgcaca agtgaacaga actgcaccag cccatttagc 780

tacaaaaatg tgctcagtct tactaataaa ggagaagtat ttaatgaact tgttggaaaa 840

cagcgcatat ctggaaattt ggattctcca gaaggtggtt tcgatgccat catgcaagtt 900

gcagtttgtg gatcactgat tggctggagg aatgttacac ggctgctggt gttttccaca 960

gatgccgggt ttcactttgc tggagatggg aaacttggtg gcattgtttt accaaatgat 1020

ggacaatgtc acctggaaaa taatatgtac acaatgagcc attattatga ttatccttct 1080 attgctcacc ttgtccagaa actgagtgaa aataatattc agacaatttt tgcagttact 1140

gaagaatttc agcctgttta caaggagctg aaaaacttga tccctaagtc agcagtagga 1200

acattatctg caaattctag caatgtaatt cagttgatca ttgatgcata caattccctt 1260

tcctcagaag tcattttgga aaacggcaaa ttgtcagaag gagtaacaat aagttacaaa 1320

tcttactgca agaacggggt gaatggaaca ggggaaaatg gaagaaaatg ttccaatatt 1380

tccattggag atgaggttca atttgaaatt agcataactt caaataagtg tccaaaaaag 1440

gattctgaca gctttaaaat taggcctctg ggctttacgg aggaagtaga ggttattctt 1500

cagtacatct gtgaatgtga atgccaaagc gaaggcatcc ctgaaagtcc caagtgtcat 1560

gaaggaaatg ggacatttga ggtggcgcg tgcaggtgca atgaagggcg tgttggtaga 162 0

cattgtgaat gcagcacaga tgaagttaac agtgaagaca tggatgctta ctgcaggaaa 1680

gaaaacagtt cagaaatctg cagtaacaat ggagagtgcg tctgcggaca gtgtgtttgt 174 0

aggaagaggg ataatacaaa tgaaatttat tctggcaaat tctgcgagtg tgataatttc 1800

aactgtgata gatccaatgg cttaatttgt ggaggaaatg gtgtttgcaa gtgtcgtgtg 1860

tgtgagtgca accccaacta cactggcagt gcatgtgact gttctttgga tactagtact 192 0

tgtgaagcca gcaacggaca gatctgcaat ggccggggca tctgcgagtg tggtgtctgt 1980

aagtgtacag atccgaagtt tcaagggcaa acgtgtgaga tgtgtcagac ctgccttggt 2 04 0

gtctgtgctg agcataaaga atgtgttcag tgcagagcct tcaataaagg agaaaagaaa 2100

gacacatgca cacaggaatg ttcctatttt aacattacca aggtagaaag tcgggacaaa 2160

ttaccccagc cggtccaacc tgatcctgtg tcccattgta aggagaagga tgttgacgac 222 0

tgttggttct attttacgta ttcagtgaat gggaacaacg aggtcatggt tcatgttgtg 2280

gagaatccag agtgtcccac tggtccagac atcattccaa ttgtagctgg tgtggttgct 2340

ggaattgttc ttattggcct tgcattactg ctgatatgga agcttttaat gataattcat 2400

gacagaaggg agtttgctaa atttgaaaag gagaaaatga atgccaaatg ggacacgggt 2460

gaaaatccta tttataagag tgccgtaaca actgtggtca atccgaagta tgagggaaaa 2520

tgagtactgc ccgtgcaaat cccacaacac tgaatgcaaa gtagcaattt ccatagtcac 2580

agttaggtag ctttagggca atattgccat ggttttactc atgtgcaggt tttgaaaatg 2640

tacaatatgt ataattttta aaatgtttta ttattttgaa aataatgttg taattcatgc 2700

cagggactga caaaagactt gagacaggat ggttattctt gtcagctaag gtcacattgt 2760

gcctttttga ccttttcttc ctggactatt gaaatcaagc ttattggatt aagtgatatt 2820

tctatagcga ttgaaagggc aatagttaaa gtaatgagca tgatgagagt ttctgttaat 2880

catgtattaa aactgatttt tagctttaca aatatgtcag tttgcagtta tgcagaatcc 2940

aaagtaaatg tcctgctagc tagttaagga ttgttttaaa tctgttattt tgctatttgc 3000

ctgttagaca tgactgatga catatctgaa agacaagtat gttgagagtt gctggtgtaa 3,060

aatacgtttg aaatagttga tctacaaagg ccatgggaaa aattcagaga gttaggaagg 3120

aaaaaccaat agctttaaaa cctgtgtgcc attttaagag ttacttaatg tttggtaact 3180

tttatgcctt cactttacaa attcaagcct tagataaaag aaccgagcaa ttttctgcta 3240

aaaagtcctt gatttagcac tatttacata caggccatac tttacaaagt.atttgctgaa 3300

tggggacctt ttgagttgaa tttattttat tatttttatt ttgtttaatg tctggtgctt 3360

tctatcacct cttctaatct tttaatgtat ttgtttgcaa ttttggggta agactttttt 3420

atgagtactt tttctttgaa gttttagcgg tcaatttgcc tttttaatga acatgtgaag 3480

ttatactgtg gctatgcaac agctctcacc tacgcgagtc ttactttgag ttagtgccat 3540

aacagaccac tgtatgttta cttctcacca tttgagttgc ccatcttgtt tcacactagt 3600

cacattcttg ttttaagtgc ctttagtttt aacagttcac tttttacagt gctatttact 3660

gaagttattt attaaatatg cctaaaatac ttaaatcgga 3700

10 798 PRT

Homo sapiens

<210> <211> <212> <213>

<2 2 0>

<221> MIS C_FEATURE

<223> Human beta-1 integrin

<4 0 0> 10 Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys 15 10 15

Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala

20 25 30

Lys Be Cys Gly Glu Cys Ile Gln Wing Gly Pro Asn Cys Gly Trp Cys

35 40 45

Thr Asn Be Thr Phe Read Gln Glu Gly Met Pro Thr Be Wing Arg Cys

50 55 60

Asp Asp Leu Glu Wing Leu Lys Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile 65 70 75 80

Glu Asn Pro Arg Gly Be Lys Asp Ile Lys Lys Asn Lys Asn Val Thr

85 90 95

Asn Arg Be Lys Gly Thr Wing Glu Lys Leu Lys Pro Glu Asp Ile Thr

100 105 110

Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro

115 120 125

Gln Thr Phe Thr Read Lys Phe Lys Arg Wing Glu Asp Tyr Pro Ile Asp

130 135 140

Leu Tyr Tyr Leu Met Asp Leu Be Tyr Ser Met Lys Asp Asu Leu Glu 145 150 155 160

Asn Val Lys Being Read Gly Thr Asp Read Le Met Asn Glu Met Arg Arg Ile

165 170 175

Thr Be Asp Phe Arg Ile Gly Phe Gly Be Phe Val Glu Lys Thr Val

180 185 190

Met Pro Tyr Ile Be Thr Thr Pro Lys Wing Read Arg Asn Pro Cys Thr

195 200 205

Be Glu Gln Asn Cys Thr Be Pro Phe Ser Tyr Lys Asn Val Leu Ser

210 215 220

Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Read Le Val Gly Lys Gln Arg 225 230 235 240

Ile Be Gly Asn Read Asp Be Pro Glu Gly Gly Phe Asp Wing Ile Met

245 250 255

Gln Val Wing Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg

260 265 270

Leu Leu Val Phe Be Thr Asp Wing Gly Phe His Phe Wing Gly Asp Gly

275 280 285

Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu

290 295 300

Asn Asn Met Tyr Thr Met Be His Tyr Tyr Asp Tyr Pro Ser Ile Wing 305 310 315 320

His Leu Val Gln Lys Leu Be Glu Asn Asn Ile Gln Thr Ile Phe Ala

325 330 335

Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile

340 345 350

Pro Lys To Be Wing Val Gly Thr Read To Be Wing Asn To Be Asn Val Ile

355 360 365

Gln Leu Ile Ile Asp Wing Tyr Asn Be Read Be Ser Be Glu Val Ile Leu

370 375 380

Glu Asn Gly Lys Read Be Glu Gly Val Thr Ile Be Tyr Lys Be Tyr 385 390 395 400

Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser

405 410 415 Asn Ile Ser Ile Gly 420

Asn Lys Cys Pro Lys 435

Gly Phe Thr Glu Glu 450

Glu Cys Gln Ser Glu 465

Asn Gly Thr Phe Glu

485

Gly Arg His Cys Glu 500

Asp Wing Tyr Cys Arg 515

Gly Glu Cys Val Cys 530

Asn Glu Ile Tyr Ser 545

Asp Arg Ser Asn Gly

565

Arg Val Cys Glu Cys 580

Ser Leu Asp Thr Ser 595

Gly Arg Gly Ile Cys 610

Phe Gln Gly Gln Thr 625

Glu Wing His Lys Glu

645

Lys Lys Asp Thr Cys 660

Val Glu Ser Arg Asp 675

Be His Cys Lys Glu 690

Tyr Ser Val Asn Gly 705

Pro Glu Cys Pro Thr

725

Val Wing Gly Ile Val 740

Leu Leu Met Ile Ile 755

Glu Lys Met Asn Wing 770

Ser Wing Val Thr Thr 785

Asp Glu Val Gln Phe 425

Lys Asp Ser Asp Ser 440

Val Glu Val Ile Leu 455

Gly Ile Pro Glu Ser 470

Cys Gly Wing Cys Arg

490

Cys Ser Thr Asp Glu 505

Lys Glu Asn Ser Ser 520

Gly Gln Cys Val Cys 535

Gly Lys Phe Cys Glu 550

Read Ile Cys Gly Gly

570

Asn Pro Asn Tyr Thr 585

Thr Cys Glu Wing 600

Glu Cys Gly Val Cys 615

Cys Glu Met Cys Gln 630

Cys Val Gln Cys Arg

650

Thr Gln Glu Cys Ser 665

Lys Leu Pro Gln Pro 680

Lys Asp Val Asp Asp 695

Asn Asn Glu Val Met 710

Gly Pro Asp Ile Ile

730

Leu Ile Gly Leu Wing 745

His Asp Arg Arg Glu 760

Lys Trp Asp Thr Gly 775

Val Val Asn Pro Lys 790

Glu Ile Ser Ile Thr Ser 430

Phe Lys Ile Arg Pro Read 445 Gln Tyr Ile Cys Glu Cys 460 Pro Lys Cys His Glu Gly 475 480 Cys Asn Glu Gly Arg Val 495 Val Asn Be Glu Asp Met 510 Glu Ile Cys Be Asn Asn 525 Arg Lys Arg Asp Asn Thr 540 Cys Asp Asn Phe Asn Cys 555 560 Asn Gly Val Cys Lys Cys 575 Gly Ser Wing Cys Asp Cys 590 Asn Gly Gln Ile Cys Asn 605 Lys Cys Thr Asp Pro Lys 620 Thr Cys Leu Gly Val Cys 635 640 Phe Asn Lys Gly Glu 655 Tyr Phe Asn Ile Thr Lys 670 Val Gln Pro Asp Pro Val 685 Cys Trp Phe Tyr Phe Thr 700 Val His Val Val Glu Asn 715 720 Pro Ile Val Wing Gly Val 735 Leu Leu Leu Ile Trp Lys 750 Phe Ala Lys Phe Glu Lys 765 Glu Asn Pro Ile Tyr Lys 780 Tyr Glu Gly Lys 795 <210> 11

<211> 226

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<22 3> Human alpha-2 domain 1

<400> 11

Be Pro Asp Phe Gln Read Be Wing Be Phe Be Pro Wing Thr Gln Pro 1 5 10 15

Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser

20 25 30

Ile Tyr Pro Trp Asp Wing Val Lys Asn Phe Leu Glu Lys Phe Val Gln

35 40 45

Gly Leu Asp Ile Gly Pro Lys Thr Thr Gln Val Gly Leu Ile Gln Tyr

50 55 60

Asn Wing Asn Pro Arg Val Val Phe Asn Read Asn Thr Tyr Lys Thr Lys 65 70 75 80

Glu Glu Met Ile Val Wing Thr Be Gln Thr Be Gln Tyr Gly Gly Asp

85 90 95

Read Thr Asn Thr Phe Gly Wing Ile Gln Tyr Wing Arg Lys Tyr Wing Tyr

100 105 110

Be Wing Wing Be Gly Gly Arg Arg Be Wing Thr Lys Val Met Val Val

115 120 125

Val Thr Asp Gly Glu Be His Asp Gly Be Met Leu Lys Wing Val Ile

130 135 140

Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Wing Val Leu 145 150 155 160

Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu

165 170 175

Ile Lys Wing Ile Wing Be Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val

180 185 190

Ser Asp Glu Wing Leu Wing Leu Glu Lys Wing Gly Thr Leu Gly Glu Gln

195 200 205

Ile Phe Ser Ile Glu Gly Thr Val Gln Gly Gly Asp

210 215 220

Glu Met 225

<210> 12

<211> 71

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MISC FEATURE <223> FW1-3 4-59 [FW = 1-25; FW2 = 26-39; FW3 = 40-71] <400> 12

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 15 10 15

Thr Read Be Read Thr Cys Thr Val Be Trp Ile Arg Gln Pro Pro Gly

20 25 30

Lys Gly Leu Glu Trp Ile Gly Arg Val Thr Ile Be Val Asp Thr Be

35 40 45

Lys Asn Gln Phe To Be Read Lys Asn To Be Read Val Thr Wing Asp Thr

50 55 60

Val Tyr Wing Tyr Cys Arg 65 Wing 70

<210> 13

<211> 11

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<223> FW4 4-59 [NCBI Entry gi / 33583] <4 0 0> 13

Trp Gly Gln Gly Thr Read Val Thr Val Ser Ser 15 10

<210> 14

<211> 27

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> VHL-for [previous primer]

<4 0 0> 14

ccatggctgt cttggggctg ctcttct 27

<210> 15

<211> 17

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature

<223> HC-rev [reverse primer]

<400> 15 ggggccagtg gatagac

<210> 16

<211> 28

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> VLL-for [previous primer]

<400> 16

ccatggattt tcaagtgcag attttcag 28

<210> 17

<211> 17

<212> DNA

<213> Homo Sapiens

<22 O>

<221> misc_feature

<223> LCkappa-rev [reverse primer] <400> 17

gttggtgcag catcagc 17

<210> 18

<211> 318

<212> DNA

<213> Homo Sapiens

<22 O>

<221> misc_feature

<223> TMC-22 06 VL

<2 2 O>

<221> CDS

<222> (1). . (318) <400> 18

caa ttt gtt ctc acc cag Gln Phe Val Leu Thr Gln 1 5

gag aag gtc acc atg acc Glu Lys Val Thr Met Thr 20

cac tgg tac cag aag his Trp Tyr Gln Gln Lys 35

gac act tcc aaa ctg gt Asp Thr Ser Lys Leu Wing 50

gga tct ggg acc tct tac Gly Ser Gly Thr Ser Tyr 65 70

gat gct gcc act tat tac Asp Wing Wing Wing Thr Tyr Tyr

85

ttc ggt gct ggg acc agg Phe Gly Wing Gly Thr Arg 100

tct cca gca ttc ttg Ser Pro Ala Phe Leu 10

tgc agt gcc aac tca Cys Ser Ala Asn Ser 25

tca ggc acc tcc ccc Ser Gly Thr Ser Pro 40

tct gga gtc cct gtt Ser Gly Val Pro Val 55

tct ctc aca till age Ser Leu Thr Ile Ser

75

cg cag cag tgg act Cys Gln Gln Trp Thr 90

gtg gag ctg aaa Val Glu Leu Lys 105

tct gct tct cca ggg 48 Ser Ala Ser Pro Gly 15

agt gtg aat tac att 96 Ser Val Asn Tyr Ile 30

aaa aaa tgg att tat 144 Lys Lys Trp Ile Tyr 45

cgc ttc agt ggc agt 192 Arg Phe Ser Gly Ser 60

age atg gag act gag 240 Ser Met Glu Thr Glu 80

act aac cca ac ct 288 Thr Asn Pro Leu Thr 95

318

<210> 19

<211> 106

<212> PRT

<213> Homo Sapiens

<400> 19

Gln Phe Val Leu Thr Gln Be Pro Wing Phe Leu Be Wing Be Pro Gly 1 5 10 15

Glu Lys Val Thr Met Thr Cys To Be Wing Asn To Be Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Being Gly Thr Being Pro Lys Lys Trp Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Val Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Be Tyr Be Read Thr Ile Be Be Met Glu Thr Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Wing Gly Thr Arg Val Glu Leu Lys 100 105

<210> 20

<211> 357

<212> DNA

<213> Homo sapiens <220>

<221> CDS

<222> (1). . (357)

<220>

<221> misc_feature

<222> (1). . (357)

<223> TMC-2206 VH

<400> 20

cag gtg cag ttg aag gag Gln Val Gln Leu Lys Glu 1 5

age ctg tcc until act tgt Ser Leu Ser Ile Thr Cys 20

ggt att cac tgg gtt cgc Gly Ile His Trp Val Arg 35

gga gtg ata tgg gct cgt Gly Val Ile Trp Wing Arg 50

tcc aga ctg until till aca Ser Arg Leu Ile Ile Thr 65 70

aaa atg aac agt cta caa Lys Met Asn Ser Leu Gln

85

aga gcg aac gac ggg gtc Arg Ala Asn Asp Gly Val 100

acc tca gtc acc gtc tcc Thr Ser Val Thr Val Ser 115

tca gga cct ggc ctg Ser Gly Pro Gly Leu 10

act gtc tet gga tt Thr Val Ser Gly Phe 25

cag cct cca gga aag Gln Pro Pro Gly Lys 40

gga ttc aca aat tat Gly Phe Thr Asn Tyr 55

aaa gac aat tcc cag Lys Asp Asn Ser Gln

75

cct gat gac tca gcc Pro Asp Asp Ser Wing 90

tat tat gct atg gac Tyr Tyr Ala Met Asp 105

tca Ser

gtg gcg ccc tca cag 48 Val Wing Pro Ser Gln 15

tca tta acc aac tat 96 Ser Leu Thr Asn Tyr 30

ggt ctg gag tgg ctg 144 Gly Leu Glu Trp Leu 45

aat tcg gct ctc atg 192 Asn Ser Ala Leu Met 60

agt caa gtc ttc tta 240 Ser Gln Val Phe Leu

80

act tac ttc tgt gcc 288 Thr Tyr Phe Cys Ala 95

tac tgg ggt cag gga 33 6 Tyr Trp Gly Gln Gly 110

357

<210> 21

<211> 119

<212> PRT

<213> Homo sapiens

<4 0 0> 21

Gln Val Gln Leu Lys Glu Ser Gly 1 5

Ser Leu Ser Ile Thr Cys Thr Val 20

Gly Ile His Trp Val Arg Gln Pro

35 40

Gly Val Ile Trp Arg Wing Gly Phe 50 55

Pro Gly Leu Val Wing Pro Ser Gln

10 15

Be Gly Phe Be Read Thr Asn Tyr 25 30

Pro Gly Lys Gly Leu Glu Trp Leu 45

Thr Asn Tyr Asn Be Wing Read Met 60 Be Arg Read Le Ile Ile Thr Lys Asp 65 70

Lys Met Asn Being Read Gln Pro Asp

85

Arg Wing Asn Asp Gly Val Tyr Tyr 100

Thr Ser Val Thr Thr Val Ser 115

Asn Be Gln Be Gln Val Phe Leu

75 80

Asp Ser Wing Thr Tyr Phe Cys Wing

90 95

Met Asp Wing Tyr Trp Gly Gln Gly 105 HO

<210> 22

<211> 31

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> TMC-2206-r51 [previous primer] <400> 22

cccgaattca caggtgcagt tgaaggagtc at 31

<210> 23

<211> 35

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> TMC-2206-r3 '[reverse primer] <400> 23

cgggatcctt aggatcattt accaggagag tggga 35

<210> 24

<211> 31

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> TMC-2206-k51 [previous primer]

<400> 24

cccgaattca caatttgttc tcacccagtc t

31 <210> 25

<211> 35

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> TMC-22 0 6-k3 '[reverse primer] <400> 25

cgggatcctt atctctaaca ctcattcctg ttgaa 35

<210> 26

<211> 22

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE

<223> Igkappa (Igk) leader sequence <400> 26

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 15 10 15

Gly Gly Being Thr Gly Asp 20

<210> 27

<211> 76

<212> DNA

<213> Homo Sapiens

<22 O>

<221> misc_feature

<223> Igkappa-S Oligonucleotides [Primer] <400> 27

tcgagccacc atggagacag acacactcct gctatgggta ctgctgctct gggttccagg 60 ttccactgga gacgcg 76

<210> 28

<211> 76

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature

<223> Igkappa-AS Oligonucleotides [Primer] <400> 28

aattcgcgtc tccagtggaa cctggaaccc agagcagcag tacccatagc aggagtgtgt 60 ctgtctccat ggtggc 76

<210> 29 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> TMC22 06VH-hIgGl / 4Fc-SalI

<400> 29

cttggtcgac gctgaggaga cggtgactga ggt 33

<210> 30

<211> 30

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hIgGl / 4Fc-SalI-F [previous primer] <400> 30

tcagcgtcga ccaagggccc atcsgtcttc 30

<210> 31 <211> 48 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hIgGl / 4Fc-NotI-R [reverse primer]

<400> 31 aagggaagcg gccgcttatc atttacccyg agacagggag aggctctt

<210> 32 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> TMC2206VL-hKc-SalI [reverse primer] <400> 32

tcgtttgatg tcgaccttgg tcccagcacc gaacgtgag

<210> 33 <211> 35 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hKc-SalI-F [previous primer] <400> 33

accaaggtcg acatcaaacg aactgtggct gcacc

<210> 34 <211> 45 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hKc-Notl-R [reverse primer]

<400> 34

aagggaagcg gccgcttatc arcactctcc cctgttgaag ctctt

<210> 35

<211> 29

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature

<223> TMC-22O6VLwt-hKc-F [previous primer] <4 0 0> 35

agggtggagc tgaaacgaac tgtggctgc 29

<210> 36

<211> 24

<212> DNA

<213> Homo Sapiens

<2 2 0>

<221> misc_feature

<223> TMC-2206VLwt-hKc-R [reverse primer] <4 00> 36

tcgtttcagc tccaccctgg tccc ??? 24

<210> 37

<211> 107

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MISC_FEATURE

<223> A14 VL germ line protein

<4 00> 37 Val Asp Val Val Met Thr Gln Be Pro Wing Phe Leu Be Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Gln Gln Lys Pro 40 Asp Gln Ala Pro Lys 45 Read Leu Ile Lys Tyr 50 Ala Ser Gln Ser Ile 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ser Ple Thr Ile Ser Sera Glu Wing 65 70 75 80 Glu Asp Wing Ward Thr 85 Tyr Tyr Cys Gln Gln 90 Trp Thr Thr Asn Pro 95 Leu Thr Phe Gly Gln

100 105 <210> 38

<211> 10

<212> PRT

<213> Homo

Sapiens

<220>

<221> MISC_FEATURE

<223> FW 4 of developed kappa light chain <400> 38

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10

<210> 39 <211> 121 <212> PRT <213> Homo Sapiens

<220>

<221> MIS C_FEATURE

<223> 4-59 VH germline protein <400> 39

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 1 5 10 15

Thr Read Be Read Thr Thr Cys Thr Val Be Gly Gly Be Ile Be Ser Tyr

20 25 30

Tyr Trp Being Trle Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Be Gly Tyr Asn Tyr Asn Pro Be Read Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80

Lys Read Ser Be Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Wing

85 90 95

Arg His Asn Be Being Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Read Val Val Val Ser Ser 115 120

<210> 40

<211> 119

<212> PRT

<213> Homo Sapiens <220>

<221> MISC_FEATURE

<223> hVHl.0

<400> 40

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 1 5 10 15 Thr Leu Be Leu 20 Thr Cys Thr Val Be 25 Gly Phe Be Leu Thr 30 Asn Tyr Gly Ile His 35 Trp Val Arg Gln Pro 40 Pro Gly Lys Gly Leu 45 Glu Trp Leu Gly Val 50 Ile Trp Arg Wing Gly 55 Phe Thr Asn Tyr Asn 60 Be Wing Leu Met Ser Arg Leu Thr Ile As Lys Asp Ser Val 85 Thr Wing Asp Thr 90 Wing Val Tyr Phe Cys 95 Wing Arg Wing Asn Asp Gly Val Tyr Tyr Wing Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val 115 Thr Val Ser Ser

<210> 41

<211> 106

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MIS C_FEATURE <223> hVLl.0

<400> 41 Gln Phe Val Leu Thr Gln Be Pro Wing Phe Leu Be Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr 20 Ile Thr Cys Be Wing 25 Asn Be Ser Val Asn 30 Tyr Ile His Trp Tyr 35 Gln Gln Lys Pro Asp 40 Gln Wing Pro Lys Lys 45 Trp Ile Tyr Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Be Leu Glu 65 70 75 80 Asp Wing Wing Thr Tyr 85 Tyr Cys Gln Gln Trp 90 Thr Asn Pro Leu 95 Thr Phe Gly Gln Gly 100 Thr Lys Val Glu Ile 105 Lys <210> 42

<211> 48

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH3.O-F [previous primer]

<400> 42

agcgtggaca ccagcaagaa ccagttcagc ctgaagctga gcagcgtg 48

<210> 43

<211> 51

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH3.0-R [reverse primer]

<400> 43

gttcttgctg gtgtccacgc tgatggtcac gcgggacatg agagcgctgt t 51

<210> 44

<211> 48

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH4.0-F [previous primer]

<400> 44

cctccaggca agggcctgga gtggatcggc gtgatatggg ctcgcggc 48

<210> 45

<211> 51

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH4.O-R [reverse primer] ctccaggccc ttgcctggag gctggcgtat ccagtggatg ccatagttgg

<210> 46 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hVL3.O-F [previous primer]

<4 0 0> 46

cccaagctcc tgatctatga cacttccaag ctg

<210> 47 <211> 42 <212> DNA <213> Homo

<22 0>

<221> misc_feature

<223> hVL3.O-R [reverse primer]

<400> 47

agtgtcatag atcaggagct tgggggcctg gtcgggcttc tg

<210> 48 <211> 45 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hVL4.O-F [previous primer]

<4 0 0> 48

gacgcgaatt cagacgtggt gatgacccag tctccagcat tcctg

<210> 49 <211> 24 <212> DNA <213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH2.O-F [previous primer]

<400> 49

gtgaccatca gcaaggacaa cagc ??? 24

<210> 50

<211> 45

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH2.O-R [reverse primer]

<400> 50

gctgttgtcc ttgctgatgg tcacgcggga catgagagcg ctgtt 45

<210> 51

<211> 27

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH5.0-F [previous primer]

<400> 51

atcggcgtga tatgggctcg cggcttc 27

<210> 52

<211> 45

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH5.0-R [reverse primer]

<400> 52

gccgcgagcc catatcacgc cgatccactc caggcccttg cctgg

45 <210> 53

<211> 24

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH6.O-F [previous primer]

<400> 53

atatgggctc gcggcttcac aaac 24

<210> 54

<211> 24

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH6.0-R [reverse primer]

<400> 54

gtttgtgaag ccgcgagccc atat 24

<210> 55

<211> 45

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH7.O-F [previous primer]

<400> 55

gccgcggaca ccgccgtgta ctactgcgcc agagccaacg acggg 45

<210> 56

<211> 27

<212> DNA

<213> Homo Sapiens

<22O> <221> misc_feature

<223> hVH7.0-R [reverse primer]

<400> 56

gtagtacacg gcggtgtccg cggcggt 2 7

<210> 57

<211> 27

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH8.O-F [previous primer]

<400> 57

atatccaact atggcatcca ctgggtt 27

<210> 58

<211> 48

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVH8.0-R [reverse primer]

<400> 58

ccagtggatg ccatagttgg atatgctaaa tccagagacg gtacaggt 48

<210> 59

<211> 45

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVL2.O-R [reverse primer]

<400> 59

cagcttggaa gtgtcataga tcaatttctt gggggcctgg tcggg

45 <210> 60 <211> 45 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hVL5.O-F [previous primer]

<400> 60

gacgcgaatt cagacttcgt gctgacccag tctccagcat tcctg 45

<210> 61

<211> 45

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVL6.O-F [previous primer]

<4 0 0> 61

gacgcgaatt cacagttcgt gatgacccag tctccagcat tcctg 45

<210> 62 <211> 45 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hVL7.O-F [previous primer]

<4 0 0> 62

gacgcgaatt cagacttcgt gatgacccag tctccagcat tcctg 45

<210> 63

<211> 27

<212> DNA

<213> Homo Sapiens

<220>

<221> misc feature <223> hVL8.O-F [previous primer] <400> 63

ttcaccttca ccatcagcag cctggag 27

<210> 64

<211> 48

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hVL8.O-R [reverse primer]

<4 0 0> 64

ctccaggctg ctgatggtga aggtgaagtc ggtgccgctg ccgctgcc 48

<210> 65

<211> 28

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hLCQ3-F [previous primer]

<4 0 0> 65

ccaatcaagc gtgaactaca ttcactgg 28

<210> 66

<211> 48

<212> DNA

<213> Homo Sapiens

<2 2 0>

<221> misc_feature

<223> hLCQ3-R [reverse primer]

<4 0 0> 66

ccagtgaatg tagttcacgc ttgattgggc gctgcaggtg atggtcac 48 <210> 67 <211> 23 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> Igk-For [previous primer]

<400> 67

actcctgcta tgggtactgc tgc ??? 23

<210> 68 <211> 22 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> hlgGlFc-CHl-R [primer

<400> 68

gaagtagtcc ttgaccaggc ag

reverse]

22

<210> 69 <211> 22 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> CI-neo-msc3 '[Primer]

<400> 69

tttcactgca ttctagttgt gg 22

<210> 70

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC FEATURE <223> hVH2.0 <400> 70

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 15 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr

20 25 30

Gly Ile His Trp Val Arg Pro Gln Gly Lys Gly Leu

35 40 45

Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Be Wing Read Met 50 55 60

Be Arg Val Thr Ile Be Lys Asp Asn Be Lys Asn Gln Val Ser Leu 65 70 75 80

Lys Read Ser Be Val Thr Wing Asp Thr Wing Val Tyr Phe Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 71

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE <223> hVH3.0

<400> 71

Gln Val Gln Leu Gln Glu Ser Gly 1 5

Thr Leu Be Leu Thr Cys Thr Val 20

Gly Ile His Trp Val Arg Gln Pro

35 40

Gly Val Ile Trp Arg Wing Gly Phe

50 55

Ser Arg Val Thr Ile Ser Val Asp 65 70

Lys Reads Being Ser Val Thr Wing

85

Arg Wing Asn Asp Gly Val Tyr Tyr 100

Thr Leu Val Thr Val Ser Ser 115

Pro Gly Leu Val Lys Pro Be Glu 10 15 Ser Gly Phe Ser Leu Thr Asn Tyr 25 30 Pro Gly Lys Gly Leu Glu Trp Leu 45 Thr Asn Tyr Asn Ser Wing Leu Met 60 Thr Ser Lys Asn Gln Phe Ser Leu 75 80 Asp Thr Val Wing Tyr Phe Cys Wing 90 95 Wing Met Asp Tyr Trp Gly Gln Gly 105 110 <210> 72

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE <223> hVH4.0

<400> 72

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 15 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr

20 25 30

Gly Ile His Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Be Wing Read Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80

Lys Read Ser Be Val Thr Wing Asp Thr Wing Val Tyr Phe Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 73

<211> 119

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MISC_FEATURE <223> hVH5.0

<4 00> 73 Gln Val Gln Leu Gln 1 5

Thr Leu Ser Leu Thr 20

Gly Ile His Trp Val 35

Gly Val Ile Trp Wing 50

Ser Arg Leu Thr Ile 65

Glu Ser Gly Pro Gly

10

Cys Thr Val Ser Gly 25

Arg Gln Pro Pro Gly 40

Arg Gly Phe Thr Asn 55

Ser Lys Asp Asn Ser 70

Read Val Lys Pro To Be Glu

15

Phe Ser Leu Thr Asn Tyr 30

Lys Gly Leu Glu Trp Ile 45

Tyr Asn Ser Ala Leu Met 60

Lys Asn Gln Val To Be Read 75 80 Lys Asu To Be To Be Val Thr Wing Asp Thr Wing Val Tyr Phe Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 74

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE <2 2 3> hVH6.0

<4 0 0> 74 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu 20 Thr Cys Thr Val Ser 25 Gly Phe Ser Leu Thr 30 Asn Tyr Gly Ile His 35 Trp Val Arg Gln Pro 40 Pro Gly Lys Gly Leu 45 Glu Trp Ile Gly Val Ile Trp Wing Arg Gly Phe Thr Asn Tyr Asn Be Wing Leu Met 50 55 60 Be Arg Val Thr Ile Be Lys Asn 70 75 80 Lys Leu Be Ser Val 85 Thr Wing Asp Wing Thr 90 Wing Val Tyr Phe Cys 95 Wing Arg Wing Asn Asp Gly Val

<210> 75

<211> 119

<212> PRT

<213> Homo Sapiens

<2 2 0>

<221> MISC_FEATURE <223> hVH7.0

<4 00> 75

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Read Val Lys Pro Be Glu 15 10 15 Thr Read Le Be Read Thr Cys Thr Val 20

Gly Ile His Trp Val Arg Gln Pro

35 40

Gly Val Ile Trp Arg Wing Gly Phe

50 55

Ser Arg Leu Thr Ile Ser Lys Asp 65 70

Lys Reads Being Ser Val Thr Wing

85

Arg Wing Asn Asp Gly Val Tyr Tyr 100

Thr Leu Val Thr Val Ser Ser 115

Ser Gly Phe Ser Leu Thr Asn Tyr 25 30 Pro Gly Lys Gly Leu Glu Trp Leu 45 Thr Asn Tyr Asn Ser Wing Leu Met 60 Asn Ser Lys Asn Gln Val Ser Leu 75 80 Asp Thr Wing Val Tyr Cyr Wing 90 95 Ala Met Asp Tyr Trp Gly Gln Gly 105 110

<210> 76

<211> 119

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_FE <223> hVH8.0

<400> 76

Gln Val Gln Leu 1

Thr Leu Ser Leu 20

Gly Ile His Trp 35

Gly Val Ile Trp 50

Ser Arg Leu Thr 65

Lys Leu Ser Ser

Arg Wing Asn Asp 100

Thr Leu Val Thr 115

Gln Glu Ser Gly 5

Thr Cys Thr Val

Val Arg Gln Pro 40

Wing Arg Gly Phe 55

Ile Ser Lys Asp 70

Val Thr Wing Ala 85

Gly Val Tyr

Pro Gly Leu Val 10

Ser Gly Phe Ser 25

Pro Gly Lys Gly

Thr Asn Tyr Asn 60

Asn Ser Lys Asn 75

Asp Thr Wing Val 90

Met Asp Tyr 105 Wing

Lys Pro Ser Glu 15

Ile Ser Asn Tyr 30

Leu Glu Trp Leu 45

Ser Ala Met

Gln Phe Ser Leu 80

Tyr Phe Cys Wing 95

Trp Gly Gln Gly 110

<210> 77

<211> 119

<212> PRT

<213> Homo Sapiens

<220> <221> MISC_FEATURE

<223> hVH9.Position 48 can be Leucine or Isoleucine <400> 77

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 15 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Be Wing Read Met

50 55 60

Ser Arg Leu Thr Ile Ser Val Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80

Lys Read Ser Be Val Thr Wing Asp Thr Wing Val Tyr Phe Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 78

<211> 119

<212> PRT

<213> Homo Sapiens

<2 2 0>

<221> MISC_FE <223> hVHIO.O

<4 0 0> 78 Gln Val Gln Leu 1

Thr Leu Ser Leu 20

Gly Ile His Trp 35

Gly Val Ile Trp 50

Ser Arg Leu Thr 65

Lys Leu Ser Ser

Arg Wing Asn Asp 100

Thr Leu Val Thr 115

Gln Glu Ser Gly 5

Thr Cys Thr Val

Val Arg Gln Pro 40

Wing Arg Gly Phe 55

Ile Ser Lys Asp 70

Val Thr Wing Ala 85

Gly Val Tyr

Pro Gly Leu Val 10

Ser Gly Phe Ser 25

Pro Gly Lys Gly

Thr Asn Tyr Asn 60

Thr Ser Lys Asn 75

Asp Thr Wing Val 90

Met Asp Tyr 105 Wing

Lys Pro Ser Glu 15

Read Thr Asn Tyr 30

Leu Glu Trp Leu 45

Ser Ala Met

Gln Val Ser Leu 80

Tyr Phe Cys Wing 95

Trp Gly Gln Gly 110 <210> 79

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<223> hVHll.Position 48 can be Leucine or Isoleucine <400> 79

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 15 10 15

Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Be Wing Read Met

50 55 60

Be Arg Leu Thr Ile Be Lys Asp Asn Be Lys Asn Gln Phe Be Leu 65 70 75 80

Lys Read Ser Be Val Thr Wing Asp Thr Wing Val Tyr Phe Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 80

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE <223> hVL2.0

<400> 80

Gln Phe Val Leu Thr Gln Phe Pro Wing Phe Leu Be Val Thr Pro Gly 15 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Asn Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Lys Pro Wing Lys Lys Read Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser 50 55 60 Gly Be Gly Thr Asp Tyr Thr Phe Thr Ile Be Be Leu Glu Wing Glu

65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 81

<211> 106

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MIS C_FEATURE <223> hVL3.0

<400> 81

Gln Phe Val Leu Thr Gln Phe Pro Wing Phe Leu Be Val Thr Pro Gly 15 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Asn Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Lys Pro Wing Lys Leu Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Serve Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 82

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE <223> hVL4.0

<400> 82

Asp Val Val Met Thr Gln Be Pro Wing Phe Read Be Val Thr Pro Gly 15 10 15

Glu Lys Val Thr Ile Thr Cys Be Ala Asn Ser Be Val Asn Tyr Ile 20 25 30 His Trp Tyr Gln Lys Pro Asp 35 40

Asp Thr Ser Lys Leu Wing Ser Gly 50 55

Gly Ser Gly Thr Asp Tyr Thr Phe 65 70

Asp Wing Wing Thr Tyr Tyr Cys Gln 85

Phe Gly Gln Gly Thr Lys Val Glu 100

Gln Wing Pro Lys Lys Trp Ile Tyr 45

Val Pro Ser Arg Phe Ser Gly Ser 60

Thr Ile Ser Serve Leu Glu Wing Glu 75 80

Gln Trp Thr Thr Asn Pro Read Thr 90 95

Ile Lys 105

<210> 83

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE

<223> hVL5.0

<400> 83

Asp Phe Val Leu Thr Gln Ser Pro 1 5

Glu Lys Val Thr Ile Thr Cys Ser 20

His Trp Tyr Gln Gln Lys Pro Asp 35 40

Asp Thr Ser Lys Leu Wing Ser Gly 50 55

Gly Ser Gly Thr Asp Tyr Thr Phe 65 70

Asp Wing Wing Thr Tyr Tyr Cys Gln 85

Phe Gly Gln Gly Thr Lys Val Glu 100

Phe Wing Read Ser Val Thr Pro Gly 10 15

Wing Asn Ser Ser Val Asn Tyr Ile 25 30

Gln Wing Pro Lys Lys Trp Ile Tyr 45

Val Pro Ser Arg Phe Ser Gly Ser 60

Thr Ile Ser Serve Leu Glu Wing Glu 75 80

Gln Trp Thr Thr Asn Pro Read Thr 90 95

Ile Lys 105

<210> 84

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE <223> hVL6.0

<400>

84 Gln Phe Val Met Thr Gln Ser Pro 1 5

Glu Lys Val Thr Ile Thr Cys Ser 20

His Trp Tyr Gln Gln Lys Pro Asp

35 40

Asp Thr Be Lys Leu Wing Be Gly

50 55

Gly Ser Gly Thr Asp Tyr Thr Phe 65 70

Asp Wing Wing Thr Tyr Tyr Cys Gln

85

Phe Gly Gln Gly Thr Lys Val Glu 100

Phe Wing Read Ser Val Thr Pro Gly

10 15

Wing Asn Ser Ser Val Asn Tyr Ile 25 30

Gln Wing Pro Lys Lys Trp Ile Tyr

45

Val Pro Ser Arg Phe Ser Gly Ser 60

Thr Ile Ser Ser Leu Glu Wing Glu

75 80

Gln Trp Thr Thr

90 95

Ile Lys 105

<210> 85

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE <223> hVL7.0

<400> 85

Asp Phe Val Met Thr Gln Be Pro Wing Phe Read Be Val Thr Pro Gly 1 5 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Asn Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Wing Pro Lys Lys Trp Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Serve Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 86

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE <223> hVL8.0 <400> 86

Gln Phe Val Leu Thr Gln Ser Pro 1 5

Glu Lys Val Thr Ile Thr Cys Ser 20

His Trp Tyr Gln Gln Lys Pro Asp

35 40

Asp Thr Be Lys Leu Wing Be Gly

50 55

Gly Ser Gly Thr Asp Phe Thr Phe 65 70

Asp Wing Wing Thr Tyr Tyr Cys Gln

85

Phe Gly Gln Gly Thr Lys Val Glu 100

Phe Wing Read Ser Val Thr Pro Gly

10 15

Wing Asn Ser Ser Val Asn Tyr Ile 25 30

Gln Wing Pro Lys Lys Trp Ile Tyr

45

Val Pro Ser Arg Phe Ser Gly Ser 60

Thr Ile Ser Ser Leu Glu Wing Glu

75 80

Gln Trp Thr Thr

90 95

Ile Lys 105

<210> 87

<211> 106

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE <223> hVL9.0

<400> 87 Gln Phe Val Leu Thr 1 5

Glu Lys Val Thr Ile 20

His Trp Tyr Gln Gln 35

Asp Thr Ser Lys Leu 50

Gly Ser Gly Thr Asp 65

Asp Wing Wing Thr Tyr

85

Phe Gly Gln Gly Thr 100

Gln Ser Pro Ala Phe

10

Thr Cys Ser Asn Wing 25

Lys Pro Asp Gln Wing 40

Ser Gly Val Pro 55 Wing

Tyr Thr Phe Thr Ile 70

Tyr Cys Gln Gln Trp

90

Lys Val Glu Ile Lys 105

Read Ser Val Thr Pro Gly

15

Ser Ser Val Asn Tyr Ile 30

Pro Lys Lys Trp Ile Lys 45

Ser Arg Phe Ser Gly Ser 60

Ser Ser Leu Glu Ala Glu 75 80

Thr Thr Asn Pro Read Thr

95

<210> 88

<211> 106

<212> PRT

<213> Homo Sapiens

<22 0> <221> MISC_FEATURE <223> hVLlO.O

<400> 88

Asp Phe Val Met Thr Gln Be Pro Wing Phe Read Be Val Thr Pro Gly 1 5 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Asn Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Lys Pro Wing Lys Lys Read Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Serve Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 89

<211> 106

<212> PRT

<213> Homo Sapiens

<2 2 O>

<221> MIS C_FEATURE <223> hVLll.O

<400> 89

Asp Phe Val Met Thr Gln Be Pro Wing Phe Read Be Val Thr Pro Gly 15 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Asn Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Wing Pro Lys Lys Trp Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Being Seru Read Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 90

<211> 106

<212> PRT

<213> Homo Sapiens <220>

<221> MIS C_FEATURE <223> hVLl.OQ

<4OO> 90

Gln Phe Val Leu Thr Gln Phe Pro Wing Phe Leu Be Val Thr Pro Gly 1 5 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Gln Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Wing Pro Lys Lys Trp Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Serve Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 91

<211> 106

<212> PRT

<213> Homo Sapiens

<22O>

<221> MISC_FEATURE <223> hVLlO.OQ

<4O0> 91 Asp Phe Val Met Thr Gln Being Pro Wing Phe Leu Being Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr 20 Ile Thr Cys Being Wing 25 Gln Being Ser Val Asn 30 Tyr Ile His Trp Tyr 35 Gln Gln Lys Pro Asp 40 Gln Wing Pro Lys Lys 45 Leu Ile Tyr Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Be Leu Glu 65 70 75 80 Asp Wing Wing Thr Tyr 85 Tyr Cys Gln Gln Trp 90 Thr Asn Pro Leu 95 Thr Phe Gly Gln Gly 100 Thr Lys Val Glu Ile 105 Lys

<210> 92 <211> 106 <212> PRT <213> Homo Sapiens

<220>

<221> MIS C_FEATURE <223> hVL12.OQ

<400> 92

Asp Phe Val Met Thr Gln Be Pro Wing Phe Read Be Val Thr Pro Gly 15 10 15

Glu Lys Val Thr Ile Thr Cys Be Wing Gln Be Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Lys Pro Asp Gln Lys Pro Wing Lys Lys Read Ile Tyr

35 40 45

Asp Thr Be Lys Leu Wing Be Gly Val Pro Be Arg Phe Be Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Being Seru Read Glu Wing Glu 65 70 75 80

Asp Wing Ward Thr Tyr Tyr Cys Gln Gln Thr Thr Thr Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 93

<211> 222

<212> PRT

<213> Rattus rattus

<220>

<221> MISC_FEATURE

<223> Mouse alpha-2 integrin domain I protein <4 0 0> 93

Be Pro Asp Phe Gln Be Read Thr Be Phe Ser Pro Wing Val Gln Asp 1 5 10 15

Val Val Val Val Val Cys Asp Glu Ser Asn Ser Ile Tyr Pro Trp Glu Wing

20 25 30

Val Lys Asn Phe Leu Glu Lys Phe Val Gln Gly Leu Asp Ile Gly Pro

35 40 45

Lys Lys Thr Gln Val Wing Read Ile Gln Tyr Wing Asn Asp Pro Arg Val

50 55 60

Val Phe Asn Read Thr Thr Tyr Lys Asn Lys Glu Asp Met Val Gln Wing 65 70 75 80

Thr Be Glu Thr Arg Gln Tyr Gly Gly Asp Read Thr Asn Thr Phe Lys

85 90 95

Wing Ile Gln Phe Wing Arg Asp Ile Wing Tyr Leu Pro Glu Ser Gly Gly

100 105 110

Arg Pro Gly Wing Thr Lys Val Val Val Val Val Val As Thr Gly Glu Ser 115 120 125 His Asp Gly Ser 130

Glu Ile Leu Arg 145

Wing Read Asp Thr

Thr Pro Thr Glu 180

Read Glu Lys Wing 195

Thr Val Gln Gly 210

Lys Leu Gln Thr 135

Phe Gly Ile Wing 150

Lys Asn Leu Ile 165

Arg Tyr Phe Phe

Gly Thr Read Gly 200

Gly Asp Asn Phe 215

Val Ile Gln Gln 140

Val Leu Gly Tyr 155

Lys Glu Ile Lys 170

Asn Val Wing Asp 185

Glu His Ile Phe

Gln Met Glu Met 220

Cys Asn Asp Asp

Read Asn Arg Asn 160

Ala Ile Ala Ser 175

Glu Wing Wing Leu 190

Ser Ile Glu Gly 205

Gln Wing

<210> 94

<211> 228

<212> PRT

<213> Mus

musculus

<220>

<221> MISC_FEATURE

<223> Mouse alpha-2 integrin domain I protein

<400> 94 Be Pro Asp Phe Gln Phe Read Thr Be Phe Pro Pro Wing Val Gln Wing 1 5 10 15 Cys Pro Be Read Val Asp Val Val Val Lys Asn Phe Leu Val Lys Phe Val 35 35 45 Gly Leu Asp Ile Gly Pro Lys Lys Thr Gln Val Wing Leu Ile Gln Tyr 50 55 60 Wing Asn Glu Pro Arg Ile Ile Phe Asn Leu Asn Pp Glu Thr Lys 65 70 75 80 Glu Asp Met Val Gln Wing Thr Thr Be Glu Thr Arg Gln His Gly Gly Asp 85 90 95 Leu Thr Asn Thr Phe Arg Wing Ile Glu Phe Wing Arg Asp Tyr Wing Tyr 100 105 110 Be Gln Thr Be Gly Gly Arg Pro Gly Wing Thr Lys Val Met Val Val 115 120 125 Val Thr Asp Gly Glu Be His Asp Gly Be Lys Leu Lys Thr Val Ile 130 135 140 Gln Cys Asn Asp Asp Glu Ile Leu Arg Phe Gly Ile Wing Val Leu 145 150 155 160 Gly Tyr Leu Asn Arg Asn Wing Read Asp Thr Lys Wing Asn Read Ile Lys Glu 165 170 175 Ile Lys Wing Ile Wing Ser Thr Thr Thr Glu Arg Tyr Phe Phe Asn Val 180 185 190 Ser Asp Glu Wing Wing Read Leu Glu Lys Wing Gly Thr Leu Gly Glu Gln 195 200 205 Ile Phe Ser Ile Glu Gly Thr Val

210 215

Glu Met Ser Gln 225

Gln Gly Gly Asp Asn Phe Gln Met 220

<210> 95

<211> 26

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> Kappa-F

<4 0 0> 95

cgaactgtgg ctgcaccatc tgtctt 26

<210> 96

<211> 41

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> Kappa-BamHI-R

<4 00> 96

aattcggatc cttactaaca ctctcccctg ttgaagctct t ??? 41

<210> 97

<211> 40

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> VH12.0- (K71V) -F

<4 0 0> 97

gcctgaccat cagcgtggac aacagcaaga accaggtgag 40

<210> 98 <211> 40 <212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> VH12.0 (K71V) -R

<400> 98

ctcacctggt tcttgctgtt gtccacgctg atggtcaggc

<210> 99 <211> 40 <212> DNA <213> Homo

<22 0>

<221> misc_feature <223> VHl3.0- (N73T) -F

<400> 99

ctgaccatca gcaaggacac cagcaagaac caggtgagcc

<210> 100 <211> 40 <212> DNA <2 13> Homo

<220>

<221> misc_feature <223> VH13.0- (N73T) R

<400> 100

ggctcacctg gttcttgctg gtgtccttgc tgatggtcag

<210> 101

<211> 41

<212> DNA

<213> Homo

<220>

<221> misc

<2 2 3> VH14

Sapiens

feature 0- (V78F) -F

<400> 101 gcaaggacaa cagcaagaac cagtttagcc tgaagctgag c 41

<210> 102

<211> 41

<212> DNA

<213> Homo Sapiens

<22 0>

<221> misc_feature <223> VH14.0 (V78F) -R

<400> 102

gctcagcttc aggctaaact ggttcttgct gttgtccttg c 41

<210> 103

<211> 648

<212> DNA

<213> Monkey

fascicularis

<220>

<221> misc_feature

<223> Cynomolugus alpha-2 domain I

<4 O O> 103

agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60

atagatgttg tggttgtgtg tgatgaatca aatagtattt atccttggga tgcagtagac 120

aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180

ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240

gaagaaatga ttgtagcaac atcccagaca tcccaatatg gtggggacct cacaaacaca 3 00

ttcggagcaa ttcaatatgc aagaaaatat gcctattcag cagcttctgg tgggcgacga 360

agtgctacga aagtaatggt agttgtaact gacggtgaat cacatgatgg ttcaatgttg 420

aaagctgtga ttgatcaatg caaccatgac aatatactga ggtttggcat agcagttctt 480

gggtacttaa acagaaacgc ccttgatact aaaaatttaa taaaagaaat aaaagcgatc 540

gctagtattc caacagaaag atactttttc aatgtgtctg atgaagcagc tctactagaa 600

aaggctggga cattaggaga acaaattttc agcattgaag gtactgtt 648

<210> 104 <211> 648 <212> DNA

<213> Mulatta macaque

<220>

<221> misc_feature

<223> Rhesus alpha-2 domain I

<4 0 0> 104 agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60

atagatgttg tggttgtgtg tgatgaatca aatagtattt atccttggga tgcagtaaag 120

aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180

ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240

gaagaaatga ttgtagcaac atcccagaca tcccaatatg gtggggacct cacaaacaca 300

ttcggagcaa ttcaatatgc aagaaaatat gcctattcag cagcttctgg tgggcgacga 360

agtgctacga aagtaatggt agttgtaact gacggtgaat cacatgatgg ttcaatgttg 420

aaagctgtga ttgatcaatg caaccatgac aatatactga ggtttggcat agcagttctt 480

gggtacttaa acagaaacgc ccttgatact aaaaatttaa taaaagaaat aaaagcgatc 540

gctagtattc caacagaaag atactttttc aatgtgtctg atgaagcagc tctactagaa 600

aaggctggga cattaggaga acaaattttc agcattgaag gtactgtt 648

<210> 105

<211> 997

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> IgG4 constant region

<220>

<221> CDS

<222> (1). . (978)

<4 0 0> 105

tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg age 48

Be Thr Lys Gly Pro Be Val Phe Pro Read Wing Pro Cys Be Arg Be

15 10 15

acc tcc gag age aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc 96

Thr Be Glu Be Thr Wing Wing Read Gly Cys Read Val Lys Asp Tyr Phe

20 25 30

ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc age ggc 144

Pro Glu Pro Val Thr Val Ser Tr Asn Ser Gly Wing Leu Thr Ser Gly

35 40 45

gtg cac acc ttc ccg gct gtc cta ctc tc tca gga ctc tcc tc 192

Val His Thr Phe Pro Wing Val Leu Gln Ser Gly Leu Tyr Ser Leu

50 55 60

age age gtg gtg acc gtg ccc cc age age ttg ggc acg aag acc tac 240

Be Ser Val Val Thr Val Pro Be Ser Leu Gly Thr Lys Thr Tyr 65 70 75 80

acc tgc aac gta gat aag ccc age aac acc aag gtg gac aag aga 288

Thr Cys Asn Val Asp His Lys Pro Be Asn Thr Cys Val Asp Lys Arg

85 90 95

gtt gag cc cc aaa tat ggt ccc cca tgc cca tca tgc cca gea cct gag 336

Val Glu Be Lys Tyr Gly Pro Pro Cys Pro Be Cys Pro Wing Pro Glu

100 105 110

ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag gac 384

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 act CtC atg to tcc cgg acc cct gg gtg gtg gtg gac 432 Thr Leu Met Ile Ser Arg Thr Pro Cys Val Val Val Asp 130 135 140 gtg age cag gaa gac CCC gag gtc cag ttc aac tgg tac gtg gat ggc 480 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 gtg gag gtg cat aat gcc aag aca aag cc g cgg gag gag cag ttc aac 528 Val Glu Val His Asn Alys Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 age acg tac cgt gtg age gtc etc acc gtc ctg cac cag gac tgg 576 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 ctg aac ggc aag gag tac ag tgc aag gtc tcc aac aaa ggc etc ccg 624 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 tcc tcc until gag aaa acc until tcc aaa gcc aaa ggg cag CCC cga gag 672 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 cca cag gtg tac acc ctg CCC cca tcc cag gag gag atg acc aag aac 720 Pro Gln Val Tyr Thr Leu Pro Pro Gln Glu Glu Met Thr Lys Asn 225 230 235 240 cag gtc age ctg acc tgc ctg gtc aaa ggc ttc tac CCC age gac up 768 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 gcc gtg gag tgg gag age aat ggg cag aac aac tac aag acc 816 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 acg cct gtg ctg gtc tcc gac tcc ttc ttc ttc ttc tac age agg 864 Thr Pro Pro Val Leu Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285 cta acc gtg gac aag age agg tgg cag gag ggg aat gtc ttc tca tgc 912 Leu Thr Val Asp Lys Ser Arg Trp Gln Gly Asn Val Phe Ser Cys 290 295 300 tcc gtg atg cat gag gct ctg cac aac cac tac aag age ctc 960 Ser Val Met His Glu Ala Leu Read His Asn His Tyr Thr cc ctg tet ctg ggt aaa tgataggatc cgcggccgc 997 Ser Leu Ser Leu Gly Lys 325 <210> 106 <211> 326 <212> Hom <Sap> <4 00> 106 Ser Thr Lys Gly Pro Val Phe Pro Cys Be Arg Be 1 5 10 15 Thr Be Glu Be Thr Wing Ward Leu Gly Cys Read Val Val Lys Asp Tyr Phe 20 25 30 Pro Glu Pro Val Thr Val Ser Tr Asn Be Gly Wing Thr Be Gly

35 40 45 Val His Thr Phe 50

Ser Ser Val Val 65

Thr Cys Asn Val

Val Glu Ser Lys 100

Phe Leu Gly Gly 115

Thr Leu Met Ile 130

Val Ser Gln Glu 145

Val Glu Val His

Ser Thr Tyr Arg 180

Read Asn Gly Lys 195

Ser Ser Ile Glu 210

Pro Gln Val Tyr 225

Gln Val Ser Leu

Val Glu Trp 260 Wing

Thr Pro Pro Val 275

Read Thr Val Asp 290

Ser Val Met His 305

Be Read Be Read

Pro Wing Val Leu 55

Thr Val Pro Ser 70

Asp His Lys Pro 85

Tyr Gly Pro Pro

Pro Ser Val Phe 120

Being Arg Thr Pro 135

Asp Pro Glu Val 150

Asn Wing Lys Thr 165

Val Val Ser Val

Glu Tyr Lys Cys 200

Lys Thr Ile Ser 215

Thr Leu Pro Pro 230

Thr Cys Leu Val 245

Glu Ser Asn Gly

Read Asp Ser Asp 280

Lys Ser Arg Trp 295

Glu Wing Leu His

310 Gly Lys 325

Gln Ser Ser Gly 60

Ser Ser Leu Gly 75

Ser Asn Thr Lys 90

Cys Pro Ser Cys 105

Read Phe Pro Pro

Glu Val Thr Cys 140

Gln Phe Asn Trp 155

Lys Pro Arg Glu 170

Leu Thr Val Leu 185

Lys Val Ser Asn

Lys Wing Lys Gly 220

Ser Gln Glu Glu 235

Lys Gly Phe Tyr 250

Gln Pro Glu Asn 265

Gly Ser Phe Phe

Gln Glu Gly Asn 300

Asn His Tyr Thr 315

Leu Tyr Ser Leu

Thr Lys Thr Tyr 80

Val Asp Lys Arg 95

Pro Wing Pro Glu 110

Lys Pro Lys Asp 125

Val Val Val Asp

Tyr Val Asp Gly 160

Glu Gln Phe Asn 175

His Gln Asp Trp 190

Lys Gly Leu Pro 205

Gln Pro Arg Glu

Met Thr Lys Asn 240

Pro Ser Asp Ile 255

Asn Tyr Lys Thr 270

Tyr Ser Arg 285

Val Phe Ser Cys

Gln Lys Ser Leu 320

<210> 107

<211> 648

<212> DNA

<213> HOMO SAPIENS

<220>

<221> misc_feature

<223> Human alpha-2 domain 1

<4 0 0> 107

agtcctgatt ttcagctctc agccagcttc tcacctgcaa ctcagccctg cccttccctc 60

atagatgttg tggtggtgtg tgatgaatca aatagtattt atccttggga tgcagtaaag 120

aattttttgg aaaaatttgt acaaggcctg gatataggcc ccacaaagac acaggtgggg 180

ttaattcagt atgccaataa tccaagagtt gtgtttaact tgaacacata taaaaccaaa 240 gaagaaatga ttcggagcaa agtgctacga aaagctgtga gggtacttaa gctagtattc aaggctggga

ttgtagcaac ttcaatatgc aagtaatggt ttgatcaatg acagaaacgc caacagaaag cattaggaga

atcccagaca aagaaaatat agttgtaact caaccatgac ccttgatact atactttttc acaaattttc

tcccaatatg gcctattcag gacggtgaat aatatactga aaaaatttaa aatgtgtctg agcattgaag

gtggggacct cacaaacaca 300 cagcttctgg tgggcgacga 360 cacatgatgg ttcaatgttg 420 ggtttggcat agcagttctt 480 taaaagaaat aaaagcgatc 540 atgaagcagc tctactagaa 600 gtactgtt 648

<210> 108

<211> 106

<212> PRT

<213> Homo Sapiens

<22 0>

<221> MISC_FEATURE <223> hVL12.0

<4 0 0> 108 Asp Phe Val Met Thr Gln Be Pro Wing Phe Leu Be Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr 20 Ile Thr Cys Be Wing 25 Asn Be Ser Val Asn 30 Tyr Ile His Trp Tyr 35 Gln Gln Lys Pro Asp 40 Gln Wing Pro Lys Lys 45 Leu Ile Tyr Asp Thr 50 Ser Lys Leu Wing To Be 55 Gly Val Pro To Be Arg 60 Phe To Be Gly To Be Gly To As Gly Thr Asp Phe Thr 70 75 80 Asp Wing Wing Thr Thr Tyr 85 Tyr Cys Gln Gln Trp 90 Thr Asn Pro Leu 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 109

<211> 119

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<223> hVH12.0 Position 48 can be Leucine or Isoleucine <400> 109

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 1 5 10 15

Thr Read Be Read Read Thr Cys Thr Val Be Gly Phe Be Read Thr Asn Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro

35 40

Gly Val Ile Trp Arg Wing Gly Phe

50 55

Ser Arg Leu Thr Ile Ser Lys Asp 65 70

Lys Reads Being Ser Val Thr Wing

85

Arg Wing Asn Asp Gly Val Tyr Tyr 100

Thr Leu Val Thr Val Ser Ser 115

Pro Gly Lys Gly Leu Glu Trp Leu 45

Thr Asn Tyr Asn Ser Ala Leu Met 60

Asn Ser Lys Asn Gln Val Ser Leu

75 80

Asp Thr Wing Val Tyr Tyr Cys Wing

90 95

Met Asp Wing Tyr Trp Gly Gln Gly 105 HO

<210> 110 <211> 119 <212> PRT <213> Homo Sapiens

<220>

<221> MISC_FEATURE <223> hVH13.0

<400> 110

Gln Val Gln Leu 1

Thr Leu Ser Leu 20

Gly Ile His Trp 35

Gly Val Ile Trp 50

Ser Arg Val Thr 65

Lys Leu Ser Ser

Arg Wing Asn Asp 100

Thr Leu Val Thr 115

Gln Glu Ser Gly 5

Thr Cys Thr Val

Val Arg Gln Pro 40

Wing Arg Gly Phe 55

Ile Ser Lys Asp 70

Val Thr Wing Ala 85

Gly Val Tyr

Pro Gly Leu Val 10

Ser Gly Phe Ser 25

Pro Gly Lys Gly

Thr Asn Tyr Asn 60

Asn Ser Lys Asn 75

Asp Thr Wing Val 90

Met Asp Tyr 105 Wing

Lys Pro Ser Glu 15

Read Thr Asn Tyr 30

Read Glu Trp Ile 45

Ser Ala Met

Gln Val Ser Leu 80

Tyr Tyr Cys Wing 95

Trp Gly Gln Gly 110

<210> 111

<211> 119 <212> PRT <213> Homo Sapiens

<220>

<221> MISC FEATURE <223> hVH14.0 Position 48 can be Leucine or Isoleucine <400> 111 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Wing Arg Gly Phe Thr Asn Tyr Asn Ser Wing Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Wing Asp Thr Wing Val Tyr Tyr Cys Wing 85 90 95 Arg Wing Asn Asp Gly Val Tyr Tyr Wing Met Asp Tyr Trp Gly Gln Gly 100 105 110

Thr Leu Val Thr Val Ser Ser

<210> 112

<211> 10

<212> PRT

<213> Homo Sapiens

<220>

<221> MISC_FEATURE

<223> LCDRl [N26Q]

<400> 112

Ser Ala Gln Ser Ser Val Asn Tyr Ile His 15 10

<210> 113

<211> 681

<212> DNA

<213> Rattus rattus

<220>

<221> misc_feature

<223> Rat alpha-2 domain I

<400> 113

agtccagact ttcagtcgtt gacaagcttc tcacctgcag ttcaagcttg cccttccctc 60 gtagatgtcg tagttgtctg tgatgaatca aacagtattt atccctggga agcagtaaag 120 aattttttgg aaaagtttgt gcaaggcctg gatataggac ctaaaaagac acaggtggcg 180 ttaattcaat atgccaacga cccaagagtt gtctttaact tgaccactta caaaaacaaa 240 gaagatatgg ttcaggccac atccgagacg cgccagtatg gtggggacct 300 cacaaacacc

ttcaaggcta tccaatttgc aagagacatt gcttatttac cggagtctgg cgggcgccca 360

ggtgctacaa aagtcatggt agttgtgact gatggggaat cccatgatgg gtcgaagctg 420

caaactgtga tccagcaatg caatgatgac gagatactga ggtttggcat agcggttctt 480

ggatatttaa acagaaatgc tcttgatact aaaaatctaa tcaaagaaat taaagcaatc 540

gctagcactc caacggagag gtactttttc aatgtggccg atgaggcggc tcttctggag 600

aaagctggca ctctagggga gcacatattc agcattgaag gcactgttca aggaggagac 660

aacttccaga tggaaatggc a 681

<210> 114

<211> 648

<212> DNA

<213> Mus

musculus

<22 0>

<221> misc_feature

<223> Mouse alpha-2 domain I clone

<4 0 0> 114

agtccagact ttcagttctt gaccagcttt tcacctgcag ttcaggcttg cccttccctc 60

gtggatgttg tagttgtatg tgatgaatca aacagtattt atccttggga agcagtaaag 120

aactttttgg taaagtttgt gacaggcctg gatataggac ctaaaaagac acaggtggcg 180

ttaattcaat atgccaatga gccgagaatt atatttaact tgaacgattt cgaaaccaaa 240

gaggatatgg tccaggccac atctgagacg cgccaacatg gtggggacct cacaaacacc 300

ttcagagcta tcgaattcgc aagagactac gcttattcac agacttctgg cgggcgcccg 360

ggtgctacaa aagtcatggt agttgtgacc gatggcgagt cccatgatgg gtcgaagctg 42 0

aaaactgtga tccagcaatg caatgatgac gagatactga ggttcggcat agcagttctt 480

gggtatttaa acagaaatgc tcttgatact aaaaatttaa tcaaagaaat aaaagcaatt 540

gctagtactc caaccgagag atactttttc aatgtggccg acgaagcggc tcttctggag 600

aaggctggaa ctctagggga gcaaatattc agcattgaag gcactgtt 648

<210> 115

<211> 6

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Example of upstream sequence from start of

gene transcription

<22 0>

<221> misc_feature

<222> (2). . (2)

<223> η is a, c, g, or t

<4 00> 115

cncaat 6 <210> 116

<211> 6

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Example of 3'-end sequence of gene <400> 116

aataaa 6

<210> 117 <211> 31 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal F

<400> 117

ggggatccag tcctgaattt tcagctctca g ??? 31

<210> 118 <211> 27 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal R

<400> 118

gggaattcaa cagtaccttc aatgctg 27

<210> 119

<211> 19

<212> DNA

<213> Homo sapiens

<22 0>

<221> misc feature <223> Human Primer Domain I Primer <400> 119

ggggatccag tcctgattt 19

<210> 120 <211> 18 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> Human Domain I Reverse Primer <4 0 0> 120

ggaattcaac agtacctt 18

<210> 121 <211> 29 <212> DNA <213> Homo

<220>

<221> misc_feature <223> maIphaI F

<4 0 0> 121

ggggatccag tccagacttt cagttcttg 29

<210> 122 <211> 28 <212> DNA <213> Homo

<220>

<221> misc_feature <223> malphal R

<4 0 0> 122

tgggaattca acagtgcctt caatgctg 28

<210> 123 <211> 31 <212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> ralphal F

<400> 123

ggggatccag tccagacttt cagtcgttga c ??? 31

<210> 124

<211> 31

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> ralphal R

<400> 124

tgggaattct gccatttcca tctggaagtt g ??? 31

<210> 125 <211> 34 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal I21V F

<400> 125

cagccctgcc cttccctcgt agatgttgtg gttg 34

<210> 126

<211> 34

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal I21V R <400> 126

caaccacaac atctacgagg gaagggcagg gctg 34

<210> 127 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal E44V F

<4 OO> 127

cagtaaagaa ttttttggta aaatttgtca agg 33

<210> 128 <211> 33 <212> DNA <213> Homo

<22 0>

<221> misc_feature <223> halphal E44V R

<4 0 0> 128

ccttgacaaa ttttaccaaa aaattcttta ctg 33

<210> 129 <211> 35 <212> DNA <213> Homo

<22 0>

<221> misc_feature <223> halphal Q48T F

<4 0 0> 129

ttttggaaaa atttgtaaca ggcctggata taggc 35

<210> 130

<211> 35

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature <223> halphal Q48T R

<400> 130

gcctatatcc aggcctgtta caaatttttc cgggg

<210> 131 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal N67E F

<400> 131

cagtatgcca atgagccaag agttgtgttt aac

<210> 132 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal N67E R

<400> 132

gttaaacaca actcttggct cattggcata ctg

<210> 133 <211> 34 <212> DNA <213> homo

<220>

<221> misc_feature <223> halphal V70I F

<400> 133

tgccaataat ccaagaattg tgtttaactt gaac <210> 134 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal V70I R

<400> 134

gttcaagtta acacaattct tggattattg gca

<210> 135 <211> 34 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal V71I F

<400> 135

ccaataatcc aagagttatc tttaacttga acac

<210> 136 <211> 34 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal V71I R

<400> 136

gtgttcaagt taaagataac tcttggatta ttgg

<210> 137 <211> 33 <212> DNA <213> Homo

<220> <221> misc feature <223> halphal T76D F <400> 137

gtgtttaact tgaacgacta taaaaccaaa gaa 33

<210> 138 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> haplhal T76D R

<400> 138

ttctttggtt ttatagtcgt tcaagttaaa cac 33

<210> 139 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Y77F F

<400> 139

tttaacttga acacatttaa aaccaaagaa gaa 33

<210> 140 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Y77F R

<400> 140

ttcttctttg gttttaaatg tgttcaagtt aaa 33

<210> 141 <211> 33 <212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal Κ78Ε F

<400> 141

aacttgaaca catatgaaac caaagaagaa atg

<210> 142 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal K78E R

<400> 142

catttcttct ttggtttcat atgtgttcaa gtt

<210> 143 <211> 33 <212> DNA <213> Homo

<22 0>

<221> misc_feature <223> halphal Y93H F

<4 0 0> 143

tcccagacat cccaacatgg tggggacctc aca

<210> 144

<211> 33

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> halphal Y93H R

<400>

144 tgtgaggtcc ccaccatgtt gggatgtctg gga

<210> 145 <211> 39 <212> DNA <213> Homo

<22O>

<221> misc_feature <223> halphal Y93F F

<4OO> 145

acatgggaga catcccaatt tggtggggac ctcacaaac

<210> 146 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Y93F R

<400> 146

gtttgtgagg tccccaccaa attgggatgt ctcccatgt

<210> 147 <211> 33 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Q105E F

<400> 147

ttcggagcaa ttgaatatgc aagaaaatat gcc

<210> 148

<211> 33

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature <223> halphal Q105E R

<400> 148

ggcatatttt cttgcatatt caattgctcc gaa 33

<210> 149 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal A114Q F

<400> 149

aaatatgcct attcacaagc ttctggtggg cgacgaagt 39

<210> 150 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal A114Q R

<400> 150

acttcgtcgc ccaccagaag cttgtgaata ggcatattt 3 9

<210> 151 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal A115T F

<400> 151

aaatatgcct attcagcaac ttctggtggg cgacgaagt

39 <210> 152

<211> 39

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal Α115Τ R

<400> 152

acttcgtcgc ccaccagaag ttgctgaata ggcatattt

<210> 153

<211> 39

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> halphal A115Q F

<4 0 0> 153

aaatatgcct attcagcaca gtctggtggg cgacgaagt

<210> 154

<211> 39

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal A115Q R

<4 0 0> 154

acttcgtcgc ccaccagact gtgctgaata ggcatattt

<210> 155

<211> 39

<212> DNA

<213> Homo Sapiens

<22 0>

<221> misc feature <223> halphal R165D F

<400> 155

gttcttgggt acttaaacga caacgccctt gatactaaa

<210> 156 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal R165D R

<400> 156

tttagtatca agggcgttgt cgtttaagta cccaagaac

<210> 157 <211> 39 <212> DNA <213> Homo

<22 O>

<221> misc_feature <223> halphal N166D F

<400> 157

cttgggtact taaacaggga cgcccttgat actaaaaat

<210> 158 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature

<223> halphal N166D R

<400> 158

atttttagta tcaagggcgt ccctgtttaa gtacccaag

<210> 159

<211> 40 <212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal E195W F

<400> 159

ttcaatgtgt ctgattgggc agctctacta gaaaaggctg 40

<210> 160 <211> 40 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal E195W R

<400> 160

cagccttttc tagtagagct gcccaatcag acacattgaa 40

<210> 161 <211> 38 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal K40D F

<400> 161

atccttggga tgcagtagac aattttttgg aaaaattt 38

<210> 162 <211> 38 <212> DNA <213> Homo Sapiens <220> <221> misc feature <223> halphal K4 0D

<400> 162 aaatttttcc aaaaaattgt ctactgcatc ccaaggat 38

<210> 163

<211> 39

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal R69D F

<400> 163

cagtatgcca ataatccaga cgttgtgttt aacttgaac 39

<210> 164

<211> 39

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> halphal R69D R

<400> 164

gttcaagtta aacacaacgt ctggattatt ggcatactg 3 9

<210> 165

<211> 36

<212> DNA

<213> Homo Sapiens

<2 2 0>

<221> misc_feature <223> halphal N73D F

<4 0 0> 165

aatccaagag ttgtgtttga cttgaacaca tataaa 36

<210> 166

<211> 36

<212> DNA

<213> Homo Sapiens <220>

<221> misc_feature <223> halphal N73D R

<400> 166

tttatatgtg ttcaagtcaa acacaactct tggatt

<210> 167 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Q89H F

<400> 167

atgattgtag caacatccca cacatcccaa tatggtggg

<210> 168 <211> 39 <212> DNA <213> Homo

<220>

<221> misc_feature <223> halphal Q89H R

<400> 168

atgattgtag caacatccca cacatcccaa tatggtggg

<210> 169 <211> 40 <212> DNA <213> Homo

<220>

<221> misc_feature <223> malphal H93Y F

<400> 169

cacatctgag acgcgccaat atggtgggga cctcacaaac <210> 170

<211> 40

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature <223> maIphaI Η93Υ R

<400> 170

gtttgtgagg tccccaccat attggcgcgt ctcagatgtg 40

<210> 171

<211> 216

<212> PRT

<213> Monkey

fascicularis

<220>

<221> MISC_FEATURE <223> Cynomologus

<400> 171

Be Pro Asp Phe Gln Read Be Wing Be Phe Be Pro Wing Thr Gln Pro 15 10 15

Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser

20 25 30

Ile Tyr Pro Trp Asp Val Wing Asp Asn Phe Leu Glu Lys Phe Val Gln

35 40 45

Gly Leu Asp Ile Gly Pro Lys Thr Thr Gln Val Gly Leu Ile Gln Tyr

50 55 60

Asn Wing Asn Pro Arg Val Val Phe Asn Read Asn Thr Tyr Lys Thr Lys 65 70 75 80

Glu Glu Met Ile Val Wing Thr Be Gln Thr Be Gln Tyr Gly Gly Asp

85 90 95

Read Thr Asn Thr Phe Gly Wing Ile Gln Tyr Wing Arg Lys Tyr Wing Tyr

100 105 110

Be Wing Wing Be Gly Gly Arg Arg Be Wing Thr Lys Val Met Val Val

115 120 125

Val Thr Asp Gly Glu Be His Asp Gly Be Met Leu Lys Wing Val Ile

130 135 140

Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Wing Val Leu 145 150 155 160

Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu

165 170 175

Ile Lys Wing Ile Wing Be Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val

180 185 190

Ser Asp Glu Wing Wing Read Leu Glu Lys Wing Gly Thr Leu Gly Glu Gln 195 200 205 Ile Phe Ser Ile Glu Gly Thr Val 210 215

<210> 172 <211> 216 <212> PRT

<213> Mulatta macaque

<220>

<221> MIS C_FEATURE <223> Rhesus

<400> 172

Be Pro Asp Phe Gln Read Be Wing Be Phe Be Pro Wing Thr Gln Pro 1 5 10 15

Cys Pro Ser Leu Ile Asp Val Val Val Val Cys Asp Glu Ser Asn Ser

20 25 30

Ile Tyr Pro Trp Asp Wing Val Lys Asn Phe Leu Glu Lys Phe Val Gln

35 40 45

Gly Leu Asp Ile Gly Pro Lys Thr Thr Gln Val Gly Leu Ile Gln Tyr

50 55 60

Asn Wing Asn Pro Arg Val Val Phe Asn Read Asn Thr Tyr Lys Thr Lys 65 70 75 80

Glu Glu Met Ile Val Wing Thr Be Gln Thr Be Gln Tyr Gly Gly Asp

85 90 95

Read Thr Asn Thr Phe Gly Wing Ile Gln Tyr Wing Arg Lys Tyr Wing Tyr

100 105 110

Be Wing Wing Be Gly Gly Arg Arg Be Wing Thr Lys Val Met Val Val

115 120 125

Val Thr Asp Gly Glu Be His Asp Gly Be Met Leu Lys Wing Val Ile

130 135 140

Asp Gln Cys Asn His Asp Asn Ile Leu Arg Phe Gly Ile Wing Val Leu 145 150 155 160

Gly Tyr Leu Asn Arg Asn Ala Leu Asp Thr Lys Asn Leu Ile Lys Glu

165 170 175

Ile Lys Wing Ile Wing Be Ile Pro Thr Glu Arg Tyr Phe Phe Asn Val

180 185 190

Ser Asp Glu Wing Leu Wing Leu Glu Lys Wing Gly Thr Leu Gly Glu Gln

195 200 205

Ile Phe Ser Ile Glu Gly Thr Val 210 215

<210> 173

<211> 1435

<212> DNA

<213> Homo Sapiens

<220> <221> misc_feature

<223> hvH14.0 IgG4 heavy chain

<220>

<221> CDS

<222> (22). . (1416)

<400> 173

tctcgagaag cttccaccat g gag aca gac aca ctc ctg cta

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 1 5 10

ctg ctc tgg gtt cca ggt tcc act gga cag gtg cag ttg cag gag tca 99 Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Valu Leu Gln Glu Ser

15 20 25

ggc cct ggc ctg gtg aag ccc age gag acc ctg age ctg acc tgt acc 147 Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr

30 35 40

gtc tet gga ttt age tta acc aac tat ggc till cac tgg ata cgc cag 195 Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Ile His Trp Ile Arg Gln

45 50 55

cct cca ggc aag ggc ctg gag tgg ctg ggc gtg ata tgg gct cgc ggc 243 Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Wing Arg Gly

60 65 70

ttc aca aac tat aac age gct ctc atg tcc cgc gtg acc until age aag 291 Phe Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys 75 80 85 90

gac aac age aag aac cag gtg age ctg age aag ctg age age gtg acc gcc 33 9 Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala

95 100 105

gcg gac acc gcc gtg tac tac tgc gcc aga gcc aac gac ggg gtc tac 3 87 Wing Asp Thr Wing Val Tyr Tyr Cys Arg Wing Asn Asp Gly Val Tyr

110 115 120

tat gcc atg gac tac tgg ggc cag gga acc ctg gtc acc gtc age tca 435 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

125 130 135

gcg tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 483 Wing Ser Thr Lys Gly Pro Ser Val Phe Pro Read Wing Pro Cys Ser Arg

140 145 150

age acc tcc gag age aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 531 Ser Thr Be Glu Ser Thr Wing Wing Leu Gly Cys Leu Val Lys Asp Tyr 155 160 165 170

tcc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 579 Phe Pro Glu Pro Val Thr Val Ser Tr Asn Ser Gly Ala Leu Thr Ser

175 180 185

ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 627 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

190 195 200

ctc age age gtg gtg acc gtg ccc tcc age age ttg ggc acg aag acc 675 Le Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr

205 210 215

tac acc tgc aac gta gat cac aag ccc age aac acc aag gtg gac aag 723 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 220 225 230 aga gtt gag tcc aaa tat ggt ccc cca tgc cca tca tgc cca gca cct 771 Arg Val Glu Be Lys Tyr Gly Pro Pro Cys Pro Be Cys Pro Wing 235 240 245 250

gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc aaa ccc aag 819 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys

255 260 265

gac act ctc atg up to tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 867 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

270 275 280

gac gtg age cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 915 Asp Val Ser Gln Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

285 290 295

ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 963 Gly Val Glu Val His Asn Alys Lys Thr Lys Pro Arg Glu Glu Gln Phe

300 305 310

aac age acg tac cgt gtg gtc age gtc ctc acc gtc ctg cac cag gac 1011 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 315 320 325 330

tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 1059 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

335 340 345

ccg tcc tcc to gag aaa acc up to tcc aaa gcc aaa ggg cag ccc cga 1107 Pro Ser Ser Ile Glu Lys Thr Ser Ile Ser Lys Ally Lys Gly Gln Pro Arg

350 355 360

gag cca cag gtg tac acc ctg ccc tcc cag gag gag gag atg acc aag 1155 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

365 370 375

aac cag gtc age ctg acc tgc ctg gtc aaa ggc ttc tac ccc age gac 1203 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

380 385 390

up to gcc gtg gag tgg gag age aat ggg cag ccg gag aac aac tac aag 1251 Ile Wing Val Glu Trp Glu Ser Asn Gly Gln Pro

acc acg cct cct gtg ctg gac tcc gac ggc tcc ttc ttc tac age 1299 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

415 420 425

agg cta acc gtg gac aag age agg tgg cag gag ggg aat gtc ttc tca 1347 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

430 435 440

tgc tcc gtg atg cat gag gct ctg cac aac cac tac cag aag age 1395 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

445 450 455

ctc tcc ctg tet ctg ggt aaa tgataggatc cgcggccgc 1435

Read Be Read Read Be Gly Lys 460 465

<210> 174

<211> 465

<212> PRT

<213> Homo Sapiens

<400> 174 Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly 15 10 15

Be Gly Gn Gln Val Gln Leu Gln Glu Gly Pro Gly Leu Val Lys

20 25 30

Pro Be Glu Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read

35 40 45

Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Gly Lys Gly Leu

50 55 60

Glu Trp Read Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Ser 65 70 75 80

Wing Read Met Be Arg Val Thr Ile Be Lys Asp Asn Be Lys Asn Gln

85 90 95

Val Be Leu Lys Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr

100 105 110

Tyr Cys Wing Arg Wing Asn Asp Gly Val Tyr Tyr Wing Met Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Read Val Val Val Be Ser Wing Be Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Read Wing Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160

Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His Thr Phe Pro

180 185 190

Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Thr

195 200 205

Val Pro Be Ser Be Read Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

210 215 220

His Lys Pro Be Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 225 230 235 240

Gly Pro Pro Cys Pro Be Cys Pro Wing Pro Glu Phe Leu Gly Gly Pro

245 250 255

Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

275 280 285

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Wing Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Be Thr Tyr Arg Val 305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro To Be Ile Glu Lys

340 345 350

Thr Ile Ser Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Pro Leu Pro Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Wing Val Glu Trp Glu 385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu

405 410 415 Asp Be Asp Gly Be Phe Phe Read Tyr Be Arg Read Leu Thr Val Asp Lys

420 425 430

Being Arg Trp Gln Glu Gly Asn Val Phe Being Cys Being Val Met His Glu

435 440 445

Wing Read His Asn His Tyr Thr Gln Lys Be Read Be Read Be Read Gly 450 455 460

Lys 465

<210> 175

<211> 1435

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> hvH12.0 IgG4 heavy chain

<220>

<221> CDS

<222> (22). . (1416)

<400> 175

tctcgagaag cttccaccat g gag aca gac aca ctc ctg cta tgg gta ctg 51

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu 15 10

ctg ctc tgg gtt cca ggt tcc act gga cag gtg cag ttg cag gag tca 99

Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Ser

15 20 25

ggc cct ggc ctg gtg aag ccc age gag acc ctg age ctg acc tgt acc 147

Gly Pro Gly Read Val Lys Pro Be Glu Thr

30 35 40

gtc tet gga ttt age tta acc aac tat ggc until cac tgg ata cgc cag 195

Val Be Gly Phe Be Read Thr Asn Tyr Gly Ile His Trp Ile Arg Gln

45 50 55

cct cca ggc aag ggc ctg gag tgg ctg ggc gtg ata tgg gct cgc ggc 243

Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Wing Arg Gly

60 65 70

ttc aca aac tat aac age gct ctc atg tcc cgc ctg acc until age aag 291

Phe Thr Asn Tyr Asn To Be Wing Read Met To Be Arg Read Le I Thr Be Lys 75 80 85 90

gac aac age aag aac cag gtg age ctg age aag ctg age age gtg acc gcc 339

Asp Asn Be Lys Asn Gln Val Be Read Lys Lys Read Be Be Val Thr Wing

95 100 105

gcg gac acc gcc gtg tac tac tgc gcc aga gcc aac gac ggg gtc tac 387

Wing Asp Thr Wing Val Tyr Tyr Cys Wing Arg Wing Asn Asp Gly Val Tyr

110 115 120

tat gcc atg gac tac tgg ggc cag gga acc ctg gtc acc gtc age tca 435

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Read Val Thr Val Ser Ser 125 130 135 gcg tcc acc aag Wing Ser Thr Lys 140

age acc tcc gag Ser Thr Ser Glu 155

tc ccc gaa ccg Phe Pro Glu Pro

ggc gtg cac acc Gly Val His Thr 190

ctc age age gtg Leu Ser Ser Val 205

tac acc tgc aac Tyr Thr Cys Asn 220

aga gtt gag tcc Arg Val Glu Ser 235

gag ttc ctg ggg Glu Phe Leu Gly

gac act ctc atg Asp Thr Leu Met 270

gac gtg age cag Asp Val Ser Gln 285

ggc gtg gag gtg Gly Val Glu Val 300

aac age acg tac Asn Ser Thr Tyr 315

tgg ctg aac ggc Trp Leu Asn Gly

ccg tcc tcc until Pro Ser Ser Ile 350

gag cca cag gtg Glu Pro Gln Val 365

aac cag age Asn Gln Val Ser 380

until gcc gtg gag Ile Ala Val Glu 395

ggc cca tcc gtc Gly Pro Ser Val 145

age aca gcc gcc Ser Thr Ala Ala 160

gtg acg gtg tcg Val Thr Val Ser 175

ttc ccg gct gtc Phe Pro Val Wing

gtg acc gtg ccc Val Thr Val Pro 210

gta gat cac aag Val Asp His Lys 225

aaa tat ggt ccc Lys Tyr Gly Pro 240

gga cca tca gtc Gly Pro Ser Val 255

until tcc cgg acc Ile Ser Arg Thr

gaa cc cc gag Glu Asp Pro Glu 290

cat aat gcc aag His Asn Ala Lys 305

cgt gtg gtc age Arg Val Val Ser 320

aag gag tac aag Lys Glu Tyr Lys 335

gag aaa acc up to Glu Lys Thr Ile

tac acc ctg ccc Tyr Thr Leu Pro 370

ctg acc tgc ctg Leu Thr Cys Leu 385

tgg gag age aat Trp Glu Ser Asn 400

cct ccc ctg gcg ccc Phe Pro Read Ala Pro 150

ctg ggc tgc ctg gtc Leu Gly Cys Leu Val 165

tgg aac tca ggc gcc Trp Asn Ser Gly Wing 180

cta cag tcc tca gga Read Gln Ser Ser Gly 195

tcc age age ttg ggc Ser Ser Ser Leu Gly 215

ccc acts aac acc aag Pro Ser Asn Thr Lys 230

cca tgc cca tca cgt Pro Cys Pro Ser Cys 245

ttc ctg ttc ccc cca Phe Leu Phe Pro Pro 260

cct gag gtc acg tgc Pro Glu Val Thr Cys 275

gtc cag ttc aac tgg Val Gln Phe Asn Trp

295

aca aag ccg cgg gag Thr Lys Pro Arg Glu 310

gtc ctc acc gtc ctg Val Leu Thr Val Leu 325

tgc aag gtc acc cys lys val ser asn 340

tcc aaa gcc aaa ggg Ser Lys Ala Lys Gly 355

cca tcc cag gag gag Pro Ser Gln Glu Glu 375

gtc aaa ggc ttc tac Val Lys Gly Phe Tyr 390

ggg cag ccg gag aac Gly Gln Pro Glu Asn 405

tgc tcc agg 483 Cys Ser Arg

aag gac tac 531 Lys Asp Tyr 170

ctg acc age 579 Leu Thr Ser 185

ctc tac tcc 627 Leu Tyr Ser 200

acg aag acc 675 Thr Lys Thr

gtg gac aag 723 Val Asp Lys

cca gea cct 771 Pro Ala Pro 250

aaa ccc aag 819 Lys Pro Lys 265

gtg gtg gtg 867 Val Val Val 280

tac gtg gat 915 Tyr Val Asp

gag cag ttc 963 Glu Gln Phe

cac cag gac 1011 His Gln Asp 330

aaa ggc ctc 1059 Lys Gly Leu 345

cag ccc cga 1107 Gln Pro Arg 360

atg acc aag 1155 Met Thr Lys

ccc age gac 1203 Pro Ser Asp

aac tac aag 1251 Asn Tyr Lys 410 acg cct ccc gtg ctg gac tcc gac gcc tcc ttc ttc tac age 1299 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

415 420 425

agg cta acc gtg gac aag age agg tgg cag gag ggg aat gtc ttc tca 1347

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

430 435 440

tgc tcc gtg atg cat gag gct ctg cac aac cac tac a cag aag age 13 95

Cys Ser Val Met His Glu Wing Read His Asn His Tyr Thr Gln Lys Ser

445 450 455

ctc tcc ctg tet ctg ggt aaa tgataggatc cgcggccgc 1435

Read Be Read Read Be Gly Lys 460 465

<210> 176

<211> 465

<212> PRT

<213> Homo Sapiens

<4 0 0> 176

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly 1 5 10 15

Be Gly Gn Gln Val Gln Leu Gln Glu Gly Pro Gly Leu Val Lys

20 25 30

Pro Be Glu Thr Read Be Read Thr Cys Thr Val Be Gly Phe Be Read

35 40 45

Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Gly Lys Gly Leu

50 55 60

Glu Trp Read Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Ser 65 70 75 80

Wing Read Met Be Arg Read Le Thr Ile Be Lys Asp Asn Be Lys Asn Gln

85 90 95

Val Be Leu Lys Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr

100 105 110

Tyr Cys Wing Arg Wing Asn Asp Gly Val Tyr Tyr Wing Met Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Read Val Val Val Be Ser Wing Be Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Read Wing Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160

Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His Thr Phe Pro

180 185 190

Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Thr

195 200 205

Val Pro Be Ser Be Read Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

210 215 220

His Lys Pro Be Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 225 230 235 240

Gly Pro Pro Cys Pro Be Cys Pro Wing Pro Glu Phe Leu Gly Gly Pro

245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

275 280 285

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Wing Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Be Thr Tyr Arg Val 305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro To Be Ile Glu Lys

340 345 350

Thr Ile Ser Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Pro Leu Pro Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Wing Val Glu Trp Glu 385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu

405 410 415

Asp Be Asp Gly Be Phe Phe Read Tyr Be Arg Read Le Thr Thr Asp Lys

420 425 430

Being Arg Trp Gln Glu Gly Asn Val Phe Being Cys Being Val Met His Glu

435 440 445

Wing Read His Asn His Tyr Thr Gln Lys Be Read Be Read Be Read Gly 450 455 460

Lys 465

<210> 177 <211> 744 <212> DNA <213> Homo Sapeins

<220>

<221> misc_feature

<223> VL10.OQ Light Chain

<220>

<221> CDS

<222> (22). . (720)

<400> 177

ctatctcgag 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 10

ctg ctg ctg tgg gtt cca ggt tcc actgga gac ttc gtg atg acc cag 99

Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Phe Val Met Thr Gln

15 20 25 tct cca gca ttc ctg age gtg acc ccc ggc gag aag gtg acc until acc 147 Ser Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys Val Thr Ile Thr

30 35 40

tgc age gcc caa tca age gtg aac tac att cac tgg tac cag cag aag 195 Cys Ser Ala Gln Ser Ser Val Asn Tyr Ile His Trp Tyr Gln Gln Lys

45 50 55

ccc gac cag gcc ccc aag aaa ttg until tat gac act tcc aag ctg gcc 243 Pro Asp Gln Ala Pro Lys Ley Leu Ile Tyr Asp Thr Ser Lys Leu Ala

60 65 70

age ggc gtg ccc age cgc ttc age ggc age ggc age ggc acc gac tac 2 91 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Thr Asp Tyr 75 80 85 90

acc ttc acc until age age ctg gag gcc gag gac gct gcc acc tat tac 33 9 Thr Phe Thr Ile Ser Serve Leu Glu Wing Glu Asp Wing Wing Thr Tyr Tyr

95 100 105

tgc cag cag tgg acc act aac cca ctg acc ttc ggc cag ggc acc aag 387 Cys Gln Gln Trp Thr Thr Asn Pro Read Thr Phe Gly Gln Gly Thr Lys

110 115 120

gtc gaa till aaa cga act gtg gct gca cca tct gtc ttc till ttc ccg 435 Val Glu Ile Lys Arg Thr Val Wing Ala Pro Ser Val Phe Ile Phe Pro

125 130 135

cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 483 Pro Ser Asp Glu Glu Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

140 145 150

ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 531 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 155 160 165 170

aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 579 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

175 180 185

age aag gac age acc tac age ctc age age acc ctg acg ctg age aaa 627 Ser Lys Asp Ser Thr Tyr Ser Read Ser Ser Thr Read Le Ser Thr

190 195 200

gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 675 Wing Asp Tyr Glu Lys His Lys Val Tyr Wing Cys Glu Val Thr His Gln

205 210 215

ggc ctg age tcg ccc gtc aca aag age ttc aac agg gga gag tgt 72 0

Gly Leu Be Ser Pro Val Thr Lys Be Phe Asn Arg Gly Glu Cys

220 225 230

tgataggatc cgcggccgca tagg 744

<210> 178

<211> 233

<212> PRT

<213> Homo Sapeins

<4 0 0> 178

Met Glu Thr Asp Thr Leu Leu Leu 1 5

Gly Ser Thr Gly Asp Phe Val Met 20

Trp Val Leu Leu Leu Trp Val Pro

10 15

Thr Gln Be Pro Wing Phe Leu Ser 25 30 Val Thr Pro Gly Glu Lys Val Thr Ile Thr Cys Be Wing Gln Be Ser 35 40 45 Val Asn Tyr Ile His Trp Tyr Gln Lys Pro Asp Gln Wing Pro Lys 50 55 60 Lys Leu Ile Tyr Asp Thr To Be Lys Leu Wing To Be Gly Val Pro To Be Arg 65 70 75 80 Phe To Be Gly Be Gly To Be Gly Thr Asp Tyr Thr To Phe Thr Ile To Be 85 90 95 Leu Glu Wing Glu Asp Wing To Thr Tyr Cys Gln Gln Trp Thr Thr 100 105 110 Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Wing Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Wing Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Be Read Be Read Thr Read Read Thr Read Lys Wing Asp Tyr Glu Lys His 195 00 205 Lys Val Tyr Cys Wing Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230

<210> 179

<211> 1460

<212> DNA

<213> Homo Sapiens

<220>

<221> misc_feature

<223> h2206-VH14 IgG1 heavy chain (DANS); Position -24 to -25 is the leading sequence; Positions -4 to -1 are extra amino acids; DANS at position -4 to -1 is preferably deleted.

<220>

<221> CDS

<222> (22). . (1440)

<220>

<221> mat_peptide <222> (94). . (1440)

<400> 179

ataggctagc ctcgagccac c atg gag aca gac aca ctc ctg cta tgg gta 51

Met Glu Thr Asp Thr Leu Leu Leu Trp Val

-20 -15 ctg ctg ctc tgg Leu Leu Leu Trp

cag ttg cag gag Gln Leu Gln Glu 5

age ctg acc tgt Ser Leu Thr Cys 20

cac tgg ata cgc His Trp Ile Arg 35

ata tgg gct cgc Ile Trp Wing Arg

gtg acc till age Val Thr Ile Ser 70

age age gtg acc Ser Ser Val Thr 85

aac gac ggg gtc Asn Asp Gly Val 100

gtc acc gtc age Val Thr Val Ser 115

gea cec tcc tcc Ala Pro Ser

ctg gtc aag gac Leu Val Lys Asp 150

ggc gcc ctg acc Gly Ala Leu Thr 165

tca gga ctc tac Ser Gly Leu Tyr 180

ttg ggc acc cag Read Gly Thr Gln 195

acc aag gtg gac Thr Lys Val Asp

aca tgc cca ccg Thr Cys Pro Pro 230

ttc CtC ttc CCC

Phe Leu Phe Pro 245

gtt cca ggt tcc Val Pro Gly Ser -10

tca ggc cct ggc Ser Gly Pro Gly 10

acc gtc tet gga Thr Val Ser Gly 25

cg cct cca ggc Gln Pro Pro Gly 40

ggc ttc aca aac Gly Phe Thr Asn 55

aag gac aac age Lys Asp Asn Ser

gcc gcg gac acc Wing Asp Thr 90

tac tat gcc atg Tyr Tyr Ala Met 105

tca gcg tcg acc Ser Ala Ser Thr 120

aag age acc tet Lys Ser Thr Ser 135

tac ttc ccc gaa Tyr Phe Pro Glu

age ggc gtg cac Ser Gly Val His 170

tcc ctc age age Ser Serve Ser Ser 185

acc tac to tgc Thr Tyr Ile Cys 200

aag aaa gtt gag Lys Lys Val Glu 215

tgc cca gea cct Cys Pro Ala Pro

cca aaa ccc aag Pro Lys Pro Lys 250

act gga gac gcg aat Thr Gly Asp Wing Asn -5

ctg gtg aag ccc age Read Val Lys Pro Ser

15

ttt age tta acc aac Phe Ser Leu Thr Asn 30

aag ggc ctg gag tgg Lys Gly Leu Glu Trp 45

tat aac age gct ctc Tyr Asn Ser Ala Leu 60

aag aac cag gtg age Lys Asn Gln Val Ser 75

gcc gtg tac tac tgc Wing Val Tyr Tyr Cys

95

gac tac tgg ggc cag Asp Tyr Trp Gly Gln 110

aag ggc cca tcg gtc Lys Gly Pro Ser Val 125

ggg ggc aca gcg gcc Gly Gly Thr Wing Wing 140

ccg gtg acg gtg Pro Val Thr Val Ser 155

acc ttc ccg gct gtc Thr Phe Pro Wing Val

175

gtg gtg acc gtg ccc Val Val Thr Val Pro 190

aac gtg aat cac aag Asn Val Asn His Lys 205

ccc aaa tet tgt gac Pro Lys Ser Cys Asp 220

gaa ctc ctg ggg gga Glu Leu Leu Gly Gly 235

gac acc ctc atg to Asp Thr Leu Met Ile

255

tca cag gtg 99

To be Gln Val

-1 1

gag acc ctg 147

Glu Thr Leu

tat ggc up to 195 Tyr Gly Ile

ctg ggc gtg 243 Leu Gly Val 50

atg tcc cgc 291 Met Ser Arg 65

ctg aag ctg 339 Leu Lys Leu 80

gcc aga gcc 3 87 Wing Arg Wing

gga acc ctg 435 Gly Thr Leu

ttc ccc ctg 483 Phe Pro Leu 130

ctg ggc tgc 531 Read Gly Cys 145

tgg aac tca 579 Trp Asn Ser 160

cta cag tcc 627 Read Gln Ser

tcc age age 675 Ser Ser Ser

ccc age aac 723 Pro Ser Asn 210

aaa act cac 771 Lys Thr His 225

ccg tca gtc 819 Pro Ser Val 240

tcc cgg acc 867 Ser Arg Thr cct gag gtc aca tgc gtg gtg gtg gac gtg age cac gaa gac cct gag 915 Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag 963 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 290

aca aag ccg gg gag gag cag tac aac age acg tac cgt gtg gtc age 1011 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

295 300 305

gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag 1059 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

310 315 320

tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc until gag aaa acc until 1107 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc 1155 Ser Lys Ally Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

cca tcc cgg gat gag ctg acc aag aac cag gtc age ctg acc tgc ctg 1203 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 370

gtc aaa ggc ttc tat ccc age gac till gcc gtg gag tgg gag age aat 1251 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

375 380 385

ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc 1299 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser

390 395 400

gac ggc tcc ttc ttc ctc tac aag ctc acc gtg gac aag age agg 1347 Asp

405 410 415

tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg 13 95 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

cac aac cac tac ag cag aag age ctc tcc ctg tet ccg ggt aaa 1440

His Asn His Tyr Thr Gln Lys Be Read Be Read Be Pro Gly Lys 435 440 445

tgataagcgg ccgcttccct 1460

<210> 180

<211> 473

<212> PRT

<213> Homo Sapiens

<4 0 0> 180

Met Glu Thr Asp Thr Leu Leu Leu

-20

Gly Ser Thr Gly Asp Wing Asn Ser

-5 -1

Pro Gly Leu Val Lys Pro Ser Glu

10 15

Be Gly Phe Be Read Thr Asn Tyr 25 30

Trp Val Leu Leu Leu Trp Val Pro

-15 -10

Gln Val Gln Leu Gln Glu Ser Gly 1 5

Thr Leu Be Leu Thr Cys Thr Val 20

Gly Ile His Trp Ile Arg Gln Pro 35 40 Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Wing Arg Gly Phe

45 50 55

Thr Asn Tyr Asn Be Wing Read Met Be Arg Val Thr Ile Be Lys Asp

60 65 70

Asn Be Lys Asn Gln Val Be Read Lys As Le Be Ser Val Thr Wing Ward

75 80 85

Asp Thr Wing Val Tyr Tyr Cys Wing Arg Wing Asn Asp Gly Val Tyr Tyr

90 95 100

Met Asp Wing Tyr Trp Gly Gln Gly Thr Read Val Val Val Ser Ser Wing 105 110 115 120

Be Thr Lys Gly Pro Be Val Phe Pro Read Wing Pro Be Ser Lys Ser

125 130 135

Thr Be Gly Gly Thr Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe

140 145 150

Pro Glu Pro Val Thr Val Ser Tr Asn Ser Gly Wing Leu Thr Ser Gly

155 160 165

Val His Thr Phe Pro Wing Val Leu Gln Ser Gly Leu Tyr Ser Leu

170 175 180

Be Ser Val Val Thr Val Pro Be Ser Leu Gly Thr Gln Thr Tyr 185 190 195 200

Ile Cys Asn Val Asn His Lys Pro Being Asn Thr Lys Val Asp Lys Lys

205 210 215

Val Glu Pro Lys Be Cys Asp Lys Thr His Cys Pro Pro Cys Pro

220 225 230

Pro Glu Wing Read Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

235 240 245

Pro Lys Asp Thr Read Met Ile Be Arg Thr Pro Glu Val Thr Cys Val

250 255 260

Val Val Asp Val Be His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 265 270 275 280

Val Asp Gly Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu

285 290 295

Gln Tyr Asn Be Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

300 305 310

Gln Asp Trp Read Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

315 320 325

Wing Leu Pro Wing Pro Ile Glu Lys Thr Ile Ser Lys Wing Lys Gly Gln

330 335 340

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Being Arg Asp Glu Leu 345 350 355 360

Thr Lys Asn Gln Val Ser Read Thr Cys Read Val Lys Gly Phe Tyr Pro

365 370 375

Be Asp Ile Wing Val Glu Trp Glu Be Asn Gly Gln Pro Glu Asn Asn

380 385 390

Tyr Lys Thr Pro Thr Val Leu Asp Be Asp Gly Be Phe Phe Leu

395 400 405

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

410 415 420

Phe Be Cys Be Val Met His Glu Wing Read His Asn His Tyr Thr Gln 425 430 435 440

Lys Be Read Be Read Be Pro Gly Lys

445 <210> 181

<211> 469

<212> PRT

<213> Homo Sapiens

<220>

<221> MIS C_FEATURE

<223> hVH14.0 IgG1 Heavy Chain [DANS-Deleted] <4OO> 181

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15

Gly Ser Thr Gly Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val

20 25 30

Lys Pro To Be Glu Thr Read To Be Read Thr Cys Thr Val To Be Gly Phe Ser

35 40 45

Read Thr Asn Tyr Gly Ile His Trp Ile Arg Gln Pro Gly Lys Gly

50 55 60

Read Glu Trp Read Gly Val Ile Trp Wing Arg Gly Phe Thr Asn Tyr Asn 65 70 75 80

Be Wing Read Met Be Arg Val Thr Ile Be Lys Asp Asn Be Lys Asn

85 90 95

Gln Val Ser Leu Lys Le Ser Ser Val Thr Wing Asp Thr Wing Val

100 105 110

Tyr Tyr Cys Wing Arg Wing Asn Asp Gly Val Tyr Tyr Wing Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Read Val Val Val Ser Be Wing Be Thr Lys Gly

130 135 140

Pro Be Val Phe Pro Read Wing Pro Be Ser Lys Ser Thr Be Gly Gly 145 150 155 160

Thr Wing Wing Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Be Trp Asn Be Gly Wing Read Thr Be Gly Val His Thr Phe

180 185 190

Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Le Ser Ser Val Val

195 200 205

Thr Val Pro Be Ser Be Read Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Being Asn Thr Lys Val Asp Lys Val Glu Pro Lys 225 230 235 240

Ser Cys Asp Lys Thr His Cys Pro Pro Cys Pro Wing Pro Glu Leu

245 250 255

Read Gly Gly Pro Be Val Phe Read Phe Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Be Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Be His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val

Asn Gly Lys Glu 340

Pro Ile Glu Lys 355

Gln Val Tyr Thr 370

Val Ser Leu Thr 385

Val Glu Trp Glu

Pro Pro Val Leu 420

Thr Val Asp Lys 435

Val Met His Glu 450

Read Ser Pro Gly 465

Val Ser Val Leu 325

Tyr Lys Cys Lys

Thr Ile Ser Lys 360

Leu Pro Pro Ser 375

Cys Leu Val Lys 390

Ser Asn Gly Gln 405

Asp Ser Asp Gly

Ser Arg Trp Gln 440

Wing Leu His Asn 455

Lys

Thr Val Leu His 330

Val Ser Asn Lys 345

Wing Lys Gly Gln

Arg Asp Glu Leu 380

Gly Phe Tyr Pro 395

Pro Glu Asn Asn 410

Ser Phe Phe Leu 425

Gln Gly Asn Val

His Tyr Thr Gln 460

Gln Asp Trp Leu 335

Wing Leu Pro Wing 350

Pro Arg Glu Pro 365

Thr Lys Asn Gln

Ser Asp Ile Wing 400

Tyr Lys Thr Thr 415

Tyr Ser Lys Leu 430

Phe Ser Cys Ser 445

Lys Ser Read Ser

<210> 182

<211> 469

<212> PRT

<213> Homo Sapeins

<220>

<221> MISC_FEATURE

<223> hVH12.0 IgGl Heavy Chain [DANS-Deleted]

<400> 182 Met Glu Thr Asp 1

Gly Ser Thr Gly 20

Lys Pro Ser Glu 35

Read Thr Asn Tyr 50

Leu Glu Trp Leu 65

Ser Ala Met

Gln Val Ser Leu 100

Tyr Tyr Cys Wing 115

Trp Gly Gln Gly 130

Thr Leu Leu Leu 5

Gln Val Gln Leu

Thr Leu Ser Leu 40

Gly Ile His Trp 55

Gly Val Ile Trp 70

Ser Arg Leu Thr 85

Lys Leu Ser Ser

Arg Wing Asn Asp 120

Thr Leu Val Thr 135

Trp Val Leu Leu 10

Gln Glu Ser Gly 25

Thr Cys Thr Val

Ile Arg Gln Pro 60

Wing Arg Gly Phe 75

Ile Ser Lys Asp 90

Val Thr Wing Wing 105

Gly Val Tyr Tyr

Val Ser Ser Ala 140

Read Trp Val Pro 15

Pro Gly Leu Val 30

Ser Gly Phe Ser 45

Pro Gly Lys Gly

Thr Asn Tyr Asn 80

Asn Ser Lys Asn 95

Asp Thr Wing Val 110

Met Asp Tyr 125 Wing

Being Thr Lys Gly Pro Being Val Phe 145

Thr Wing Wing Leu

Thr Val Ser Trp 180

Pro Wing Val Leu 195

Thr Val Pro Ser 210

Asn His Lys Pro 225

Be Cys Asp Lys

Read Gly Gly Pro 260

Read Met Ile Ser 275

Be His Glu Asp 290

Glu Val His Asn 305

Thr Tyr Arg Val

Asn Gly Lys Glu 340

Pro Ile Glu Lys 355

Gln Val Tyr Thr 370

Val Ser Leu Thr 385

Val Glu Trp Glu

Pro Pro Val Leu 420

Thr Val Asp Lys 435

Val Met His Glu 450

Read Ser Pro Gly 465

Pro Leu Wing Pro 150

Gly Cys Leu Val 165

Asn Ser Gly Wing

Gln Ser Ser Gly 200

Ser Ser Leu Gly 215

Ser Asn Thr Lys 230

Thr His Thr Cys 245

Ser Val Phe Leu

Arg Thr Pro Glu 280

Pro Glu Val Lys 295

Wing Lys Thr Lys 310

Val Ser Val Leu 325

Tyr Lys Cys Lys

Thr Ile Ser Lys 360

Leu Pro Pro Ser 375

Cys Leu Val Lys 390

Ser Asn Gly Gln 405

Asp Ser Asp Gly

Ser Arg Trp Gln 440

Wing Leu His Asn 455

Lys

Ser Ser Lys Ser 155

Lys Asp Tyr Phe 170

Read Thr Ser Gly 185

Leu Tyr Ser Leu

Thr Gln Thr Tyr 220

Val Asp Lys Lys 235

Pro Pro Cys Pro 250

Phe Pro Pro Lys 265

Val Thr Cys Val

Phe Asn Trp Tyr 300

Pro Arg Glu Glu 315

Thr Val Leu His 330

Val Ser Asn Lys 345

Wing Lys Gly Gln

Arg Asp Glu Leu 380

Gly Phe Tyr Pro 395

Pro Glu Asn Asn 410

Ser Phe Phe Leu 425

Gln Gly Asn Val

His Tyr Thr Gln 460

Thr Ser Gly Gly 160

Pro Glu Pro Val 175

Val His Thr Phe 190

Ser Ser Val Val 205

Ile Cys Asn Val

Val Glu Pro Lys 240

Pro Wing Glu Leu 255

Pro Lys Asp Thr 270

Val Val Asp Val 285

Val Asp Gly Val

Gln Tyr Asn Ser 320

Gln Asp Trp Leu 335

Wing Leu Pro Wing 350

Pro Arg Glu Pro 365

Thr Lys Asn Gln

Ser Asp Ile Wing 400

Tyr Lys Thr Thr 415

Tyr Ser Lys Leu 430

Phe Ser Cys Ser 445

Lys Ser Read Ser

<210> 183

<211> 24

<212> PRT

<213> Homo sapiens

<400> 183

His Asn Be Ser Trp Tyr Gly Arg Tyr Phe Asp Tyr Trp Gly Gln 1 5 10 15 Gly Thr Read Val Thr Val Ser Ser 20

<210> 184

<211> 19

<212> PRT

<213> Homo sapiens

<400> 184

Glu Glu Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 15 10 15

Glu Ile Lys

<210> 185

<211> 119

<212> PRT

<213> Artificial Sequence <220>

<223> Humanized VH region <220>

<221> VARIANT

<222> (30). . (30)

<223> Xaa at position 30 can be Thr or Ile <220>

<221> VARIANT

<222> (31). . (31)

<223> Xaa at position 31 can be Asn or Ser <220>

<221> VARIANT

<222> (37). . (37)

<223> Xaa at position 37 can be Val or Ile <220>

<221> VARIANT

<222> (48). . (48)

<223> Xaa at position 48 can be Leu or Ile <2 2 0>

<221> VARIANT

<222> (67). . (67)

<223> Xaa at position 67 can be Val or Leu <220>

<221> VARIANT <222> (71). . (71)

<223> Xaa in position

<220>

<221> VARIANT

<222> (73). . (73)

<223> Xaa in position

<220>

<221> VARIANT

<222> (78). . (78)

<223> Xaa in position

<220>

<221> VARIANT

<222> (94). . (94)

<223> Xaa in position

71 can be Lys or Val

73 can be Asn or Thr

78 can be Val or Phe

94 can be Phe or Tyr

<400> 185

Gln Val Gln Leu Gln Glu Be Gly Pro Gly Leu Val Lys Pro Be Glu 1 5 10 15

Thr Leu Be Leu Thr Cys Thr Val Be Gly Phe Be Leu Xaa Xaa Tyr

20 25 30

Gly Ile His Trp Xaa Arg Gln Pro Gly Lys Gly Leu Glu Trp Xaa

35 40 45

Gly Val Ile Trp Arg Wing Gly Phe Thr Asn Tyr Asn Be Wing Read Met

50 55 60

Ser Arg Xaa Thr Ile Ser Xaa Asp Xaa Ser Lys Asn Gln Xaa Ser Leu 65 70 75 80

Lys Leu Be Ser Val Thr Wing Asp Thr Wing Val Tyr Xaa Cys Wing

85 90 95

Arg Wing Asn Asp Gly Val Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 115

<210> 186

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Humanized VL Region

<220>

<221> VARIANT

<222> (1). . (1)

<223> Xaa at position 1 can be either Gln or Asp <220>

<221> VARIANT

<222> (2). . (2)

<223> Xaa at position 2 can be Phe or Val

<220>

<221> VARIANT

<222> (4). . (4)

<223> Xaa at position 4 can be either Leu or Met

<220>

<221> VARIANT

<222> (45). . (45)

<223> Xaa at position 48 can be either Lys or Leu

<220>

<221> VARIANT

<222> (46). . (46)

<223> Xaa at position 46 can be Trp or Leu

<220>

<221> VARIANT

<222> (48). . (48)

<223> Xaa at position 48 can be Tyr or Lys

<220>

<221> VARIANT

<222> (70). . (70)

<223> Xaa at position 70 can be either Tyr or Phe

<400> 186

Xaa Xaa Val Xaa Thr Gln Ser Pro 1 5

Glu Lys Val Thr Ile Thr Cys Ser 20

His Trp Tyr Gln Gln Lys Pro Asp

35 40

Asp Thr Be Lys Leu Wing Be Gly

50 55

Gly Ser Gly Thr Asp Xaa Thr Phe 65 70

Asp Wing Wing Thr Tyr Tyr Cys Gln

85

Phe Gly Gln Gly Thr Lys Val Glu 100

Phe Wing Read Ser Val Thr Pro Gly

10 15

Wing Asn Ser Ser Val Asn Tyr Ile 25 30

Gln Wing Pro Lys Xaa Xaa Ile Xaa

45

Val Pro Ser Arg Phe Ser Gly Ser 60

Thr Ile Ser Ser Leu Glu Wing Glu

75 80

Gln Trp Thr Thr

90 95

Ile Lys 105

Claims (111)

1. Humanized anti-integrin α2 antibody, characterized in that it is composed of a heavy chain variable region containing the amino acid sequence (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2), (b) HCDR1 (GFSLTNYGIH, SEQ ID N0: 1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO: 3), or (c) SEQ ID NO: 40 Humanized anti-integrin α2 antibody according to claim 1, characterized in that the heavy chain variable region is composed of the amino acid sequence SEQ ID NO: 185. Humanized anti-integrin α2 antibody according to claim 2, characterized in that the heavy chain variable region is composed of the amino acid sequence and the amino acid sequence 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). Humanized anti-integrin α2 antibody according to claim 1, characterized in that the heavy chain variable region is composed of the amino acid sequence selected from SEQ ID NOs: 70-79 and SEQ ID NOs: 109-111. Humanized anti-integrin α2 antibody according to claim 1, characterized in that it is also composed of the FW4 region formed by the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 13. 6. Humanized anti-integrin a2 antibody. Claim 5, characterized in that it is composed of the amino acid sequence of HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3). Humanized anti-integrin α2 antibody according to any one of claims 1-6, characterized in that it also contains a light chain. Humanized anti-integrin α2 antibody characterized in that it is composed of a light chain variable region composed of amino acid sequence (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). Humanized anti-integrin α2 antibody according to claim 8, characterized in that the light chain variable region is composed of the amino acid sequence SEQ ID NO: 186. Humanized anti-integrin α2 antibody according to claim 8, characterized in that the light chain variable region is composed of the amino acid sequence 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). Humanized anti-integrin oc2 antibody according to claim 8, characterized in that the light chain variable region is composed of the amino acid sequence selected from SEQ ID NO: 41, SEQ ID NOs: 80-92 and SEQ ID NO: 108. Humanized anti-integrin α2 antibody according to claim 8, characterized in that it is also composed of the FW4 region formed by the amino acid sequence FGQGTKVEIK of SEQ ID NO: 38 Humanized anti-integrin oc2 antibody according to claim 12, characterized in that it is composed of the amino acid sequence of LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO : 6). Humanized anti-integrin α2 antibody according to any one of claims 8-13, characterized in that it also contains a heavy chain. Humanized anti-integrin α2 antibody, characterized in that it is composed of: (i) a heavy chain variable region composed of the amino acid sequence (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2), (b) HCDRl ( 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 composed of the amino acid sequence by (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). Humanized anti-integrin α2 antibody according to claim 15, characterized in that (a) the heavy chain variable region is composed of the amino acid sequence SEQ ID NO: 185, (b) the light chain variable region be composed of the amino acid sequence f SEQ ID NO: 186, or (c) both (a) and (b). Humanized anti-integrin α2 antibody according to claim 15, characterized in that (i) the heavy chain variable region is composed of the amino acid sequence 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 is composed of the amino acid sequence 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). Humanized anti-integrin α2 antibody according to claim 15, characterized in that (a) the heavy chain variable region is composed of the amino acid sequence selected from SEQ ID NOs: 70-79 and SEQ ID NOs: 109 -111; (b) the light chain variable region is composed of the 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). Humanized anti-Î ± 2 integrin antibody according to claim 1, characterized in that the antibody recognizes the human Î ± 2 integrin domain I domain. Humanized anti-Î ± 2 integrin antibody according to claim 8, characterized in that the antibody recognizes the human Î ± 2 integrin domain I domain. Humanized anti-Î ± 2 integrin antibody according to claim 15, characterized in that the antibody recognizes the human Î ± 2 integrin domain I domain. Humanized anti-integrin cc2 antibody according to claim 1, characterized in that the antibody binds to α2β1 integrin. Humanized anti-integrin α2 antibody according to Claim 8, characterized in that the antibody binds to α2β1 integrin. Humanized anti-integrin α2 antibody according to Claim 15, characterized in that the antibody binds to α2β1 integrin. Humanized α2 integrin anti-antibody according to claim 1, characterized in that the antibody binds to an α2 integrin epitope, which is composed of: (a) a Lys residue corresponding to position 192 of the amino acid sequence the cx2 integrin expressed in SEQ ID NO: 8 or position 40 of the domain I amino acid sequence of a2 integrin expressed in SEQ ID NO: 11; (b) an Asn residue corresponding to position 225 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 89 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (d) a Lys residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 93 of the a.2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (e) an Arg residue corresponding to position 317 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 165 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the α2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 166 of the α2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). Humanized anti-integrin α2 antibody according to claim 8, characterized in that the antibody binds to an α2 integrin epitope, which is composed of: (a) a Lys residue corresponding to position 192 of the amino acid sequence a2 integrin expressed in SEQ ID NO: 8 or position 40 of the domain I amino acid sequence of the cc2 integrin expressed in SEQ ID NO: 11; (b) an Asn residue corresponding to position 225 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the α2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 89 of the α2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (d) a Lys residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 93 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (e) an Arg residue corresponding to position 317 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 165 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the α2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 166 of the α1 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). Humanized anti-Î ± 2 integrin antibody according to claim 15, characterized in that the antibody binds to an Î ± 2 integrin epitope, which epitope comprises: (a) a Lys residue corresponding to position 192 of the amino acid sequence a2 integrin expressed in SEQ ID NO: 8 or position 40 of the domain I amino acid sequence of a2 integrin expressed in SEQ ID NO: 11; (b) an Asn residue corresponding to position 225 of the cx2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 89 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (d) a Tyr residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 93 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (e) an Arg residue corresponding to position 317 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 165 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 166 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). 28. Humanized anti-integrin α2 antibody, characterized in that the antibody binds to an α2 integrin epitope, which is composed of: (a) a Lys residue corresponding to position 192 of the α2 integrin amino acid sequence expressed in SEQ ID. NO: 8 or position 40 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (b) an Asn residue corresponding to position 225 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 73 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 89 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (d) a Tyr residue corresponding to position 245 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 93 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (e) an Arg residue corresponding to position 317 of the α2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 165 of the α2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the a2 integrin amino acid sequence expressed in SEQ ID NO: 8 or position 166 of the a2 integrin domain I amino acid sequence expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). Humanized anti-integrin α2 antibody according to claim 1, characterized in that it is a complete antibody. Humanized anti-integrin α2 antibody according to claim 8, characterized in that it is a complete antibody. Humanized anti-integrin α2 antibody according to claim 15, characterized in that it is a complete antibody. Humanized anti-integrin α2 antibody according to claim 28, characterized in that it is a complete antibody. Humanized anti-integrin α2 antibody according to claim 1, characterized in that it is an antibody fragment. The humanized anti-integrin α2 antibody according to claim 8, cpfd being an antibody fragment. Humanized anti-integrin α2 antibody according to claim 15, characterized in that it is an antibody fragment. Humanized anti-integrin α2 antibody according to claim 28, characterized in that it is an antibody fragment. Humanized anti-integrin α2 antibody according to claim 1, characterized in that it is bound to a detectable marker. Humanized anti-integrin α2 antibody according to claim 8, cpfd be bound to a detectable label. Humanized anti-integrin α2 antibody according to claim 15, characterized in that it is bound to a detectable marker. Humanized anti-integrin α2 antibody according to claim 28, characterized in that it is linked to a detectable marker. Humanized anti-integrin α2 antibody according to Claim 1, characterized in that it is immobilized on the solid phase. Humanized anti-integrin α2 antibody according to claim 8, characterized in that it is immobilized on the solid phase. Humanized anti-integrin α2 antibody according to claim 15, characterized in that it is immobilized on the solid phase. Humanized anti-integrin α2 antibody according to claim 28, characterized in that it is immobilized on the solid phase. Humanized anti-α2 integrin antibody according to Claim 1, characterized in that the antibody inhibits the binding of α2 or α2β1 integrin to an α2β1 integrin ligand. Humanized anti-α2 integrin antibody according to Claim 8, characterized in that the antibody inhibits the binding of α2 or α2β1 integrin to an α2β1 integrin ligand. Humanized α2-integrin anti-antibody according to Claim 15, characterized in that the antibody inhibits the binding of α2 or α2β1 integrin to an α2β1 integrin ligand. Humanized anti-integrin a2 antibody according to claim 28, characterized in that the antibody inhibits the binding of α2 or α2β1 integrin to an α2β1 integrin ligand. Humanized anti-integrin α2 antibody according to Claim 45, characterized in that the α2β1 integrin ligand is selected from collagen, laminin, Ecovirus 1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, collectin and complement protein Clq. Humanized α2-integrin anti-integrin antibody according to claim 46, characterized in that the α2β1 integrin ligand is selected from collagen, laminin, Ecovirus 1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, collectin and complement protein Clq. Humanized anti-integrin α2 antibody according to claim 47, characterized in that the α2β1 integrin ligand is selected from collagen, laminin, Ecovirus 1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, collectin and complement protein Clq. Humanized anti-integrin α2 antibody according to claim 48, characterized in that the α2β1 integrin ligand is selected from collagen, laminin, Ecovirus 1, decorin, E-cadherin, matrix metalloproteinase I (MMP-I), endorepelin, collectin and complement protein Clq. Method for determining whether a sample contains cc2 integrin, α2β1 integrin, or both, comprising contacting the humanized anti-integrin a2 antibody as defined in claim 1 and determining whether The antibody binds to the sample, the binding being an indication that the sample contains the α2 integrin, α2β1 integrin or both. Method for determining whether a sample contains α2 integrin, α2β1 integrin, or both, comprising contacting the humanized anti-integrin α2 antibody as defined in claim 8 and determining whether The antibody binds to the sample, the binding being an indication that the sample contains α2 integrin, α2β1 integrin or both. Method for determining whether a sample contains α2 integrin, α2α1 integrin, or both, comprising contacting the humanized anti-integrin α2 antibody as defined in claim 15 and determining whether The antibody binds to the sample, the binding being an indication that the sample contains α2 integrin, α2β1 integrin or both. Method for determining whether a sample contains α2 integrin, α2β1 integrin, or both, characterized in that the sample is brought into contact with the humanized α2 integrin antibody as defined in claim 28 and determined whether antibody binds to the sample, the binding being an indication that the sample contains α2 integrin, α2β1 integrin or both. Kit comprising the humanized anti-integrin α2 antibody as defined in claim 1 and instructions on how to use it to detect α2 or α2β1 integrin protein. 58. A kit comprising the humanized anti-integrin α2 antibody as defined in claim 8 and instructions on how to use it to detect α2 or α2β1 integrin protein. Kit comprising the humanized anti-integrin α2 antibody as defined in claim 15 and instructions on how to use it to detect α2 or α2β1 integrin protein. Kit comprising the humanized anti-integrin α2 antibody as defined in claim 28 and instructions on how to use it to detect the α2 or α2β1 integrin protein. An isolated nucleic acid characterized in that it encodes the humanized anti-integrin α2β1 antibody as defined in claim 1. Isolated nucleic acid encoding the humanized anti-integrin α2β1 antibody as defined in claim 8. 63. A vector characterized in that it is composed of nucleic acid as defined in claim 61. 64. A vector characterized in that it is composed of nucleic acid as defined in claim 62. Host cell, characterized in that it comprises: (a) nucleic acid as defined in claim -61; (b) nucleic acid as defined in claim -62; (c) vector as defined in claim 63; (d) vector as defined in claim 64; or (e) any combination of (a) to (d). A process for producing a humanized anti-integrin? X2 antibody, comprising culturing the host cell of claim 65 under conditions that allow expression of the antibody. 67. The method of claim 66 further comprising recovering the humanized anti-integrin α 2 antibody from the host cell. Process according to Claim 67, characterized in that the humanized anti-integrin α2β1 antibody is recovered from the culture medium of the host cell. 69. Screening method, characterized in that it comprises: (a) detecting binding of α2 or α2β1 integrin to an antibody composed of the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21 in the presence versus in the absence of a test antibody; and (b) selecting a test antibody if its presence correlates with reduced binding of α2 or α2β1 integrin to an antibody composed of the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21. Method according to claim 69, characterized in that the integrin α2 or α2β1 is immobilized on a solid support. 71. Screening method, characterized in that it comprises: (a) detecting α2β1 integrin binding to collagen in the presence of a test antibody, wherein a test antibody refers to an antibody that binds to an a2 domain I ; (b) detecting binding of the test antibody to a2 domain I in the presence of Mg ++ ions; (c) detecting binding of the test antibody to α2 domain I in the presence of Ca ++ ions; (d) detecting binding of the test antibody to α2 domain I in the presence of cation free medium; and (e) selecting the test antibody if it inhibits binding of α2β1 integrin to collagen and binds to ia2 domain I in the presence of Mg ++ ion and Ca ++ ions and cation-free medium. Composition, characterized in that it comprises the humanized anti-integrin α2 antibody as defined in claim 1 and a pharmacologically acceptable carrier. Composition, characterized in that it comprises the humanized anti-integrin α2 antibody as defined in claim 8 and a pharmacologically acceptable carrier. Composition, characterized in that it comprises the humanized anti-integrin α2 antibody as defined in claim 15 and a pharmacologically acceptable carrier. 75. A composition comprising the humanized anti-integrin α2 antibody as defined in claim 28 and a pharmacologically acceptable carrier. A method for treating α2β1 integrin-associated disorders in patients, which comprises administering a therapeutically effective amount of the anti-integrin α2 antibody as defined in any one of claims 1-52 or the composition as defined in any one of these. claims 72-75. A method for inhibiting leukocyte binding to collagen, which comprises administering a sufficient amount of anti-integrin α2β1 antibody as defined in claim 28 to inhibit leukocyte binding to collagen in a patient. A method according to claim 76, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory disease, autoimmune disease and a disease characterized by altered or increased angiogenesis. 79. The method of claim 76, wherein the α2β1 integrin-associated disorder is selected from: inflammatory bowel disease, Crohn's disease, ulcerative colitis, transplant rejections, optic neuritis, spinal trauma, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, juvenile diabetes, age-related macular degeneration, cardiovascular disease, psoriasis, cancer as well as infections that induce an inflammatory reaction. 80. Method according to claim 76, characterized in that the α2β1 integrin-associated disorder is selected from: multiple sclerosis, rheumatoid arthritis, optic neuritis and spinal trauma. 81. The method according to claim 7, wherein the method is not associated with: (a) platelet activation, (b) platelet aggregation, (c) reduction in circulating platelet count, (d) hemorrhagic complications or (e) any combination of (a) to (d) Method according to claim 76, characterized in that the above-mentioned anti-integrin α2 antibody contains a heavy chain composed of SEQ ID NO: 174 or SEQ ID NO: 176 and a light chain composed of SEQ ID NO: 178 83. The method of claim 76, wherein the anti-integrin α 2 antibody competitively inhibits binding of an antibody composed of a VL region of SEQ ID NO: 19 and a VH region of SEQ ID NO: 21 to human α2β1 integrin or domain I thereof. Method according to claim 80, characterized in that the multiple sclerosis is characterized by recurrence. 85. The method of claim 80, wherein the method is associated with a relief of a neurological exacerbation or sequelae associated with multiple sclerosis. 86. The method of claim 76, wherein the anti-integrin α2 antibody inhibits the binding of α2β1 integrin to collagen and is not a ligand mimetic. A method of directing a molecule, composition or complex to a site defined by the presence of an α2β1 integrin ligand, comprising comprising attaching or binding the molecule, composition or complex to the humanized α2-integrin α2 antibody as defined in the claim. 1. 88. A method of directing a molecule, composition or complex to a site defined by the presence of an α2β1 integrin ligand, comprising comprising attaching or binding the molecule, composition or complex to the humanized anti-integrin α2 antibody as defined in claim 8. A method of directing a molecule, composition or complex to a site defined by the presence of an α2β1 integrin ligand, comprising comprising attaching or binding the molecule, composition or complex to the humanized anti-integrin α2 antibody as defined in the claim. 15 A method of directing a molecule, composition or complex to a site defined by the presence of an α2β1 integrin ligand, comprising comprising attaching or binding the molecule, composition or complex to the humanized anti-integrin α2 antibody as defined in the claim. 28 Use of a humanized anti-integrin α2 antibody as defined in any one of claims 1-52, characterized in that it is used as a medicament. Use of a humanized anti-integrin cc2 antibody as defined in any one of claims 1 to 52 or composition as defined in claim 44 characterized in that it is used for the treatment of an α2β1 integrin-associated disorder. Use of a humanized anti-integrin α2 antibody as defined in any one of claims 1 to 52, characterized in that it is used for the preparation of a medicament for the treatment of an α2β1 integrin-associated disorder. Use according to any one of claims -91 to 93, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory disease, autoimmune disease and a disease characterized by inadequate angiogenesis. Use according to any one of claims 91 to 93, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory bowel disease, Crohn's disease, ulcerative colitis, transplant rejection, optic neuritis, spinal trauma, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, juvenile diabetes, diabetic retinopathy, age-related macular degeneration, cardiovascular disease, psoriasis, cancer as well as infections that induce inflammatory reaction. Use according to any one of claims 91 to 93, characterized in that the α2β1 integrin-associated disorder is selected from: multiple sclerosis, rheumatoid arthritis, optic neuritis and spinal trauma. Composition for the treatment of an α2β1 integrin-associated disorder characterized in that it is formed by the humanized anti-integrin α2 antibody as defined in any one of claims 1 to 52 and a pharmaceutically acceptable carrier or diluent. Composition according to Claim 97, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory disease, autoimmune disease and a disease characterized by inadequate angiogenesis. Composition according to Claim 97, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory bowel disease, Crohn's disease, ulcerative colitis, transplant rejection, optic neuritis, spinal trauma, arthritis rheumatoid, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, juvenile diabetes, retinal disease, age-related macular degeneration, cardiovascular disease, psoriasis, cancer as well as infections that induce inflammatory reaction. Composition according to Claim 97, characterized in that the α2β1 integrin-associated disorder is selected from: multiple sclerosis, rheumatoid arthritis, optic neuritis and spinal trauma. A package comprising the humanized anti-integrin α2 antibody as defined in any one of claims 1 to 52 or the composition according to any one of claims 72 to 75, together with the instructions for treating a α2β1 integrin-associated disorder. Packaging according to Claim 101, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory disease, autoimmune disease and a disease characterized by inadequate angiogenesis. Packaging according to Claim 101, characterized in that the α2β1 integrin-associated disorder is selected from: inflammatory bowel disease, Crohn's disease, ulcerative colitis, transplant rejection, optic neuritis, spinal trauma, arthritis rheumatoid, systemic lupus erythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, juvenile diabetes, retinal disease, age-related macular degeneration, cardiovascular disease, psoriasis, cancer as well as infections that induce inflammatory reaction. Packaging according to Claim 101, characterized in that the α2β1 integrin-associated disorder is selected from multiple sclerosis, rheumatoid arthritis, optic neuritis and spinal trauma. 105. α2 integrin epitope which binds an anti-α2 integrin antibody, characterized in that the epitope is not composed of the α2 integrin ligand binding site: Epitope according to claim 105, characterized in that binding to the epitope is not associated with (a) platelet activation, (b) platelet aggregation, (c) decreased circulating platelet count, (d) hemorrhagic complications. , (e) α 2 integrin activation or (f) any combination of (a) to (e). Humanized anti-integrin α2 antibody according to claim 8, characterized in that LCDR1 is SAQSSVNYIH (SEQ ID NO: 112). Humanized anti-integrin α2 antibody according to claim 4, characterized in that the heavy chain variable region comprises SEQ ID NO: 109. 109. Humanized anti-integrin α2 antibody according to claim 11, characterized in that the light chain variable region comprises SEQ ID NO: 91. 110. Humanized α2 integrin anti-integrin antibody according to claim 15, characterized in that: (a) heavy chain variable region comprises SEQ ID NO: 109; and (b) light chain variable region comprises SEQ ID NO: 91. Humanized anti-integrin α2 antibody according to claim 9, characterized in that asparagine (N) at amino acid of position 26 in SEQ ID NO: -186 is replaced by glutamine (Q).