OA20299A - Antibodies to MAdCAM. - Google Patents

Abstract

The present disclosure relates to antibodies including human antibodies and antigen- binding portions thereof that specifically bind to MAdCAM, preferably human MAdCAM and that function to inhibit MAdCAM. The disclosure also relates to human anti-MAdCAM antibodies and antigenbinding portions thereof. The disclosure also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The disclosure also relates to isolated heavy and light chain immunoglobulins derived from human anti-MAdCAM antibodies and nucleic acid molecules encoding such immunoglobulins. The present disclosure also relates to methods of making human antiMAdCAM antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions for diagnosis and treatment. The disclosure also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-MAdCAM antibodies. The disclosure also relates to transgenic animals or plants comprising nuclpeic acid molecules of the disclosure.

Description

¹ ANTIBODIES TO MAdCAM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application daims the benefit of, and priority to U.S.S.N. 62/532,809 filed on July 14, 2017, the content of which is incorporated herein in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The content of the text fde name “SHR-1258A_ST25.txt”, which was created on July 14, 2017 and is 197 KB in size, is hereby incorporated-by-reference in its entirety.

BACKGROUND OF THE INVENTION

Mucosal addressin cell adhesion molécule (MAdCAM) is a member of the immunoglobulin superfamily of cell adhesion receptors. The selectivity of lymphocyte homing to specialized lymphoid tissue and mucosal sites of the gastrointestinal tract is determined by the endothélial expression of MAdCAM (Berlin, C. et al., Cell, 80:413- 422(1994); Berlin, C., et al., Cell, 74:185-195 (1993); and Erle, D.J., et al., J. Immunol., 153: 517-528 (1994)). MAdCAM is uniquely expressed on the cell surface of high endothélial vendes of organized intestinal lymphoid tissue, such as Peyer’s patches and mesenteric lymph nodes (Streeter et al., Nature, 331:41-6 (1988); Nakache et al., Nature, 337:179-81 (1989); Briskin et al., Am. J. Pathol. 15197-110 (1997)), but also in other lymphoid organs, such as pancréas, gall bladder and splenic vendes and marginal sinus of the splenic white pulp (Briskin et al(1997), supra', Kraal et al., Am. J. Path., 147: 763-771 (1995)).

While MAdCAM plays a physiological rôle in gut immune surveillance, it appears to facilitate excessive lymphocyte extravasation in inflammatory bowel disease under conditions of chronic gastrointestinal tract inflammation. TNFa and other pro-inflammatory cytokines increase endothélial MAdCAM expression and, in biopsy specimens taken from patients with Crohn’s disease and ulcerative colitis, there is an approximate 2-3 fold focal increase in MAdCAM expression at sites of inflammation (Briskin et al. (1997), Souza et al., Gut, 45:856-63 (1999); Arihiro et al., Pathol Int., 52:367-74 (2002)). Similar patterns of elevated expression hâve been observed in experimental models of colitis (Hesterberg et al., Gastroenterology, 111:1373-1380 (1997); Picarella et al., J. Immunol., 158: 2099-2106 (1997); Connor et al., JLeukoc Biol., 65:349-55 (1999); Kato et al., JPharmacol Exp Ther., 295:183-9 (2000); Hokari et al., Clin Exp Immunol., 26:259-65 (2001); Shigematsu et al., Am JPhysiol Gastrointest Liver Physiol., 281:G1309-15 (2001)). In other pre-clinical models for inflammatory conditions, such as insulin-dependent diabètes (Yang et al. Diabètes, 46:1542-7 (1997); Hânninen et al., JImmunol., 160:6018-25 (1998)), graft versus host disease (Fujisaki et al., Scand J Gastroenterol., 38:437-42

-2 (2003), Murai et al., Nat Immunol., 4:154-60 (2003)), chronic liver disease (Hillan et al., Liver, 19:509-18 (1999); Grant et al., Hepatology, 33:1065-72 (2001)), inflammatory encephalopathy (Stalder et al., Am J Pathol., 153:767-83 (1998); Kanawar et al., Immunol Cell Biol., 78:641-5 (2000)), and gastritis (Barrett et al., JLeukoc Biol., 67:169-73 (2000); Hatanaka et al., Clin Exp Immunol., 130:183-9 (2002)), there is also reawakening of fêtai MAdCAM expression and participation of activated cuP?⁺lymphocytes in disease pathogenesis. In these inflammatory models as well as hapten- mediated (e.g., TNBS, DSS, etc.) or adoptive transfer (CD4 ^rCD45Rb^hlgh) mouse colitic models, the rat anti-mouse MAdCAM monoclonal antibody (mAb), MECA-367, which blocks the binding of α4β?⁺lymphocytes to MAdCAM, reduces the lymphocyte recruitment, tissue extravasation, inflammation and disease severity. Mouse monoclonal antibodies (mAbs) against human MAdCAM also hâve been reported (see, e.g., WO 96/24673 and WO 99/58573).

Given the rôle of MAdCAM in inflammatory bowel disease (IBD) and other inflammatory diseases associated with the gastrointestinal tract or other tissues, a means for inhibiting ΟΜβγ binding and MAdCAM-mediated leukocyte recruitment is désirable. It further would be désirable to hâve such therapeutic means with advantageous properties including but not limited to the absence of unwanted interactions with other médications in patients and favorable physico-chemical properties such as pK/pD values in humans, solubility, stability, shelf-life and in vivo half-life. A therapeutic protein, such as an antibody, would advantageously be free of unwanted post-translational modifications or aggregate formation. Accordingly, there is a critical need for therapeutic anti-MAdCAM antibodies.

SUMMARY OF THE INVENTION

Provided herein is an isolated antibody that specifically binds MAdCAM, wherein at least the CDR sequences of said antibody are human CDR sequences, or an antigen-binding portion of said antibody. In embodiments the antibody is a human antibody, preferably an antibody that acts as a MAdCAM antagonist. Also provided are compositions comprising said antibodies or portions.

The disclosure also provides a composition comprising the heavy and/or light chain of said antiMAdCAM antagonist antibody or the variable région or other antigen-binding portion thereof or nucleic acid molécules encoding any of the foregoing and a pharmaceutically acceptable carrier. Compositions of the invention may further comprise another component, such as a therapeutic agent or a diagnostic agent. Diagnostic and therapeutic methods are also provided by the invention.

- 3 The disclosure further provides an isolated cell line, that produces said anti- MAdCAM antibody or antigen-binding portion thereof.

The disclosure also provides nucleic acid molécules encoding the heavy and/or light chain of said anti-MAdCAM antibody or the variable région thereof or antigen-binding portion thereof.

The disclosure provides vectors and host cells comprising said nucleic acid molécules, as well as methods of recombinantly producing the polypeptides encoded by the nucleic acid molécules.

Non-human transgenic animais or plants that express the heavy and/or light chain of said antiMAdCAM antibody, or antigen-binding portion thereof, are also provided.

In embodiments, a human monoclonal antibody or an antigen-binding portion thereof is provided that specifically binds to Mucosal Adressin Cell Adhesion Molécule (MAdCAM).

In embodiments, the human monoclonal antibody or antigen-binding portion possesses at least one of the following properties: (a) binds to human cells; (b) has a selectivity for MAdCAM over VCAM or fibronectin of at least 100 fold; (c) binds to human MAdCAM with a Kaof 3 x 10‘¹⁰ M or less; or (d) inhibits the binding of ο^βγ expressing cells to human MAdCAM. (e) inhibits the recruitment of lymphocytes to gastrointestinal lymphoid tissue.

In embodiments, the human monoclonal antibody or antigen-binding portion binds human MAdCAM with a Kaof 3 x 10'¹⁰ M or less and inhibits «4 β?binding to human MAdCAM.

In embodiments, the heavy chain comprises an amino acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 148.

In embodiments, the heavy chain comprises an amino acid sequence identical to SEQ ID NO: 148.

In embodiments, heavy chain comprises between 1 and 25 amino acid substitutions as compared to SEQIDNO: 148.

In embodiments, the heavy chain comprises between 1 and 10 amino acid substitutions as compared to SEQ ID NO: 148.

In embodiments, the light chain comprises an amino acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 150.

In embodiments, the light chain comprises an amino acid sequence identical to SEQ ID NO: 150.

In embodiments, the light chain comprises between 1 and 25 amino acid substitutions as compared to SEQ ID NO: 150.

-4In embodiments, the light chain comprises between 1 and 10 amino acid substitutions as compared to SEQ I DNO: 150.

In embodiments, the heavy chain comprises an amino acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 148, and the light chain comprises an amino acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 150.

In embodiments, the heavy chain comprises an amino acid sequence identical to SEQ ID NO: 148, and the light chain comprises an amino acid sequence identical to SEQ ID NO: 150.

In embodiments, a nucleic acid sequence encoding the amino acid sequence of a MAdCAM antibody is provided.

In embodiments, a cell producing a human monoclonal antibody that binds MAdCAM is provided.

In embodiments, a cell comprising a nucleic acid sequence encoding a MAdCAM antibody is provided.

In embodiments, a hybridoma cell line that produces a human monoclonal MAdCAM antibody is provided. In embodiments, the hybridoma is selected from the group consisting of 1.7.2 (ECACC Accession No. 03090901), 1.8.2 (ECACC Accession No. 03090902), 6.14.2 (ECACC Accession No. 03090903), 6.22.2 (ECACC Accession No. 03090904), 6.34.2 (ECACC Accession No. 03090905), 6.67.1 (ECACC Accession No. 03090906), 6.73.2 (ECACC Accession No. 03090907), 6.77.1 (ECACC Accession No. 03090908), 7.16.6 (ECACC Accession No. 03090909), 7.20.5 (ECACC Accession No. 03090910), 7.26.4 (ECACC Accession No. 03090911), and 9.8.2 (ECACC Accession No. 03090912).

In embodiments, the human monoclonal antibody produced by the hybridoma cell line or an antigen-binding portion of said monoclonal antibody is provided.

In embodiments, the heavy chain C-terminal lysine is cleaved.

In embodiments, said antibody or antigen-binding portion inhibits binding of human MAdCAM to ο^βγ, and wherein the antibody or portion thereof has at least one of the following properties: (a) cross-competes with a reference antibody for binding to MAdCAM;

(b) competes with a reference antibody for binding to MAdCAM; (c) binds to the same epitope of MAdCAM as a reference antibody; (d) binds to MAdCAM with substantially the same Kd as a reference antibody; (e) binds to MAdCAM with substantially the same off rate as a reference antibody; wherein the reference antibody is selected from the group consisting of: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody

6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, X481.2 monoclonal antibody, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2- mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

In embodiments, the antibody is selected from the group consisting of: (a) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4, without the signal sequences; (b) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 8, without the signal sequences; (c) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 12, without the signal sequences; (d) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 16, without the signal sequences; (e) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 20, without the signal sequences; (f) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24, without the signal sequences; (g) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 26 and SEQ ID NO: 28, without the signal sequences; (h) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 30 and SEQ ID NO: 32, without the signal sequences; (i) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 34 and SEQ ID NO: 36, without the signal sequences; (j) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 40, without the signal sequences; (k) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 42 and SEQ ID NO: 44, without the signal sequences; (1) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 46 and SEQ ID NO: 48, without the signal sequences; (m) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 52 and SEQ ID NO: 54, without the signal sequences; (n) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 58, without the signal sequences; (o) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 60 and SEQ ID NO: 62, without the signal sequences; (p) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 64 and SEQ ID NO: 66, without the signal sequences; and (q) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 42 and SEQ ID NO: 68, without the signal sequences (r) an antibody comprising the amino acid sequences set forth in SEQ ID NO: 148 and SEQ ID NO: 150, without the signal sequence.

In embodiments, the heavy chain of said antibody or portion thereof comprises the heavy chain CDR1, CDR2 and CDR3 or wherein the light chain comprises the light chain CDR1, CDR2 and CDR3 of a monoclonal antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2,

6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod.

In embodiments, said antibody or portion comprises a heavy chain that utilizes a human VH 1-18 gene, a human VH 3-15 gene, a human VH 3-21 gene, a human VH 3-23 gene, a human VH 330 gene, a human VH 3-33 gene or a human VH 4-4 gene.

In embodiments, said antibody or portion comprises a light chain that utilizes a human Vk A2 gene, a human Vk A3 gene, a human Vk A26 gene, a human VkB3 gene, a human VkO12 gene or a human Vk 018 gene.

In embodiments, the heavy chain variable région, the light chain variable région or both are at least 90% identical in amino acid sequence to the corresponding région or régions of a monoclonal antibody selected from the group consisting of: monoclonal antibody 1.7.2, monoclonal antibody 1.8.2, monoclonal antibody 6.14.2, monoclonal antibody 6.22.2, monoclonal antibody 6.34.2, monoclonal antibody 6.67.1, monoclonal antibody 6.73.2, monoclonal antibody 6.77.1, monoclonal antibody 7.16.6, monoclonal antibody 7.20.5, monoclonal antibody 7.26.4, monoclonal antibody 9.8.2, monoclonal antibody X481.2, monoclonal antibody 6.22.2-mod, monoclonal antibody 6.34.2-mod, monoclonal antibody 6.67.1-mod, monoclonal antibody 6.77.1-mod and monoclonal antibody 7.26.4-mod.

In embodiments, a monoclonal antibody or an antigen-binding portion thereof is provided that specifically binds MAdCAM, wherein: (a) the heavy chain comprises the heavy chain CDR1, CDR2 and CDR3 amino acid sequences of a reference antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod (b) the light chain comprises the light chain CDR1, CDR2 and CDR3 amino acid sequences of a reference antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod (c) the antibody comprises a heavy chain of (a) and a light chain of (b); and (d) the antibody of (c) wherein the heavy chain and light chain CDR amino acid sequences are selected from the same reference antibody.

In embodiments, the heavy chain, the light chain or both comprise the amino acid sequence from the beginning of the CDR1 through the end of the CDR3 of the heavy chain, the light chain or both, respectively, of the reference antibody.

In embodiments, said antibody comprises: (a) a heavy chain comprising the heavy chain variable région amino acid sequence of an antibody selected from the group consisting of: 1.7.2 (SEQ ID

-7 NO: 2); 1.8.2 (SEQ ID NO: 6); 6.14.2 (SEQ ID NO: 10); 6.22.2 (SEQID NO: 14); 6.34.2 (SEQ ID NO: 18); 6.67.1 (SEQ ID NO: 22); 6.73.2 (SEQ ID NO: 26); 6.77.1 (SEQ ID NO: 30); 7.16.6 (SEQ ID NO: 34); 7.20.5 (SEQ ID NO: 38); 7.26.4 (SEQ ID NO: 42); and 9.8.2 (SEQ ID NO: 46); X481.2 (SEQ ID NO: 148), 6.22.2-mod (SEQ ID NO: 52); 6.34.2-mod (SEQ ID NO: 56); 6.67.1-mod (SEQ ID NO: 60); 6.77.1-mod (SEQ ID NO: 64); and 7.26.4-mod (SEQ ID NO: 42); (b) a light chain comprising the light chain variable région amino acid sequence of an antibody selected from the group consisting of: 1.7.2 (SEQ ID NO: 4); 1.8.2 (SEQ ID NO: 8); 6.14.2 (SEQ ID NO: 12); 6.22.2 (SEQ ID NO: 16); 6.34.2 (SEQ ID NO: 20); 6.67.1 (SEQ ID NO: 24); 6.73.2 (SEQ ID NO: 28); 6.77.1 (SEQ ID NO:

32); 7.16.6 (SEQ ID NO: 36); 7.20.5 (SEQ ID NO: 40); 7.26.4 (SEQ ID NO: 44); and 9.8.2 (SEQ ID NO: 48); X481.2 (SEQ ID NO: 150), 6.22.2-mod (SEQ ID NO: 54); 6.34.2-mod (SEQ ID NO: 58); 6.67.1-mod (SEQ ID NO: 62); 6.77.1-mod (SEQ ID NO: 66); and 7.26.4-mod (SEQ ID NO: 68); or (c) the heavy chain of (a) and the light chain of (b).

In embodiments, the monoclonal antibody is an immunoglobulin G (IgG), an IgM, an IgE, and IgA or an IgD molécule, a humanized antibody, a chimeric antibody or a bispecific antibody.

In embodiments, the antigen-binding portion is an Fab fragment, an F(ab’)2 fragment, an Fv fragment or a single chain antibody.

In embodiments, a pharmaceutical composition is provided comprising an effective amount of the monoclonal antibody or antigen-binding portion thereof and a pharmaceutically acceptable camer.

In embodiments, a method of treating inflammatory disease in a subject in need thereof is provided, comprising the step of administering to said subject the monoclonal antibody or antigen-binding portion thereof wherein said antibody or antigen-binding portion inhibits binding of MAdCAM to ο^β?.

In embodiments, the inflammatory disease is inflammatory disease of the gastrointestinal tract.

In embodiments, the inflammatory disease of the gastrointestinal tract is selected from the group consisting of inflammatory bowel disease, Crohn’s disease, ulcerative colitis, diverticula disease, gastritis, liver disease, primary biliary sclerosis and sclerosing cholangitis.

In embodiments, the inflammatory bowel disease is Crohn’s disease, ulcerative colitis or both.

In embodiments, the inflammatory diseases are insulin-dependent diabètes and graft versus host disease.

- 8 In embodiments, an isolated cell line is provided that produces the monoclonal antibody or antigen-binding portion or the heavy chain or light chain of said antibody or of said portion thereof. In embodiments, the cell line produces an antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5,

7.26.4, 9.8.2, and X481.2 or an antibody comprising the amino acid sequences of one of said antibodies. In embodiments, the cell line produces a monoclonal antibody selected from the group consisting of: 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, 7.26.4-mod, and X481.2 or an antibody comprising the amino acid sequences of one of said antibodies. In embodiments, an isolated nucleic acid molécule is provided comprising a nucléotide sequence that encodes the heavy chain or an antigen-binding portion thereof or the light chain or an antigen-binding portion thereof of an antibody.

In embodiments, a vector is provided comprising a nucleic acid molécule, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molécule. In embodiments, a host cell is provided comprising the vector or the nucleic acid molécule.

In embodiments, a host cell is provided comprising a nucleic acid molécule encoding the heavy chain or an antigen-binding portion thereof and a nucleic acid molécule encoding the light chain or an antigen-binding portion thereof of an antibody or antigen- binding portion.

In embodiments, a method is provided for producing a human monoclonal antibody or antigenbinding portion thereof that specifically binds MAdCAM, comprising culturing the host cell or the cell line under suitable conditions and recovering said antibody or antigen- binding portion.

In embodiments, a non-human transgenic animal or transgenic plant is provided comprising (a) nucleic acid molécule encoding the heavy chain or an antigen-binding portion thereof; (b) a nucleic acid molécule encoding the light chain or an antigen-binding portion thereof; or (c) both (a) and (b) of an antibody, wherein the non-human transgenic animal or transgenic plant expresses said heavy chain or light chain or both.

In embodiments, a method is provided of isolating an antibody or antigen-binding portion thereof that specifically binds to MAdCAM, comprising the step of isolating the antibody from the non-human transgenic animal or transgenic plant.

In embodiments, a method is provided of treating a subject in need thereof with a human antibody or antigen-binding portion thereof that specifically binds to MAdCAM and inhibits binding to α₄ β? comprising the steps of: (a) administering an effective amount of an isolated nucleic acid molécule encoding the heavy chain or an antigen-binding portion thereof, an

- 9 isolated nucleic acid molécule encoding the light chain or an antigen-binding portion thereof, or nucleic acid molécules encoding the light chain and the heavy chain or antigen-binding portions thereof; and (b) expressing the nucleic acid molécule.

In embodiments, a method for producing a human monoclonal antibody that specifically binds MAdCAM is provided, comprising the steps of: (a) immunizing a non- human transgenic animal that is capable of producing human antibodies with MAdCAM, with an immunogenic portion of MAdCAM or a with cell or tissue expressing MAdCAM; and (b) allowing the transgenic animal to mount an immune response to MAdCAM. In embodiments, a human monoclonal antibody is produced as above.

In embodiments, a method is provided of inhibiting «4 β? binding to cells expressing human MAdCAM comprising contacting the cells with the monoclonal antibody or an antigen-binding portion thereof.

In embodiments, a method for inhibiting MAdCAM-mediated leukocyte-endothelial cell adhesion is provided comprising contacting the endothélial cells with the monoclonal antibody or an antigen-binding portion thereof.

In embodiments, a method is provided for inhibiting MAdCAM-mediated leukocyte adhesion, migration and infiltration into tissues comprising the step of contacting the endothélial cells with the monoclonal antibody or an antigen-binding portion thereof.

In embodiments, a method is provided for inhibiting «4 βγ /MAdCAM-dependent cellular adhesion comprising the step of contacting cells expressing human MAdCAM with the monoclonal antibody or antigen-binding portion thereof.

In embodiments, a method is provided for inhibiting the MAdCAM-mediated recruitment of lymphocytes to gastrointestinal lymphoid tissue comprising the step of contacting cells expressing human MAdCAM with the monoclonal antibody or antigen- binding portion thereof.

In embodiments, a monoclonal antibody or an antigen-binding portion thereof is provided that specifically binds MAdCAM, wherein said antibody or portion thereof comprises one or more of an FRI, FR2, FR3 or FR4 amino acid sequence of a human monoclonal antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod.

In embodiments, the human monoclonal antibody or antigen-binding portion comprises: (a) a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence of a monoclonal antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2

- 10mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod; (b) a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence of a monoclonal antibody selected from the group consisting of: 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod; (c) both (a) and (b); or (d) either (a), (b) or (c), with or without the signal sequence.

In embodiments, a method is provided for diagnosing a disorder characterized by circulating soluble human MAdCAM comprising the steps of: (1) contacting a biological sample with the monoclonal antibody or antigen-binding portion and (2) detecting binding.

In embodiments, a method is provided for detecting inflammation in a subject comprising the steps of: (1) administering to said subject the monoclonal antibody or antigen- binding portion wherein said antibody or portion thereof is detectably labeled and (2) detecting binding.

In embodiments, a diagnostic kit is provided comprising the monoclonal antibody or antigenbinding portion.

In embodiments, the pharmaceutical composition is provided further comprising one or more additionai anti-inflammatory or immunomodulatory agents. In embodiments, the one or more additionai anti-inflammatory or immunomodulatory agents are selected from the group consisting of: corticosteroids, aminosalicylates, azathioprine, methotrexate, cyclosporin, FK506, IL-10, GM-CSF, rapamycin, anti-TNFa agents and adhesion molécule antagonists.

In embodiments, a vaccine is provided comprising an effective amount of the human antibody thereof or antigen-binding portion and a pharmaceutically acceptable carrier. In embodiments, the vaccine is mucosal.

In embodiments, a method is provided of detecting the effect of administration of an inhibitory anti-MAdCAM antibody or antigen-binding portion thereof to a subject comprising the steps of: (a) administering to a subject a human monoclonal antibody that specifically binds to MAdCAM; and (b) determining whether there is an increase in the levels of circulating α.4β7expressing leukocytes. In embodiments, said leukocytes are lymphocytes. In embodiments, said increase in the levels of circulating oo^7-expressing leukocytes is determined by FACS analysis.

In embodiments, a monoclonal antibody, or antigen-binding portion thereof, is provided that binds MAdCAM comprising the variable région of the light chain of SEQ ID NO: 150 and the variable région of the heavy chain of SEQ ID NO: 148.

- 11 In embodiments, a monoclonal antibody, or antigen-binding fragment thereof, is provided that binds MAdCAM comprising a heavy chain variable région encoded by nucléotide SEQ ID NO: 149, and a light chain variable région encoded by nucléotide SEQID NO: 35.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (i.e. Figure IA - Figure 1T) is an alignment of the predicted amino acid sequences of the heavy and kappa light chain variable régions of twelve human anti- MAdCAM monoclonal antibodies with the germline amino acid sequences of the corresponding human genes.

Figure IA shows an alignment of the predicted amino acid sequence of the heavy chain for antibodies 1.7.2 and 1.8.2 with the germline human VH 3-15 gene product.

Figure IB shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.14.2 with the germline human VH 3-23 gene product.

Figure IC shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.22.2 with the germline human VH 3-33 gene product.

Figure ID shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.34.2 with the germline human VH 3-30 geneproduct.

Figure 1E shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.67.1 with the germline human VH 4-4 geneproduct.

Figure 1F shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.73.2 with the germline human VH 3-23 geneproduct.

Figure IG shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 6.77.1 with the germline human VH 3-21 geneproduct.

Figure 1H shows an alignment of the predicted amino acid sequence of the heavy chain for antibodies 7.16.6 and 7.26.4 with the germline human VH 1-18 gene product.

Figure 11 shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 7.20.5 with the germline human VH 4-4 geneproduct.

Figure 1J shows an alignment of the predicted amino acid sequence of the heavy chain for antibody 9.8.2 with the germline human VH 3-33 geneproduct.

Figure 1K shows an alignment of the predicted amino acid sequence of the light kappa chain for antibodies 1.7.2 and 1.8.2 with the germline human A3 gene product.

Figure 1L shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.14.2 with the germline human 012 geneproduct.

- 12 Figure IM shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.22.2 with the germline human A26 gene product.

Figure IN shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.34.2 with the germline human 012 gene product.

Figure 1O shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.67.1 with the germline human B3 gene product.

Figure IP shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.73.2 with the germline human 012 gene product.

Figure 1Q shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 6.77.1 with the germline human A2 gene product.

Figure IR shows an alignment of the predicted amino acid sequence of the kappa light chain for antibodies 7.16.6 and 7.26.4 with the germline human A2 gene product.

Figure 1S shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 7.20.5 with the germline human A3 gene product.

Figure IT shows an alignment of the predicted amino acid sequence of the kappa light chain for antibody 9.8.2 with the germline human 018 gene product.

Figure 2 (i.e. Figure 2A and Figure 2B) are CLUSTAL alignments of the predicted heavy and kappa light chain amino acid sequences of human anti-MAdCAM antibodies.

Figure 2A is a CLUSTAL alignment and radial tree of the predicted kappa light chain amino acid sequences, showing the degree of similarity between the anti-MAdCAM antibody kappa light chains.

Figure 2B is a CLUSTAL alignment and radial tree of the predicted heavy amino acid sequences, showing the degree of similarity between the anti-MAdCAM antibody heavy chains.

Figure 3 is an amino acid sequence CLUSTAL alignment of the 2 N-terminal domains of cynomolgus and human MAdCAM which form the «4β? binding domain. The β- strands are aligned according to Tan et al., Structure (1998) 6:793-801.

Figure 4 is a graph representing the dose effects of purified biotinylated 1.7.2 and 7.16.6 on the adhesion of human peripheral blood lymphocytes to sections of MAdCAM- expressing frozen human liver endothélium.

Figure 5 shows a two dimensional graphical représentation based on the data captured in Table 7 ofthe diversity of MAdCAM epitopes to which the anti-MAdCAM antibodies, 1.7.2, 6.22.2,

6.34.2, 6.67.1, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2 bind. Anti-MAdCAM antibodies within the same circle show the same reactivity pattern, belong in the same epitope bin and are likely to recognize the same epitope on MAdCAM. Anti- MAdCAM antibody clones within overlapping circles are unable to bind simultaneously and are, therefore, likely to recognize an overlapping epitope on MAdCAM. Non-integrating circles represent anti-MAdCAM antibody clones with distinct spatial epitope séparation.

Figure 6 shows sandwich ELISA data with anti-MAdCAM antibodies 1.7.2 and an Alexa 488labelled 7.16.6, showing that two antibodies that are able to detect different epitopes on MAdCAM could be used to detect soluble MAdCAM for diagnostic purposes.

Figure 7 shows the effect of an inhibitory anti-MAdCAM antibody (1 mg/kg) on the number of circulating peripheral α4β?⁺lymphocytes, expressed as a fold increase over control IgG2a mAb or vehicle, using anti-MAdCAM mAb 7.16.6 in a cynomolgus monkeymodel.

DETAILED DESCRIPTION OF THE INVENTION

Définitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used in connection with the présent disclosure shall hâve the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular tenus shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the présent disclosure are generally performed according to conventional methods well known in the art and as described in varions general and more spécifie référencés that are cited and discussed throughout the présent spécification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer’s spécifications, as commonly accomplished in the art or as described herein. Standard techniques are used for Chemical synthèses, Chemical analyses, pharmaceutical préparation, formulation, and delivery, and treatment of patients.

- 14The following terms, unless otherwise indicated, shall be understood to hâve the following meanings:

The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of dérivation (1) is not associated with naturally associated components that accompany it in its native State, (2) is free of other proteins from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature.

Thus, a polypeptide that is chemically synthesized or synthesized in a cellular System different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

A protein or polypeptide is “substantially pure,” “substantially homogeneous” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptide that has an aminoterminal and/or carboxy-terminal délétion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, even more preferably at least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptide that comprises a segment of at least 25 amino acids that has substantial identity to a portion of an amino acid sequence and that has at least one of the following properties: (1) spécifie binding to MAdCAM under suitable binding conditions, (2) ability to inhibit ouP? integrin and/or L- selectin binding to MAdCAM, or (3) ability to reduce MAdCAM cell surface expression in vitro or in vivo. Typically, polypeptide

- 15 analogs comprise a conservative amino acid substitution (or insertion or délétion) with respect to the naturally-occurring sequence.

Analogs typically are at least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a ftill-length naturally- occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, or (5) confer or modify other physicochemical or fiinctional properties of such analogs. Analogs can include various muteins of a sequence other than the naturallyoccurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a hélix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thomton et al., Nature, 354:105 (1991), which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res., 15:29(1986); Veber and Freidinger, TINS, p.392(1985); and Evans et al., J. Med. Chem., 30:1229(1987), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an équivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a desired biochemical property or pharmacological activity), such as a human antibody, but hâve one or more peptide linkages optionally replaced by a linkage such as: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and CH2SO-, by methods well known in the art.

Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable

- 16 peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference); for example, by adding internai cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

An “immunoglobulin” is a tetrameric molécule. In a naturally-occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable région of about 100 to 110 or more amino acids primarily responsible for antigen récognition. The carboxy-terminal portion of each chain defines a constant région primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, a, or ε, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant régions are joined by a “J” région of about 12 or more amino acids, with the heavy chain also including a “D” région of about 10 or more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for ail purposes). The variable régions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

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

An “antibody” refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for spécifie binding. In some embodiments, an antibody is an antigen-binding portion thereof. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or Chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab’, F(ab’)2, Fv, dAb, and complementarity determining région (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer

- 17 spécifie antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge région; a Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature, 341:544-546(1989)) consists of a VH domain.

As used herein, an antibody that is referred to as, e.g., 1.7.2, 1.8.2, 6.14.2, 6.34.2, 6.67.1, 6.77.2, 7.16.6, 7.20.5, 7.26.4, 9.8.2 or X481.2 is a monoclonal antibody that is produced by the hybridoma of the same name. For example, antibody 1.7.2 is produced by hybridoma 1.7.2. An antibody that is referred to as 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, 7.26.4-mod, or X481.2 is a monoclonal antibody whose sequence has been modified from its corresponding parent by site-directed mutagenesis.

A single-chain antibody (scFv) is an antibody in which VL and VH régions are paired to form a monovalent molécule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:58795883 (1988)). Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) and Poljak, R. J., et al., Structure, 2:1121-1123 (1994)). One or more CDRs from an antibody of the disclosure may be incorporated into a molécule either covalently or noncovalently to make it an immunoadhesin that specifically binds to MAdCAM. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.

An antibody may hâve one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturallyoccurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispecific” or “bifunctional” antibody (diabody) has two different binding sites.

An “isolated antibody” is an antibody that (1) is not associated with naturally- associated components, including other naturally-associated antibodies, that accompany it in its native State, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different

- 18species, or (4) does not occur in nature. Examples of isolated antibodies include an antiMAdCAM antibody that has been affinity purified using MAdCAM, an anti-MAdCAM antibody that has been produced by a hybridoma or other cell line in vitro, and a human anti-MAdCAM antibody derived from a transgenic mammal or plant.

As used herein, the term “human antibody” means an antibody in which the variable and constant région sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which hâve been changed, e.g., to decrease possible immunogenicity, increase affinity, eliminate cysteines or glycosylation sites that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in nonhuman cells which might impart glycosylation not typical of human cells. The term also emcompasses antibodies which hâve been raised in a transgenic mouse which comprises some or ail of the human immunoglobulin heavy and light chain loci.

In one aspect, the disclosure provides a humanized antibody. In some embodiments, the humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains hâve been mutated so as to avoid or abrogate an immune response in humans. In some embodiments, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species. Examples of how to make humanized antibodies may be found in United States Patent Nos. 6,054,297, 5,886,152 and 5,877,293. In some embodiments, a humanized anti-MAdCAM antibody of the disclosure comprises the amino acid sequence of one or more framework régions of one or more human anti-MAdCAM antibodies of the disclosure.

In another aspect, the disclosure provides a “chimeric antibody”. In some embodiments the chimeric antibody refers to an antibody that contains one or more régions from one antibody and one or more régions from one or more other antibodies. In a preferred embodiment, one or more of the CDRs are derived from a human anti-MAdCAM antibody of the disclosure. In a more preferred embodiment, ail of the CDRs are derived from a human anti-MAdCAM antibody of the disclosure. In another preferred embodiment, the CDRs from more than one human antiMAdCAM antibody of the disclosure are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human antiMAdCAM antibody may be combined with CDR2 and CDR3 from the light chain of a second human anti-MAdCAM antibody, and the CDRs from the heavy chain may be derived from a third anti-MAdCAM antibody. Further, the framework régions may be derived from one of the

- 19same anti-MAdCAM antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.

A “neutralizing antibody,” “an inhibitory antibody” or antagonist antibody is an antibody that inhibits the binding of οαβ? or ouβ7-expressing cells, or any other cognate ligand or cognate ligand-expressing cells, to MAdCAM by at least about 20%. In apreferred embodiment, the antibody reduces and/or inhibits the binding of οωβγ integrin or α4β?- expressing cells to MAdCAM by at least 40%, more preferably by 60%, even more preferably by 80%, 85%, 90%, 95% or 100%. The binding réduction may be measured by any means known to one of ordinary skill in the art, for example, as measured in an in vitro compétitive binding assay. An example of measuring the réduction in binding of ατβ?- expressing cells to MAdCAM is presented in Example I.

Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art following the teachings of this spécification. Preferred amino- and carboxy- termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucléotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known (Bowie et al., Science, 253:164 (1991)).

The term “surface plasmon résonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by détection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore System (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et a\.,Ann. Biol. Clin., 51:19-26 (1993); Jonsson, U., et al., Biotechniques, 11:620-627 (1991); Johnsson, B., et al., J. Mol. Recognit., 8:125-131 (1995); and Johnnson, B., et al., Anal. Biochem., 198:268-277 (1991).

The term “k_Off” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

The term “Kd” refers to the dissociation constant of a particular antibody-antigen interaction. An antibody is said to bind an antigen when the dissociation constant is < 1 μΜ, preferably <100 nM and most preferably < 10 nM.

The term “epitope” includes any protein déterminant capable of spécifie binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molécule. Epitopic

-20déterminants usually consist of chemically active surface groupings of molécules such as amino acids or carbohydrate side chains and usually hâve spécifie three dimensional structural characteristics, as well as spécifie charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, ail of the points of interaction between the protein and the interacting molécule (such as an antibody) occur linearally along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by référencé. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the présent disclosure. Examples of unconventional amino acids include: 4- hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Νtrimethyllysine, ε-Ν-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3methylhistidine, 5-hydroxylysine, s- N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

The term “polynucleotide” as referred to herein means a polymeric form of nucléotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucléotide. The term includes single and double stranded forms of DNA.

The tenu “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with ail or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturally occurring, and modified nucléotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the

-21 construction of a gene mutant. Oligonucleotides of the disclosure can be either sense or antisense oligonucleotides.

The term “naturally occurring nucléotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucléotides” referred to herein includes nucléotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077(1984); Stein et al., Nucl. Acids Res., 16:3209(1988); Zon et al., Anti-Cancer Drug Design 6:539(1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England(1991)); Stec étal., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews, 90:543(1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for détection, if desired.

“Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The tenu “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, tenuination, promoter and enhancer sequences; efficient RNA processing signais such as splicing and polyadenylation signais; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein sécrétion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, ail components whose presence is essential for expression and processing, and can also include additionai components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term “vector”, as used herein, is intended to refer to a nucleic acid molécule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additionai DNA segments may be ligated. Another type of vector is a viral vector, wherein additionai DNA segments may be

-22ligated into the viral genome. Certain vectors are capable of autonomous réplication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of réplication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the présent spécification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., réplication defective rétro viruses, adenoviruses and adeno- associated viruses), which serve équivalent functions.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such tenus are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding générations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the tenu “host cell” as used herein.

The term “selectively hybridize” referred to herein means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the disclosure selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appréciable amounts of détectable binding to nonspecific nucleic acids. “High stringency” or “highly stringent” conditions can be used to achieve sélective hybridization conditions as known in the art and discussed herein. An example of “high stringency” or “highly stringent” conditions is a method of incubating a polynucleotide with another polynucleotide, wherein one polynucleotide may be affixed to a solid surface such as a membrane, in a hybridization buffer of 6X SSPE or SSC, 50% formamide, 5X Denhardt’s reagent, 0.5% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA at a hybridization température of 42°C for 12-16 hours, followed by twice washing at 55°C using a wash buffer of IX SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucléotide sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucléotides, usually at

-23 least about 18 nucléotides, more usually at least about 24 nucléotides, typically at least about 28 nucléotides, more typically at least about 32 nucléotides, and preferably at least about 36, 48 or more nucléotides. There are a number of different algorithme known in the art which can be used to measure nucléotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.3, Accelrys, San Diego, CA. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the régions of the best overlap between the query and search sequences (Pearson, Methods Enzymol., 183: 63-98 (1990); Pearson, Methods Mol. Biol., 132: 185-219 (2000); Pearson, Methods Enzymol., 266: 227-258 (1996); Pearson, J. Mol. Biol., 276: 71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucléotide sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in Wisconsin Package Version 10.3, herein incorporated by reference.

A reference to a nucléotide sequence encompasses its complément unless otherwise specified. Thus, a reference to a nucleic acid molécule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

In the molecular biology art, researchers use the terms “percent sequence identity”, “percent sequence similarity” and “percent sequence homology” interchangeably. In this application, these terms shah hâve the same meaning with respect to nucléotide sequences only.

The term “substantial similarity” or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucléotide insertions or délétions with another nucleic acid (or its complementary strand), there is nucléotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucléotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue

-24having a side chain (R group) with similar Chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol., 24: 307-31 (1994), herein incorporated by reference. Examples of groups of amino acids that hâve side chains with similar Chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) sulfurcontaining side chains are cysteine and méthionine. Preferred conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate- aspartate, and asparagine-glutamine.

Altematively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science, 256: 1443-45 (1992), herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, délétions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfif ’ which can be used with default parameters to détermine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., Wisconsin package Version 10.3. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in Wisconsin package Version 10.3. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the régions of the best overlap between the query and search sequences (Pearson (1990); Pearson (2000)). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

-25 The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is préférable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporation of another molécule in the antibody. In one embodiment, the label is a détectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimétrie methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.

Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, Te, ^1HIn, ^l25I, ^l31I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, βgalactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, métal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, Vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The terni “agent” is used herein to dénoté a Chemical compound, a mixture of Chemical compounds, a biological macromolecule, or an extract made from biological materials. The term “pharmaceutical agent or drug” as used herein refers to a Chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporated herein by reference).

The term “anti-inflammatory” or “immuno-modulatory” agent is used herein to refer to agents that hâve the functional property of inhibiting inflammation, including inflammatory disease in a subject, including in a human. In various embodiments of this disclosure, the inflammatory

-26disease may be, but is not limited to inflammatory diseases of the gastrointestinal tract including Crohn’s disease, ulcerative colitis, diverticula disease, gastritis, liver disease, primary biliary sclerosis, sclerosing cholangitis. Inflammatory diseases also include but are not limited to abdominal disease (including peritonitis, appendicitis, biliary tract disease), acute transverse myelitis, allergie dermatitis (including allergie skin, allergie eczema, skin atopy, atopie eczema, atopie dermatitis, cutaneous inflammation, inflammatory eczema, inflammatory dermatitis, flea skin, miliary dermatitis, miliary eczema, house dust mite skin), ankylosing spondylitis (Reiters syndrome), asthma, airway inflammation, atherosclerosis, arteriosclerosis, biliary atresia, bladder inflammation, breast cancer, cardiovascular inflammation (including vasculitis, rheumatoid nailfold infarcts, leg ulcers, polymyositis, chronic vascular inflammation, pericarditis, chronic obstructive pulmonary disease), chronic pancreatitis, perineural inflammation, colitis (including amoebic colitis, infective colitis, bacterial colitis, Crohn’s colitis, ischémie colitis, ulcerative colitis, idiopathic proctocolitis, inflammatory bowel disease, pseudomembranous colitis), collagen vascular disorders (rheumatoid arthritis, SLE, progressive systemic sclerosis, mixed connective tissue disease, diabètes mellitus), Crohn’s disease (régional enteritis, granulomatous ileitis, ileocolitis, digestive System inflammation), demyelinating disease (including myelitis, multiple sclerosis, disseminated sclerosis, acute disseminated encephalomyelitis, perivenous demyelination, vitamin B12 deficiency, Guillain-Barre syndrome, MS-associated rétro virus), dermatomyositis, diverticulitis, exudative diarrhea, gastritis, granulomatous hepatitis, granulomatous inflammation, cholecystitis, insulin- dépendent diabètes mellitus, liver inflammatory diseases (liver fibrosis primary biliary cirrhosis, hepatitis, sclerosing cholangitis), lung inflammation (idiopathic pulmonary fibrosis, éosinophilie granuloma of the lung, pulmonary histiocytosis X, peribronchiolar inflammation, acute bronchitis), lymphogranuloma venereum, malignant melanoma, mouth/tooth disease (including gingivitis, periodontal disease), mucositis, musculoskeletal System inflammation (myositis), nonalcoholic steatohepatitis (nonalcoholic fatty liver disease), ocular & orbital inflammation (including uveitis, optic neuritis, peripheral rheumatoid ulcération, peripheral comeal inflammation,), osteoarthritis, osteomyelitis, pharyngeal inflammation, polyarthritis, proctitis, psoriasis, radiation injury, sarcoidosis, sickle cell necropathy, superficial thrombophlebitis, systemic inflammatory response syndrome, thyroiditis, systemic lupus erythematosus, graft versus host disease, acute bum injury, Behçet’s syndrome, Sjôgren’s syndrome.

The tenus patient and subject include human and veterinary subjects.

-27Human Anti-MAdCAM Antibodies and Characterization Thereof

In one embodiment, the disclosure provides anti-MAdCAM antibodies comprising human CDR sequences. In a preferred embodiment, the disclosure provides human anti- MAdCAM antibodies. In some embodiments, human anti-MAdCAM antibodies are produced by immunizing a non-human transgenic animal, e.g., a rodent, whose genome comprises human immunoglobulin genes so that the transgenic animal produces human antibodies. In some embodiments, the disclosure provides an anti-MAdCAM antibody that does not bind complément.

In a preferred embodiment, the anti-MAdCAM antibody is 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the anti-MAdCAM antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66,, 68 or 150 (with or without the signal sequence) or the variable région of any one of said amino acid sequences, or one or more CDRs from these amino acid sequences. In another preferred embodiment, the anti-MAdCAM antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148 (with or without the signal sequence) or the amino acid sequence of the variable région, or of one or more CDRs from said amino acid sequences. Also included in the disclosure are human anti-MAdCAM antibodies comprising the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any one of the above-mentioned sequences. The disclosure further provides an anti-MAdCAM antibody comprising one or more FR régions of any of the above-mentioned sequences.

The disclosure further provides an anti-MAdCAM antibody comprising one of the aforementioned amino acid sequences in which one or more modifications hâve been made. In some embodiments, cysteines in the antibody, which may be chemically reactive, are substituted with another residue, such as, without limitation, alanine or serine. In one embodiment, the substitution is at a non-canonical cysteine. The substitution can be made in a CDR or framework région of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical.

In some embodiments, an amino acid substitution is made to eliminate potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework région of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the heterogeneity in the antibody product. In some embodiments, asparagineglycine pairs, which form potential deamidation sites, are eliminated by altering one or both of

-28the residues. In some embodiments, an amino acid substitution is made to add or to remove potential glycosylation sites in the variable région of an antibody of the disclosure.

In some embodiments, the C-terminal lysine of the heavy chain of the anti- MAdCAM antibody of the disclosure is cleaved. In various embodiments of the disclosure, the heavy and light chains of the anti-MAdCAM antibodies may optionally include a signal sequence.

In one aspect, the disclosure provides twelve inhibitory human anti-MAdCAM monoclonal antibodies and the hybridoma cell lines that produce them. Table 1 lists the sequence identifiers (SEQ ID NO:) of the nucleic acids encoding the full-length heavy and light chains (including signal sequence), and the corresponding full-length deduced amino acid sequences.

Table 1

HUMAN ANTI-MAdCAM ANTIBODIES

Monoclonal Antibody	SEQUENCE IDENTIFIER (SEQ ID NO:)
Full Length
Heavy	Light
DNA	Protein	DNA	Protein
1.7.2	1	2	3	4
1.8.2	5	6	7	8
6.14.2	9	10	11	12
6.22.2	13	14	15	16
6.34.2	17	18	19	20
6.67.1	21	22	23	24
6.73.2	25	26	27	28
6.77.1	29	30	31	32
7.16.6	33	34	35	36
7.20.5	37	38	39	40
7.26.4	41	42	43	44
9.8.2	45	46	47	48
X481.2	149	148	35	150

In another aspect, the disclosure provides a modified version of certain of the above- identified human anti-MAdCAM monoclonal antibodies. Table 2 lists the sequence identifiers for the DNA and protein sequences of the modified antibodies.

-29Table 2

HUMAN ANTI-MAdCAM ANTIBODIES

Modified Monoclonal Antibody	SEQUENCE IDENTIFIER (SEQ ID NO:)
Full Length
Heavy	Light
DNA	Protein	DNA	Protein
6.22.2-mod	51	52	53	54
6.34.2-mod	55	56	57	58
6.67.1-mod	59	60	61	62
6.77.1-mod	63	64	65	66
7.26.4-mod	41	42	67	68
X481.2	149	148	35	150

Class and Subclass of anti-MAdCAM Antibodies

The antibody may be an IgG, an IgM, an IgE, an IgA or an IgD molécule. In a preferred embodiment, the antibody is an IgG class and is an IgGi, IgG?, IgGa or IgG4 subclass. In a more preferred embodiment, the anti-MAdCAM antibody is subclass IgGi or IgG4. In another preferred embodiment, the anti-MAdCAM antibody is the same class and subclass as antibody 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod which is IgG2, or 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1 or 9.8.2, which is IgG₄.

The class and subclass of anti-MAdCAM antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are spécifie for a particular class and subclass of antibody. Such antibodies are available commercially. ELISA, Western Blot as well as other techniques can détermine the class and subclass. Alternative!y, the class and subclass maybe determined by sequencing ail or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies as the class showing the highest sequence identity.

Species and Molécule Selectivity

In another aspect of the disclosure, the anti-MAdCAM antibody demonstrates both species and molécule selectivity. In one embodiment, the anti-MAdCAM antibody binds to human, cynomolgus or dog MAdCAM. In some embodiments, the anti-MAdCAM antibody does not

-30bind to a New World monkey species such as a marmoset. Following the teachings of the spécification, one may détermine the species selectivity for the anti-MAdCAM antibody using methods well known in the art. For instance, one may détermine species selectivity using Western blot, FACS, ELISA or immunohistochemistry. In a preferred embodiment, one may détermine the species selectivity using immunohistochemistry.

In some embodiments, an anti-MAdCAM antibody that specifically binds MAdCAM has selectivity for MAdCAM over VCAM, fîbronectin or any other antigen that is at least 10 fold, preferably at least 20, 30, 40, 50, 60, 70, 80 or 90 fold, most preferably at least 100 fold. In a preferred embodiment, the anti-MAdCAM antibody does not exhibit any appréciable binding to VCAM, fîbronectin or any other antigen other than MAdCAM. One may déterminé the selectivity of the anti-MAdCAM antibody for MAdCAM using methods well known in the art following the teachings of the spécification. For instance, one may détermine the selectivity using Western blot, FACS, ELISA, or immunohistochemistry.

Binding Affinity of anti-MAdCAM antibodies to MAdCAM

In another aspect ofthe disclosure, the anti-MAdCAM antibodies specificallybind to MAdCAM with high affinity. In one embodiment, the anti-MAdCAM antibody specifically binds to -8

MAdCAM with a Kd of 3 x 10' M or less, as measured bysurface plasmon résonance, such as BIAcore. In more preferred embodiments, the antibody specifically binds to MAdCAM with a Kd of 1 x 10' or less or 1 x 10' M or less. In an even more preferred embodiment, the antibody specifically binds to MAdCAM with a Kdor 1 x 10'¹⁰M or less. In other preferred embodiments, an antibody of the disclosure specifically binds to MAdCAM with a Kdof 2.66 x 10 ¹⁰M or less, 2.35 x 10 “M or less or 9 x 10 ¹²M or less. In another preferred embodiment, the antibody specifically binds to MAdCAM with a Kdor 1 x 10'¹¹ M or less. In another preferred embodiment, the antibody specifically binds to MAdCAM with substantially the same Kd as an antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. An antibody with “substantially the same Kd” as a reference antibody has a Kd that is ± 100 pM, preferably ± 50 pM, more preferably ± 20 pM, still more preferably ± 10 pM, ± 5 pM or ± 2 pM, compared to the Kd of the reference antibody in the same experiment. In another preferred embodiment, the antibody binds to MAdCAM with substantially the same Kd as an antibody that comprises one or more variable domains or one or more CDRs from an antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In still another preferred embodiment, the antibody binds to MAdCAM with substantially the same K_d as an antibody that

-31 comprises one of the amino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52, 54, 56, 58, 62, 64, 66, 68, 148 or 450 (with or without the signal sequence), or the variable domain thereof. In another preferred embodiment, the antibody binds to MAdCAM with substantially the same Ka as an antibody that comprises one or more CDRs from an antibody that comprises an amino acid sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52, 54, 56, 58, 62, 64, 66, 68, 148 or 450.

The binding affmity of an anti-MAdCAM antibody to MAdCAM may be determined by any method known in the art. In one embodiment, the binding affmity can be measured by compétitive ELISAs, RIAs or surface plasmon résonance, such as BIAcore. In a more preferred embodiment, the binding affmity is measured by surface plasmon résonance. In an even more preferred embodiment, the binding affmity and dissociation rate is measured using a BIAcore. An example of determining binding affmity is described below in Example IL

Half-Life of Anti-MAdCAM Antibodies

According to another object of the disclosure, the anti-MAdCAM antibody has a half-life of at least one day in vitro or in vivo. In a preferred embodiment, the antibody or portion thereof has a half-life of at least three days. In a more preferred embodiment, the antibody or portion thereof has a half-life of four days or longer. In another embodiment, the antibody or portion thereof has a half-life of eight days or longer. In another embodiment, the antibody or antigen-binding portion thereof is derivatized or modified such that it has a longer half-life, as discussed below. In another preferred embodiment, the antibody may contain point mutations to increase sérum half life, such as described WO 00/09560, published February 24, 2000.

The antibody half-life may be measured by any means known to one having ordinary skill in the art. For instance, the antibody half life may be measured by Western blot, ELISA or RIA over an appropriate period of time. The antibody half-life may be measured in any appropriate animal, such as a primate, e.g., cynomolgus monkey, or a human.

Identification of MAdCAM Epitopes Recognized by Anti-MAdCAM Antibody

The disclosure also provides a human anti-MAdCAM antibody that binds the same antigen or epitope as a human anti-MAdCAM antibody provided herein. Further, the disclosure provides a human anti-MAdCAM antibody that competes or cross-competes with a human anti-MAdCAM antibody. In a preferred embodiment, the human anti-MAdCAM antibody is 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the human anti

- 32 MAdCAM antibody comprises one or more variable domains or one or more CDRs from an antibody selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In still another preferred embodiment, the human anti-MAdCAM antibody comprises one of the amino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52, 54, 56, 58, 62, 64, 66, 68, 148 or 150 (with or without the signal sequence), or a variable domain thereof. In another preferred embodiment, the human anti-MAdCAM antibody comprises one or more CDRs from an antibody that comprises one of the amino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 48, 52, 54, 56, 58, 62, 64, 66, 68, 148 or 150. In a highly preferred embodiment, the anti-MAdCAM antibody is another human antibody.

One may détermine whether an anti-MAdCAM antibody binds to the same antigen as another anti-MAdCAM antibody using a variety of methods known in the art. For instance, one can use a known anti-MAdCAM antibody to capture the antigen, elute the antigen from the antiMAdCAM antibody, and then détermine whether the test antibody will bind to the eluted antigen. One may détermine whether an antibody competes with an anti- MAdCAM antibody by binding the anti-MAdCAM antibody to MAdCAM under saturating conditions, and then measuring the ability of the test antibody to bind to MAdCAM. If the test antibody is able to bind to the MAdCAM at the same time as the anti-MAdCAM antibody, then the test antibody binds to a different epitope than the anti- MAdCAM antibody. However, if the test antibody is not able to bind to the MAdCAM at the same time, then the test antibody competes with the human anti-MAdCAM antibody. This experiment may be performed using ELISA, or surface plasmon résonance or, preferably, BIAcore. To test whether an anti-MAdCAM antibody crosscompetes with another anti-MAdCAM antibody, one may use the compétition method described above in two directions, i.e. determining if the known antibody blocks the test antibody and vice versa.

Light and Heavy Chain Gene Usage

The disclosure also provides an anti-MAdCAM antibody that comprises a light chain variable région encoded by a human κ gene. In a preferred embodiment, the light chain variable région is encoded by a human Vk A2, A3, A26, B3, 012 or 018 gene family. In various embodiments, the light chain comprises no more than eleven, no more than six or no more than three amino acid substitutions from the germline human Vk A2, A3, A26, B3, 012 or 018 sequence. In a preferred embodiment, the amino acid substitutions are conservative substitutions.

-33 SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48 and 150 provide the amino acid sequences of the full-length kappa light chains of thirteen anti-MAdCAM antibodies, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 andX481.2. Figures 1K-1T are alignments of the amino acid sequences of the light chain variable domains of twelve anti-MAdCAM antibodies with the germline sequences from which they are derived. Figure 2A shows an alignment of the amino acid sequences of the light chain variable domains of the kappa light chains of twelve anti-MAdCAM antibodies to each other. Following the teachings of this spécification, one of ordinary skill in the art could détermine the différences between the germline sequences and the antibody sequences of additional anti-MAdCAM antibodies. SEQ ID NOS: 54, 58, 62, 66 or 68 provide the amino acid sequences of the full length kappa light chains of five additional anti-MAdCAM antibodies, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, modified by amino acid substitution from their parent anti-MAdCAM antibodies, 6.22.2, 6.34.2, 6.67.1, 6.77.1 or 7.26.4, respectively.

In a preferred embodiment, the VL of the anti-MAdCAM antibody contains the same mutations, relative to the germline amino acid sequence, as any one or more of the VL of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The disclosure includes an antiMAdCAM antibody that utilizes the same human Vk and human Jk genes as an exemplified antibody. In some embodiments, the antibody comprises one or more of the same mutations from germline as one or more exemplified antibodies. In some embodiments, the antibody comprises different substitutions at one or more of the same positions as one or more of the exemplified antibodies. For example, the VL of the anti- MAdCAM antibody may contain one or more amino acid substitutions that are the same as those présent in antibody 7.16.6, and another amino acid substitution that is the same as antibody 7.26.4. In this manner, one can mix and match different features of antibody binding in order to alter, e.g., the affmity of the antibody for MAdCAM or its dissociation rate from the antigen. In another embodiment, the mutations are made in the same position as those found in any one or more of the VL of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, but conservative amino acid substitutions are made rather than using the same amino acid. For example, if the amino acid substitution compared to the germline in one of the antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1mod, 6.77.1-mod or 7.26.4-mod is glutamate, one may conservatively substitute aspartate.

Similarly, if the amino acid substitution is serine, one may conservatively substitute threonine.

-34In another preferred embodiment, the light chain comprises an amino acid sequence that is the same as the amino acid sequence of the VL of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another highly preferred embodiment, the light chain comprises amino acid sequences that are the same as the CDR régions of the light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the light chain comprises an amino acid sequence with at least one CDR région of the light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1,

7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the light chain comprises amino acid sequences with CDRs from different light chains that use the same Vk and Jk genes. In a more preferred embodiment, the CDRs from different light chains are obtained from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the light chain comprises an amino acid sequence selected from SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 64, 66, 68, or 150 with or without the signal sequence. In another embodiment, the light chain comprises an amino acid sequence encoded by a nucléotide sequence selected from SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67 (with or without the signal sequence), or a nucléotide sequence that encodes an amino acid sequence having 1-11 amino acid insertions, délétions or substitutions therefrom. Preferably, the amino acid substitutions are conservative amino acid substitutions. In another embodiment, the antibody or portion thereof comprises a lambda light chain.

The présent disclosure also provides an anti-MAdCAM antibody or portion thereof that comprises a human VH gene sequence or a sequence derived from a human VH gene. In one embodiment, the heavy chain amino acid sequence is derived from a human VH 1-18, 3- 15, 321, 3-23, 3-30, 3-33 or 4-4 gene family. In varions embodiments, the heavy chain comprises no more than fifteen, no more than six or no more than three amino acid changes from germline human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4 gene sequence.

SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, and 148 provide the amino acid sequences of the full-length heavy chains of thirteen anti-MAdCAM antibodies. Figures 1 A-l J are alignments of the amino acid sequences of the heavy chain variable régions of twelve antiMAdCAM antibodies with the germline sequences from which they are derived. Figure 2B shows the alignments of the amino acid sequences of the heavy chain variable régions of twelve

- 35 anti-MAdCAM antibodies to each other. Following the teachings of this spécification and the nucléotide sequences of the disclosure, one of ordinary skill in the art could détermine the encoded amino acid sequence of the twelve anti-MAdCAM heavy chains and the germline heavy chains and détermine the différences between the germline sequences and the antibody sequences. SEQ ID NOS: 52, 56, 60 and 64 provide the amino acid sequences of the full length heavy chains of anti-MAdCAM antibodies, 6.22.2-mod, 6.34.2- mod and 6.67.1-mod, modified by amino acid substitution from their parent anti-MAdCAM antibodies, 6.22.2, 6.34.2 and 6.67.1 respectively. One further modified anti-MAdCAM antibody, 7.26.4-mod, has a full length heavy chain amino acid sequence which is SEQ ID NO: 42.

In a preferred embodiment, the VH of the anti-MAdCAM antibody contains the same mutations, relative to the germline amino acid sequence, as any one or more of the VH of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Similar to that discussed above, the antibody comprises one or more of the same mutations from germline as one or more exemplified antibodies. In some embodiments, the antibody comprises different substitutions at one or more of the same positions as one or more of the exemplified antibodies. For example, the VH of the anti-MAdCAM antibody may contain one or more amino acid substitutions that are the same as those présent in antibody 7.16.6, and another amino acid substitution that is the same as antibody 7.26.4. In this manner, one can mix and match different features of antibody binding in order to alter, e.g., the affinity of the antibody for MAdCAM or its dissociation rate from the antigen. In another embodiment, an amino acid substitution compared to germline is made at the same position as a substitution from germline as found in any one or more of the VH of reference antibody 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, but the position is substituted with a different residue, which is a conservative substitution compared to the reference antibody.

In another preferred embodiment, the heavy chain comprises an amino acid sequence that is the same as the amino acid sequence of the VH of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another highly preferred embodiment, the heavy chain comprises amino acid sequences that are the same as the CDR régions of the heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the heavy chain comprises an amino acid sequence from at least one CDR région of the heavy chain of 1.7.2,

1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.4, 7.26.4, 9.8.2, X481.2, 6.22.2mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the heavy chain comprises amino acid sequences with CDRs from different heavy chains. In a more preferred embodiment, the CDRs from different heavy chains are obtained from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the heavy chain comprises an amino acid sequence selected from SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148 with or without the signal sequence. In another embodiment, the heavy chain comprises an amino acid sequence encoded by a nucléotide sequence selected from SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, 63 or 149 or a nucléotide sequence that encodes an amino acid sequence having 1-15 amino acid insertions, délétions or substitutions therefrom. In another embodiment, the substitutions are conservative amino acid substitutions.

Methods of Producing Antibodies and Antibody-Producing Cell Lines

Immunization

In one embodiment of the instant disclosure, human antibodies are produced by immunizing a non-human animal comprising some or ail of the human immunoglobulin heavy and light chain loci with an MAdCAM antigen. In a preferred embodiment, the non- human animal is a XENOMOUSE™ animal, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is déficient in mouse antibody production. See, e.g., Green et al., Nature Genetics 7:13-21 (1994) and United States Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 09560 and WO 00/037504. The XENOMOUSE ™ animal produces an adult-like human répertoire of fully human antibodies and generates antigenspecific human mAbs. A second génération XENOMOUSE ™ animal contains approximately 80% of the human antibody V gene répertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and κ light chain loci. In other embodiments, XENOMOUSE ™mice contain approximately ail of the human heavy chain and λ light chain locus. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits, J. Exp. Med. 188:483-495 (1998), the disclosures of which are hereby incorporated by reference.

The disclosure also provides a method for making anti-MAdCAM antibodies from non-human, non-mouse animais by immunizing non-human transgenic animais that comprise human immunoglobulin loci. One may produce such animais using the methods described immediately

- 37 above. The methods disclosed in these documents can be modified as described in U.S. Patent 5,994,619 (the ‘“619 patent”), which is here in incorporated by reference. The ‘619 patent describes methods for producing novel cultured inner cell mass (CICM) cells and cell lines, derived from pigs and cows, and transgenic CICM cells into which heterologous DNA has been inserted. CICM transgenic cells can be used to produce cloned transgenic embryos, fetuses, and offspring. The ‘619 patent also describes methods of producing transgenic animais that are capable of transmitting the heterologous DNA to their progeny.

In a preferred embodiment, the non-human animais may be rats, sheep, pigs, goats, cattle or horses.

In another embodiment, the non-human animal comprising human immunoglobulin loci are animais that hâve a “minilocus” of human immunoglobulins. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of individual genes from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant domain(s), and a second constant domain(s) (preferably a gamma constant domain(s) are formed into a construct for insertion into an animal. This approach is described, inter alia, in U.S. Patent No. 5,545,807, 5,545,806, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

An advantage of the minilocus approach is the rapidity with which constructs including portions of the Ig locus can be generated and introduced into animais. However, a potential disadvantage of the minilocus approach is that there may not be sufficient immunoglobulin diversity to support full B-cell development, such that there may be lower antibody production.

To produce a human anti-MAdCAM antibody, a non-human animal comprising some or ail of the human immunoglobulin loci is immunized with a MAdCAM antigen and an antibody or the antibody-producing cell is isolated from the animal. The MAdCAM antigen may be isolated and/or purified MAdCAM and is preferably a human MAdCAM. In another embodiment, the MAdCAM antigen is a fragment of MAdCAM, preferably the extracellular domain of MAdCAM. In another embodiment, the MAdCAM antigen is a fragment that comprises at least one epitope of MAdCAM. In another embodiment, the MAdCAM antigen is a cell that expresses MAdCAM on its cell surface, preferably a cell that overexpresses MAdCAM on its cell surface.

Immunization of animais may be done by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press (1990). Methods for immunizing non-human animais such as mi ce, rats, sheep, goats, pigs, cattle and horses are

- 38 well known in the art. See, e.g., Harlow and Lane and United States Patent 5,994,619. In a preferred embodiment, the MAdCAM antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complété or incomplète Freund’s adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersai by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune System. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.

Example I provides a protocol for immunizing a XENOMOUSE ™ animal with full-length human MAdCAM in phosphate-buffered saline.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a MAdCAM antigen, antibodies and/or antibodyproducing cells may be obtained from the animal. An anti-MAdCAM antibody- containing sérum is obtained from the animal by bleeding or sacrificing the animal. The sérum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the sérum, or the anti-MAdCAM antibodies may be purified from the sérum.

In another embodiment, antibody-producing immortalized cell lines may be prepared from the immunized animal. After immunization, the animal is sacrificed and B cells are immortalized using methods well-known in the art. Methods of immortalizing cells include, but are not limited to, transfecting them with oncogenes, infecting them with an oncogenic virus and cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. In embodiments involving the myeloma cells, the myeloma cells do not secrete immunoglobulin polypeptides (a non-secretory cell line). After immortalization and antibiotic sélection, the immortalized cells, or culture supematants thereof, are screened using MAdCAM, a portion thereof, or a cell expressing MAdCAM. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. WO 00/37504, herein incorporated by reference.

In another embodiment, antibody-producing cells may be prepared from a human who has an auto immune disorder and who expresses anti-MAdCAM antibodies. Cells expressing the antiMAdCAM antibodies may be isolated by isolating white blood cells and subjecting them to

- 39 fluorescence-activated cell sorting (FACS) or by panning on plates coated with MAdCAM or a portion thereof. These cells may be fused with a human non-secretory myeloma to produce human hybridomas expressing human anti-MAdCAM antibodies. In general, this is a less preferred embodiment because it is likely that the anti-MAdCAM antibodies will hâve a low affmity for MAdCAM.

Anti-MAdCAM antibody-producing cells, e.g., hybridomas are selected, cloned and further screened for désirable characteristics, including robust cell growth, high antibody production and désirable antibody characteristics, as discussed further below. Hybridomas may be cultured and expanded in vivo in syngeneic animais, in animais that lack an immune System, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

Preferably, the immunized animal is a non-human animal that expresses human immunoglobulin genes and the splenic B cells are fused to a myeloma derived from the same species as the nonhuman animal. More preferably, the immunized animal is a XENOMOUSE™ animal and the myeloma cell line is a non-secretory mouse myeloma, such as the myeloma cell line is P3-X63AG8-653 (ATCC). See, e.g., Example I.

Thus, in one embodiment, the disclosure provides methods for producing a cell line that produces a human monoclonal antibody or a fragment thereof directed to MAdCAM comprising (a) immunizing a non-human transgenic animal described herein with MAdCAM, a portion of MAdCAM or a cell or tissue expressing MAdCAM; (b) allowing the transgenic animal to mount an immune response to MAdCAM; (c) isolating antibody- producing cells from transgenic animal; (d) immortalizing the antibody-producing cells; (e) creating individual monoclonal populations of the immortalized antibody-producing cells; and (f) screening the immortalized antibody-producing cells or culture supematants thereof to identify an antibody directed to MAdCAM.

In one aspect, the disclosure provides hybridomas that produce human anti- MAdCAM antibodies. In a preferred embodiment, the hybridomas are mouse hybridomas, as described above. In another embodiment, the hybridomas are produced in a non-human, non- mouse species such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell expressing an anti-MAdCAM antibody.

-40Nucleic Acids, Vectors, Host Cells and Recombinant Methods of Making Antibodies

Nucleic Acids

Nucleic acid molécules encoding anti-MAdCAM antibodies of the disclosure are provided. In one embodiment, the nucleic acid molécule encodes a heavy and/or light chain of an antiMAdCAM immunoglobulin. In a preferred embodiment, a single nucleic acid molécule encodes a heavy chain of an anti- MAdCAM immunoglobulin and another nucleic acid molécule encodes the light chain of an anti-MAdCAM immunoglobulin. In a more preferred embodiment, the encoded immunoglobulin is a human immunoglobulin, preferably a human IgG. The encoded light chain may be a λ chain or a κ chain, preferably a κ chain.

In a preferred embodiment the nucleic acid molécule encoding the variable région of the light chain comprises the germline sequence of a human Vk the A2, A3, A26, B3, 012 or O18 gene or a variant of said sequence. In a preferred embodiment, the nucleic acid molécule encoding the light chain comprises a sequence derived from a human JkI, Jk2, Jk3, Jk4 or Jk5 gene. In a preferred embodiment, the nucleic acid molécule encoding the light chain encodes no more than eleven amino acid changes from the germline A2, A3, A26, B3, 012 or 018 Vk gene, preferably no more than six amino acid changes, and even more preferably no more than three amino acid changes. In a more preferred embodiment, the nucleic acid encoding the light chain is the germline sequence.

The disclosure provides a nucleic acid molécule that encodes a variable région of the light chain (VL) containing up to eleven amino acid changes compared to the germline sequence, wherein the amino acid changes are identical to amino acid changes from the germline sequence from the VL of one of the antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2,6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The disclosure also provides a nucleic acid molécule comprising a nucléotide sequence that encodes the amino acid sequence of the variable région of the light chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The disclosure also provides a nucleic acid molécule comprising a nucléotide sequence that encodes the amino acid sequence of one or more of the CDRs of any one of the light chains of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In a preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of ail of the CDRs of any one of the light chains of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another embodiment, the

-41 nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, 150 or comprises a nucléotide sequence of one of SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67. In another preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of one or more of the CDRs of any one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, 150 or comprises a nucléotide sequence of one or more of the CDRs of any one of SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65, or 67. In amore preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of ail of the CDRs of any one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, 150 or comprises a the nucléotide sequence of ail the CDRs of any one of SEQ ID NOS: 3, 7, 11,15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65, or 67.

The disclosure also provides a nucleic acid molécule that encodes an amino acid sequence of a VL that has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VL described above, particularly to a VL that comprises an amino acid sequence of one of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, , 68 or 150. The disclosure also provides a nucléotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucléotide sequence of one of SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67.

In another embodiment, the disclosure provides a nucleic acid molécule that hybridizes under highly stringent conditions to a nucleic acid molécule encoding a VL as described above, particularly a nucleic acid molécule that comprises a nucléotide sequence encoding an amino acid sequence of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, 150. The disclosure also provides a nucleic acid molécule that hybridizes under highly stringent conditions to a nucleic acid molécule comprising a nucléotide sequence of one of SEQ ID NOS: 3,7, 11, 15, 19,23,27,31,35,39,43,47, 53,57,61,65 or 67.

The disclosure also provides a nucleic acid molécule encoding a heavy chain variable région (VH) that utilizes a human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4 VH gene. In some embodiments, the nucleic acid molécule encoding the VH gene further utilizes a human JH4 or JH6 family gene. In some embodiments, the nucleic acid molécule encoding the VH gene utilize the human JH4b or JH6b gene. In another embodiment, the nucleic acid molécule comprises a sequence derived from a human D 3-10, 4-23, 5-5, 6-6 or 6-19 gene.

In an even more preferred embodiment, the nucleic acid molécule encoding the VH contains no more than fifteen amino acid changes from the germline VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33

-42or 4-4 genes, preferably no more than six amino acid changes, and even more preferably no more than three amino acid changes. In a highly preferred embodiment, the nucleic acid molécule encoding the VH contains at least one amino acid change compared to the germline sequence, wherein the amino acid change is identical to an amino acid change from the germline sequence from the heavy chain of one of the antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In an even more preferred embodiment, the VH contains no more than fifteen amino acid changes compared to the germline sequences, wherein the changes are identical to those changes from the germline sequence from the VH of one of the antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod.

In one embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of the VH of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of one or more of the CDRs of the heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In a preferred embodiment, the nucleic acid molécule comprises nucléotide sequences that encode the amino acid sequences of ail of the CDRs of the heavy chain of 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148 or that comprises a nucléotide sequence of one of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, 63, or 149. In another preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequence of one or more ofthe CDRs of any one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148 or comprises a nucléotide sequence of one or more of the CDRs ofanyoneofSEQ IDNOS: 1,5,9, 13, 17,21,25,29,33,37,41,45,51,55, 59,63 or 149. In a preferred embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes the amino acid sequences of ail of the CDRs of any one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64, 148 or comprises a nucléotide sequence of ail ofthe CDRs of any one ofSEQ ID NOS: 1,5,9, 13, 17,21,25,29,33,37,41 45,51,55, 59, 63 or 149.

-43 In some embodiments the nucleic acid molécule comprises a nucléotide sequence encoding a contiguous région from the beginning of CDR1 to the end of CDR3 of a heavy or light chain of any of the above-mentioned anti-MAdCAM antibodies.

In another embodiment, the nucleic acid molécule encodes an amino acid sequence of a VH that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences encoding a VH as described immediately above, particularly to a VH that comprises an amino acid sequence of one of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or 64. The disclosure also provides a nucléotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucléotide sequence of one of SEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, 63 or 149.

In another embodiment, the nucleic acid molécule encoding a VH is one that hybridizes under highly stringent conditions to a nucléotide sequence encoding a VH as described above, particularly to a VH that comprises an amino acid sequence of one ofSEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148. The disclosure also provides a nucléotide sequence encoding a VH that hybridizes under highly stringent conditions to a nucleic acid molécule comprising a nucléotide sequence of one ofSEQ ID NOS: 1, 5, 9, 13, 17, 21, 25, 29, 33,37,41,45,51,55, 59, 63, 149.

The nucléotide sequence encoding either or both of the entire heavy and light chains of an antiMAdCAM antibody or the variable régions thereof may be obtained from any source that produces an anti-MAdCAM antibody. Methods of isolating mRNA encoding an antibody are well-known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one embodiment of the disclosure, the nucleic acid molécules may be obtained from a hybridoma that expresses an anti-MAdCAM antibody, as described above, preferably a hybridoma that has as one of its fusion partners a transgenic animal cell that expresses human immunoglobulin genes, such as a XENOMOUSE animal, a non-human mouse transgenic animal or a non-human, non-mouse transgenic animal. In another embodiment, the hybridoma is derived from anon-human, non-transgenic animal, which may be used, e.g., for humanized antibodies.

A nucleic acid molécule encoding the entire heavy chain of an anti-MAdCAM antibody may be constructed by fusing a nucleic acid molécule encoding the entire variable domain of a heavy chain or an antigen-binding domain thereof with a constant domain of a heavy chain. Similarly, a nucleic acid molécule encoding the light chain of an anti- MAdCAM antibody may be

-44constructed by fusing a nucleic acid molécule encoding the variable domain of a light chain or an antigen-binding domain thereof with a constant domain of a light chain. Nucleic acid molécules encoding the VH and VL régions may be converted to full-length antibody genes by inserting them into expression vectors already encoding heavy chain constant and light chain constant régions, respectively, such that the VH segment is operatively linked to the heavy chain constant région (CH) segment(s) within the vector and the VL segment is operatively linked to the light chain constant région (CL) segment within the vector. Altematively, the nucleic acid molécules encoding the VH or VL chains are converted into full-length antibody genes by linking, e.g., ligating, the nucleic acid molécule encoding a VH chain to a nucleic acid molécule encoding a CH chain using standard molecular biological techniques. The same may be achieved using nucleic acid molécules encoding VL and CL chains. The sequences of human heavy and light chain constant région genes are known in the art. See, e.g., Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242 (1991). Nucleic acid molécules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they hâve been introduced and the anti-MAdCAM antibody isolated.

In a preferred embodiment, the nucleic acid encoding the variable région of the heavy chain encodes the variable région of amino acid sequences ofSEQ IDNOS:2, 6, 10, 14, 18,22,26, 30, 34, 38, 42, 46, 52, 56, 60, 64 or 148, and the nucleic acid molécule encoding the variable région of the light chains encodes the variable région of amino acid sequence of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68 or 150.

In one embodiment, a nucleic acid molécule encoding either the heavy chain of an antiMAdCAM antibody or an antigen-binding portion thereof, or the light chain of an antiMAdCAM antibody or an antigen-binding portion thereof may be isolated from a non- human, non-mouse animal that expresses human immunoglobulin genes and has been immunized with a MAdCAM antigen. In other embodiment, the nucleic acid molécule may be isolated from an anti-MAdCAM antibody-producing cell derived from a non-transgenic animal or from a human patient who produces anti-MAdCAM antibodies. mRNA from the anti-MAdCAM antibodyproducing cells may be isolated by standard techniques, cloned and/or amplified using PCR and library construction techniques, and screened using standard protocols to obtain nucleic acid molécules encoding anti-MAdCAM heavy and light chains.

The nucleic acid molécules may be used to recombinantly express large quantities of antiMAdCAM antibodies, as described below. The nucleic acid molécules may also be used to produce chimeric antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody dérivatives, as described further below. If the nucleic acid molécules are

-45 derived from a non-human, non-transgenic animal, the nucleic acid molécules may be used for antibody humanization, also as described below.

In another embodiment, the nucleic acid molécules of the disclosure may be used as probes or PCR primers for spécifie antibody sequences. For instance, a nucleic acid molécule probe may be used in diagnostic methods or a nucleic acid molécule PCR primer may be used to amplify régions of DNA that could be used, inter alia, to isolate nucléotide sequences for use in producing variable domains of anti-MAdCAM antibodies. In a preferred embodiment, the nucleic acid molécules are oligonucleotides. In a more preferred embodiment, the oligonucleotides are from highly variable régions of the heavy and light chains of the antibody of interest. In an even more preferred embodiment, the oligonucleotides encode ail or a part of one or more of the CDRs.

Vectors

The disclosure provides vectors comprising the nucleic acid molécules of the disclosure that encode the heavy chain or the antigen-binding portion thereof. The disclosure also provides vectors comprising the nucleic acid molécules of the disclosure that encode the light chain or antigen-binding portion thereof. The disclosure also provides vectors comprising nucleic acid molécules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

To express the antibodies, or antibody portions of the disclosure, DNAs encoding partial or fulllength light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are présent).

A convenient vector is one that encodes a functionally complété human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL

-46sequence can be easily inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J région and the splice acceptor site preceding the human C région, and also at the splice régions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding régions. The recombinant expression vector can also encode a signal peptide that facilitâtes sécrétion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the sélection of regulatory sequences may dépend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral éléments that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomégalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory éléments, and sequences thereof, see e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615, each of which is hereby incorporated by référencé. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants are known in the art. See, e.g, United States Patent 6,517,529. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additionai sequences, such as sequences that regulate réplication of the vector in host cells (e.g., origins of réplication) and selectable marker genes. The selectable marker gene facilitâtes sélection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers résistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.

-47Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr'host cells with methotrexate selection/amplification) and the neo gene (for G418 sélection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molécules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of an anti-MAdCAM antibody, and vectors comprising these nucleic acid molécules, can be used for transformation of a suitable mammalian plant, bacterial or yeast host cell. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate précipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molécules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods of transforming bacterial and yeast cells are also well known in the art.

Mammalian cell Unes available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alla, Chinese hamster ovary (CHO) cells, NS0, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines hâve high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof, the light chain and/or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, sécrétion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include,

-48 e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

Further, expression of antibodies of the disclosure (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression System (the GS System) is a common approach for enhancing expression under certain conditions. The GS System is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, 0 338 841 and 0 323 997.

It is likely that antibodies expressed by different cell lines or in transgenic animais will hâve different glycosylation from each other. However, ail antibodies encoded by the nucleic acid molécules provided herein, or comprising the amino acid sequences provided herein are part of the instant disclosure, regardless of the glycosylation of the antibodies.

Transgenic Animais and Plants

The disclosure also provides transgenic non-human animais and transgenic plants comprising one or more nucleic acid molécules of the disclosure that may be used to produce antibodies of the disclosure. Antibodies can be produced in and recovered from tissue or bodily fluids, such as milk, blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, e.g, U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957. As described above, non-human transgenic animais that comprise human immunoglobulin loci can be immunized with MAdCAM or a portion thereof. Methods for making antibodies in plants are described, e.g., in U.S. Patents 6,046,037 and 5,959,177, incorporated herein by reference.

In another embodiment, non-human transgenic animais and transgenic plants are produced by introducing one or more nucleic acid molécules of the disclosure into the animal or plant by standard transgenic techniques. See Hogan, supra. The transgenic cells used for making the transgenic animal can be embryonic stem cells, somatic cells or fertilized egg cells. The transgenic non-human organisms can be chimeric, nonchimeric hétérozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al.„ Manipulating the Mouse Embryo: A Laboratory Manual 2ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); andPinkert, Transgenic Animal Technology: A Laboratory Handbook, Academie Press (1999).

In another embodiment, the transgenic non-human organisms may hâve a targeted disruption and replacement that encodes a heavy chain and/or a light chain of interest. In a preferred embodiment, the transgenic animais or plants comprise and express nucleic acid molécules

-49encoding heavy and light chains that combine to bind specifically to MAdCAM, preferably human MAdCAM. In another embodiment, the transgenic animais or plants comprise nucleic acid molécules encoding a modified antibody such as a single-chain antibody, a chimeric antibody or a humanized antibody. The anti-MAdCAM antibodies may be made in any transgenic animal. In a preferred embodiment, the non-human animais are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries

The disclosure provides a method for producing an anti-MAdCAM antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with a MAdCAM or a portion thereof, isolating phage that bind MAdCAM, and obtaining the antibody from the phage. One method to préparé the library of antibodies comprises the steps of immunizing a non-human host animal comprising a human immunoglobulin locus with MAdCAM or an antigenic portion thereof to create an immune response, extracting cells from the host animal the cells that are responsible for production of antibodies; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using a primer, and inserting the cDNA into phage display vector such that antibodies are expressed on the phage. Recombinant anti- MAdCAM antibodies of the disclosure may be obtained in this way.

Recombinant anti-MAdCAM human antibodies of the disclosure in addition to the antiMAdCAM antibodies disclosed herein can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA isolated from human lymphocytes. Méthodologies for preparing and screening such libraries are known in the art. There are commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There are also other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; Fuchs et al. (1991), Biotechnology, 9:1369-1372; Hay et al., Plum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J, 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature,

- 50352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrad et al., Biotechnology, 9:1373-1377 (1991); Hoogenboom et al., Nue Acid Res, 19:4133-4137 (1991); and Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991).

In a preferred embodiment, to isolate human anti-MAdCAM antibodies with the desired characteristics, a human anti-MAdCAM antibody as described herein is first used to select human heavy and light chain sequences having similar binding activity toward MAdCAM, using the epitope imprinting methods described in Hoogenboom et al., PCT Publication No. WO 93/06213. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described in McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al., Nature, 348:552-554 (1990); and Griffiths et al., EMBOJ, 12:725-734 (1993). The scFv antibody libraries preferably are screened using human MAdCAM as the antigen.

Once initial human VL and VH segments are selected, “mix and match” experiments, in which different pairs of the initially selected VL and VH segments are screened for MAdCAM binding, are performed to select preferred VL/VH pair combinations. Additionally, to further improve the quality of the antibody, the VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the CDR3 région of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VH and VL régions using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively, which primers hâve been “spiked” with a random mixture of the four nucléotide bases at certain positions such that the résultant PCR products encode VH and VL segments into which random mutations hâve been introduced into the VH and/or VL CDR3 régions. These randomly mutated VH and VL segments can be rescreened for binding to MAdCAM.

Following screening and isolation of an anti-MAdCAM antibody of the disclosure from a recombinant immunoglobulin display library, nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the disclosure, as described below. To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the instant disclosure is to provide a mechanism by which the class of an antiMAdCAM antibody may be switched with another. In one aspect of the disclosure, a nucleic

- 51 acid molécule encoding VL or VH is isolated using methods well-known in the art such that it does not include any nucléotide sequences encoding CL or CH. The nucleic acid molécule encoding VL or VH is then operatively linked to a nucléotide sequence encoding a CL or CH from a different class of immunoglobulin molécule. This may be achieved using a vector or nucleic acid molécule that comprises a CL or CH encoding sequence, as described above. For example, an anti-MAdCAM antibody that was originally IgM may be class switched to an IgG. Further, the class switching may be used to couvert one IgG subclass to another, e.g., from IgG4 to IgGa. A preferred method for producing an antibody of the disclosure comprising a desired isotype or antibody subclass comprises the steps of isolating a nucleic acid encoding the heavy chain of an anti-MAdCAM antibody and a nucleic acid encoding the light chain of an antiMAdCAM antibody, obtaining the variable région of the heavy chain, ligating the variable région of the heavy chain with the constant domain of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and collecting the anti-MAdCAM antibody with the desired isotype.

Antibody Dérivatives

One may use the nucleic acid molécules described above to generate antibody dérivatives using techniques and methods known to one of ordinary skill in the art.

Humanized Antibodies

The immunogenicity of non-human antibodies can be reduced to some extent using techniques of humanization, potentially employing display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See, e.g., Winter and Harris, Immunol Today, 14:43-46 (1993) and Wright et al., Crit. Reviews in Immunol., 12125- 168 (1992). The antibody of interest may be engineered by recombinant DNA techniques to substitute the ChI, Ch2, Ch3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). In another embodiment, a non- human anti-MAdCAM antibody can be humanized by substituting the ChI, hinge domain, Ch2, Ch3, and/or the framework domains with the corresponding human sequence of a anti- MAdCAM antibody of the disclosure.

Mutated Antibodies

In another embodiment, the nucleic acid molécules, vectors and host cells may be used to make mutated anti-MAdCAM antibodies. The antibodies may be mutated in the variable domains of the heavy and/or light chains to alter a binding property of the antibody. For example, a

-52mutation may be made in one or more of the CDR régions to increase or decrease the Kd of the antibody for MAdCAM. Techniques in site-directed mutagenesis are well-known in the art. See, e.g., Sambrook et al., and Ausubel et al., supra. In a preferred embodiment, mutations are made at an amino acid residue that is known to be changed compared to germline in a variable région of an anti-MAdCAM antibody. In a more preferred embodiment, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in a variable région or CDR région of oneof the anti-MAdCAM antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another embodiment, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in a variable région or CDR région whose amino acid sequence is presented in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66, 68 148 or 150 or whose nucléotide sequence is presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 53, 55, 57, 61, 63, 65, 67 or 149.

In another embodiment, the nucleic acid molécules are mutated in one or more of the framework régions. A mutation may be made in a framework région or constant domain to increase the half-life of the anti-MAdCAM antibody. See, e.g., WO 00/09560, published February 24, 2000, herein incorporated by reference. In one embodiment, there may be one, three or five or ten point mutations and no more than fifteen point mutations. A mutation in a framework région or constant domain may also be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molécule, or to alter such properties as complément fixation. Mutations may be made in each of the framework régions, the constant domain and the variable régions in a single mutated antibody. Altematively, mutations may be made in only one of the framework régions, the variable régions or the constant domain in a single mutated antibody.

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

- 53 Modifîed Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be made which comprises ail or a portion of an anti-MAdCAM antibody linked to another polypeptide. In a preferred embodiment, only the variable régions of the anti-MAdCAM antibody are linked to the polypeptide. In another preferred embodiment, the VH domain of an anti-MAdCAM antibody are linked to a first polypeptide, while the VL domain of an anti-MAdCAM antibody are linked to a second polypeptide that associâtes with the first polypeptide in a manner in which the VH and VL domains can interact with one another to form an antibody binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (see below under Single Chain Antibodies). The VH-linker-VL antibody is then linked to the polypeptide of interest. The fusion antibody is useful to directing a polypeptide to a MAdCAM-expressing cell or tissue. The polypeptide may be a therapeutic agent, such as a toxin, growth factor or other regulatory protein, or may be a diagnostic agent, such as an enzyme that may be easily visualized, such as horseradish peroxidase. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody.

To create a single chain antibody, (scFv) the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH régions joined by the flexible linker (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); McCafferty et al., Nature, 348:552-554 (1990)). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.

In another embodiment, other modifîed antibodies may be prepared using anti- MAdCAMencoding nucleic acid molécules. For instance, “Kappa bodies” (111 et al., Protein Eng, 10: 94957(1997)), “Minibodies” (Martin et al., EMBO J, 13: 5303-9(1994)), “Diabodies” (Holliger et al., PNAS USA, 90: 6444-6448(1993)), or “Janusins” (Traunecker et al., EMBO J, 10:3655-3659 (1991) and Traunecker et al., “Janusin: new molecular design for bispecific reagents,” Int J Cancer Suppl, 7:51-52 (1992)) may be prepared using standard molecular biological techniques following the teachings of the spécification.

In another aspect, chimeric and bispecific antibodies can be generated. A chimeric antibody may be made that comprises CDRs and framework régions from different antibodies. In a preferred

-54embodiment, the CDRs of the chimeric antibody comprises ail of the CDRs of the variable région of a light chain or heavy chain of a human anti-MAdCAM antibody, while the framework régions are derived from one or more different antibodies. In a more preferred embodiment, the CDRs of the chimeric antibody comprise ail of the CDRs of the variable régions of the light chain and the heavy chain of a human anti-MAdCAM antibody. The framework régions may be from another species and may, in a preferred embodiment, be humanized. Altematively, the framework régions may be from another human antibody.

A bispecific antibody can be generated that binds specifically to MAdCAM through one binding domain and to a second molécule through a second binding domain. The bispecific antibody can be produced through recombinant molecular biological techniques, or may be physically conjugated together. In addition, a single chain antibody containing more than one VH and VL may be generated that binds specifically to MAdCAM and to another molécule. Such bispecific antibodies can be generated using techniques that are well known for example, in connection with (i) and (ii) see, e.g., Fanger et al., ImmunolMethods 4: 72-81 (1994) and Wright and Harris, supra, and in connection with (iii) see, e.g., Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52 (1992). In a preferred embodiment, the bispecific antibody binds to MAdCAM and to another molécule expressed at high level on endothélial cells. In a more preferred embodiment, the other molécule is VCAM, ICAM or L-selectin.

In various embodiments, the modified antibodies described above are prepared using one or more of the variable régions or one or more CDR régions from one of the antibodies selected from 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1,7.16.6, 7.20.5, 7.26.4, 9.8.2, X481.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another embodiment, the modified antibodies are prepared using one or more of the variable régions or one or more CDR régions whose amino acid sequence is presented in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 54, 56, 58, 62, 64, 66, 68, 148 or 150 or whose nucléotide sequence is presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 53, 55, 57, 61, 63, 65, 67 or 149.

Derivatized and Labeled Antibodies

An antibody or antibody portion of the disclosure can be derivatized or linked to another molécule (e.g., another peptide or protein). In general, the antibodies or portions thereof are derivatized such that the MAdCAM binding is not affected adversely by the derivatization or labeling. Accordingly, the antibodies and antibody portions of the disclosure are intended to include both intact and modified forms of the human anti-MAdCAM antibodies described herein.

- 55 For example, an antibody or antibody portion of the disclosure can be functionally linked (by Chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a détection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can médiate association of the antibody or antibody portion with another molécule (such as a streptavidin core région or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111.

Another type of derivatized antibody is a labeled antibody. Useful détection agents with which an antibody or antibody portion of the disclosure may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-lnapthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. An antibody may also be labeled with enzymes that are useful for détection, such as horseradish peroxidase, βgalactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a détectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discemed. For example, when the agent horseradish peroxidase is présent, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is détectable. An antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. An antibody may be labeled with a magnetic agent, such as gadolinium. An antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, métal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

An anti-MAdCAM antibody may also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect MAdCAM-expressing tissues by x-ray or other diagnostic techniques. Further, the radiolabel may be used therapeutically as a toxin for diseased tissue or MAdCAM expressing tumors. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides — ³H, ^l4C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹'in, i25j i3ij

- 56 An anti-MAdCAM antibody may also be derivatized with a Chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g., to increase sérum half-life or to increase tissue binding. This methodology would also apply to any antigen-binding fragments or versions of anti-MAdCAM antibodies.

Pharmaceutical Compositions and Kits

In a further aspect, the disclosure provides compositions comprising an inhibitory human antiMAdCAM antibody and methods for treating subjects with such compositions. In some embodiments, the subject of treatment is human. In other embodiments, the subject is a veterinary subject. In some embodiments, the veterinary subject is a dog or a non-human primate.

Treatment may involve administration of one or more inhibitory anti-MAdCAM monoclonal antibodies of the disclosure, or antigen-binding fragments thereof, alone or with a pharmaceutically acceptable carrier. Inhibitory anti-MAdCAM antibodies of the disclosure and compositions comprising them, can be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. Additional therapeutic agents include antiinflammatory or immunomodulatory agents. These agents include, but are not limited to, the topical and oral corticosteroids such as prednisolone, méthylprednisolone, NCX-1015 or budesonide; the aminosalicylates such as mesalazine, olsalazine, balsalazide or NCX-456; the class of immunomodulators such as azathioprine, 6-mercaptopurine, methotrexate, cyclosporin, FK506, IL-10 (Ilodecakin), IL-11 (Oprelevkin), IL-12, MIF/CD74 antagonists, CD40 antagonists, such as TNX-100/5-D12, OX40L antagonists, GM-CSF, pimecrolimus or rapamycin; the class of anti-TNFoc agents such as infliximab, adalimumab, CDP-870, onercept, etanercept; the class of anti-inflammatory agents, such as PDE-4 inhibitors (roflumilast, etc), TACE inhibitors (DPC-333, RDP-58, etc) and ICE inhibitors (VX-740, etc) as well as IL-2 receptor antagonists, such as daclizumab, the class of sélective adhesion molécule antagonists, such as natalizumab, MLN-02, or alicaforsen, classes of analgésie agents such as, but not limited to, COX-2 inhibitors, such as rofecoxib, valdecoxib, celecoxib, P/Q-type volatge senstize channel (α2δ) modulators, such as gabapentin and pregabalin, NK-1 receptor antagonists, cannabinoid receptor modulators, and delta opioid receptor agonists, as well as anti-neoplastic, anti-tumor, anti-angiogenic or chemotherapeutic agents Such additional agents may be included in the same composition or administered separately. In some embodiments, one or more inhibitory anti-MAdCAM antibodies of the disclosure can be used as a vaccine or as adjuvants to a vaccine. In particular, because MAdCAM is expressed in lymphoid tissue, vaccine antigens

-57can be advantageously targeted to lymphoid tissue by conjugating the antigen to an antiMAdCAM antibody of the disclosure.

As used herein, “pharmaceutically acceptable carrier” means any and ail solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonie and absorption enhancing or delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, Polyethylene glycol, éthanol and the like, as well as combinations thereof. In many cases, it will be préférable to include isotonie agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additionai examples of pharmaceutically acceptable substances are surfectants, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

The compositions of this disclosure may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, lyophilized cake, dry powders, liposomes and suppositories. The preferred form dépends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parentéral (e.g., intravenous, subeutaneous, intraperitoneal, intramuscular, intradermal). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular, intradermal or subeutaneous injection.

Therapeutic compositions typically must be stérile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, lyophilized cake, dry powder, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Stérile injectable solutions can be prepared by incorporating the anti-MAdCAM antibody in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, followed by sterilization. In the case of stérile powders for the préparation of stérile injectable solutions, the preferred methods of préparation are vacuum drying and freeze-drying that yields a powder of the active ingrédient plus any any additionai desired ingrédient from a previously stérile solution thereof. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients from those enumerated above. The desired characteristics of a solution can be maintained, for example, by the use of surfactants and the

- 58 required particle size in the case of dispersion by the use of surfactants, phospholipids and polymers. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts, polymeric materials, oils and gelatin.

The antibodies of the présent disclosure can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, intradermal or intravenous infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

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

Biodégradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the préparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978)).

In certain embodiments, an anti-MAdCAM antibody of the disclosure can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingrédients, if desired) can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject’s diet. For oral therapeutic administration, the anti-MAdCAM antibodies can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, élixirs, suspensions, syrups, wafers, and the like. To administer a compound of the disclosure by other than parentéral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

The compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antigen-binding portion of the disclosure. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic resuit. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease State, âge, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically

-59bénéficiai effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic resuit. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parentéral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrète units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a pre-determined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The spécification for the dosage unit forms of the disclosure are dictated by and directly dépendent on (a) the unique characteristics of the anti-MAdCAM antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inhérent in the art of compounding such an antibody for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the disclosure is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In some embodiments, a formulation contains 5 mg/mL of antibody in a buffer of 20 mM sodium acetate, pH 5.5, 140 mM NaCl, and 0.2 mg/mL polysorbate 80. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be fùrther understood that for any particular subject, spécifie dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Another aspect of the présent disclosure provides kits comprising an anti-MAdCAM antibody or antibody portion of the disclosure or a composition comprising such an antibody. A kit may include, in addition to the antibody or composition, diagnostic or therapeutic agents. A kit can also include instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit includes the antibody or a composition comprising it and a diagnostic agent that can be used in a method described below. In another preferred embodiment, the kit includes

-60the antibody or a composition comprising it and one or more therapeutic agents that can be used in a method described below.

Gene Therapy

The nucleic acid molécules of the instant disclosure can be administered to a patient in need thereof via gene therapy. The therapy may be either in vivo or ex vivo. In a preferred embodiment, nucleic acid molécules encoding both a heavy chain and a light chain are administered to a patient. In a more preferred embodiment, the nucleic acid molécules are administered such that they are stably integrated into chromosomes of B cells because these cells are specialized for producing antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and re-transplanted into a patient in need thereof. In another embodiment, precursor B cells or other cells are infected in vivo using a recombinant virus known to infect the cell type of interest. Typical vectors used for gene therapy include liposomes, plasmids and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. After infection either in vivo or ex vivo, levels of antibody expression can be monitored by taking a sample from the treated patient and using any immunoassay known in the art or discussed herein.

In a preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molécule encoding the heavy chain or an antigen- binding portion thereof of an anti-MAdCAM antibody and expressing the nucleic acid molécule. In another embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molécule encoding the light chain or an antigen- binding portion thereof of an anti-MAdCAM antibody and expressing the nucleic acid molécule. In a more preferred method, the gene therapy method comprises the steps of administering of an isolated nucleic acid molécule encoding the heavy chain or an antigen- binding portion thereof and an isolated nucleic acid molécule encoding the light chain or the antigen-binding portion thereof of an anti-MAdCAM antibody of the disclosure and expressing the nucleic acid molécules. The gene therapy method may also comprise the step of administering another anti-inflammatory or immunomodulatory agent.

Diagnostic Methods of Use

The anti-MAdCAM antibodies may be used to detect MAdCAM in a biological sample in vitro or in vivo. The anti-MAdCAM antibodies may be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprécipitation. The anti-MAdCAM antibodies of the disclosure may be used to detect MAdCAM from humans. In another embodiment, the anti-MAdCAM antibodies may be used to detect MAdCAM from Old World primates such as cynomolgus and rhésus monkeys,

-61 chimpanzees and apes. The disclosure provides a method for detecting MAdCAM in a biological sample comprising contacting a biological sample with an anti-MAdCAM antibody of the disclosure and detecting the antibody bound to MAdCAM. In one embodiment, the antiMAdCAM antibody is directlyderivatized with a détectable label. In another embodiment, the anti-MAdCAM antibody (the first antibody) is unlabeled and a second antibody or other molécule that can bind the anti-MAdCAM antibody is labeled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the spécifie species and class of the first antibody. For example, if the anti-MAdCAM antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molécules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary hâve been disclosed supra, and include varions enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; an example of a magnetic agent includes gadolinium; and examples of suitable radioactive material include¹²⁵!, ¹³¹I,³⁵S or ³H.

In an alternative embodiment, MAdCAM can be assayed in a biological sample by a compétition immunoassay utilizing MAdCAM standards labeled with a détectable substance and an unlabeled anti-MAdCAM antibody. In this assay, the biological sample, the labeled MAdCAM standards and the anti-MAdCAM antibody are combined and the amount of labeled MAdCAM standard bound to the unlabeled antibody is determined. The amount of MAdCAM in the biological sample is inversely proportional to the amount of labeled MAdCAM standard bound to the anti-MAdCAM antibody.

One may use the immunoassays disclosed above for a number of purposes. In one embodiment, the anti-MAdCAM antibodies may be used to detect MAdCAM in cells in cell culture. In a preferred embodiment, the anti-MAdCAM antibodies may be used to détermine the level of cell surface MAdCAM expression after treatment of the cells with varions compounds. This method can be used to test compounds that may be used to activate or inhibit MAdCAM. In this method, one sample of cells is treated with a test compound for a period of time while another sample is left untreated, cell surface expression could then be determined by flow cytometry,

- 62 immunohistochemistry, Western blot, ELISA or RIA. In addition, the immunoassays may be sealed up for high throughput screening in order to test a large number of compounds for either activation or inhibition of MAdCAM.

The anti-MAdCAM antibodies of the disclosure may also be used to détermine the levels of MAdCAM on a tissue or in cells derived from the tissue. In a preferred embodiment, the tissue is a diseased tissue. In a more preferred embodiment, the tissue is inflamed gastrointestinal tract or a biopsy thereof. In a preferred embodiment of the method, a tissue or a biopsy thereof is excised from a patient. The tissue or biopsy is then used in an immunoassay to détermine, e.g., MAdCAM levels, cell surface levels of MAdCAM, or localization of MAdCAM by the methods discussed above. The method can be used to détermine if an inflamed tissue expresses MAdCAM at a high level.

The above-described diagnostic method can be used to détermine whether a tissue expresses high levels of MAdCAM, which may be indicative that the tissue will respond well to treatment with anti-MAdCAM antibody. Further, the diagnostic method may also be used to détermine whether treatment with anti-MAdCAM antibody (see below) is causing a tissue to express lower levels of MAdCAM and thus can be used to détermine whether the treatment is successful.

The antibodies of the présent disclosure may also be used in vivo to localize tissues and organs that express MAdCAM. In a preferred embodiment, the anti-MAdCAM antibodies can be used to localize inflamed tissue. The advantage of the anti-MAdCAM antibodies of the présent disclosure is that they will not generate an immune response upon administration. The method comprises the steps of administering an anti-MAdCAM antibody or a pharmaceutical composition thereof to a patient in need of such a diagnostic test and subjecting the patient to imaging analysis déterminé the location of the MAdCAM-expressing tissues. Imaging analysis is well known in the medical art, and includes, without limitation, ray analysis, gamma scintigraphy, magnetic résonance imaging (MRI), positron émission tomography or computed tomography (CT). In another embodiment of the method, a biopsy is obtained from the patient to détermine whether the tissue of interest expresses MAdCAM rather than subjecting the patient to imaging analysis. In a preferred embodiment, the anti- MAdCAM antibodies may be labeled with a détectable agent that can be imaged in a patient. For example, the antibody may be labeled with a contrast agent, such as barium, which can be used for x-ray analysis, or a magnetic contrast agent, such as a gadolinium chelate, which can be used for MRI or CT. Other labeling agents include, without limitation, radioisotopes, such as Te. In another embodiment, the antiMAdCAM antibody will be unlabeled and will be imaged by administering a second antibody or other molécule that is détectable and that can bind the anti-MAdCAM antibody.

-63The anti-MAdCAM antibodies of the disclosure may also be used to détermine the levels of soluble MAdCAM présent in donor blood, sérum, plasma, or other biofluid, including, but not limited to, stool, urine, sputum or biopsy sample. In a preferred embodiment, the biofluid is plasma. The biofluid is then used in an immunoassay to détermine levels of soluble MAdCAM. Soluble MAdCAM could be a surrogate marker for ongoing gastrointestinal inflammation and the method of détection could be used as a diagnostic marker to measure disease severity.

The above-described diagnostic method can be used to détermine whether an individual expresses high levels of soluble MAdCAM, which may be indicative that the individual will respond well to treatment with an anti-MAdCAM antibody. Further, the diagnostic method may also be used to détermine whether treatment with anti-MAdCAM antibody (see below) or other pharmaceutical agent of the disease is causing an individual to express lower levels of MAdCAM and thus can be used to déterminé whether the treatment is successful.

Inhibition of OM37/MAdCAM-dependent adhesion by anti-MAdCAM antibody:

In another embodiment, the disclosure provides an anti-MAdCAM antibody that binds MAdCAM and inhibits the binding and adhesion of ouP7-integrin bearing cells to MAdCAM or other cognate ligands, such as L-selectin, to MAdCAM. In a preferred embodiment, the MAdCAM is human and is either a soluble form, or expressed on the surface of a cell. In another preferred embodiment, the anti-MAdCAM antibody is ahuman antibody. In another embodiment, the antibody or portion thereof inhibits binding between α4βγ and MAdCAM with an IC50 value of no more than 50 nM. In a preferred embodiment, the IC50 value is no more than 5 nM. In a more preferred embodiment, the IC50 value is less than 5 nM. In a more preferred embodiment, the IC50 value is less than 0.05 pg/mL, 0.04 pg/mL or 0.03 pg/mL. In another preferred embodiment the IC50 value is less than 0.5 pg/mL, 0.4 pg/mL or 0.3 pg/mL. The IC50 value can be measured by any method known in the art. Typically, an IC50 value can be measured by ELISA or adhesion assay. In a preferred embodiment, the IC50 value is measured by adhesion assay using either cells or tissue which natively express MAdCAM or cells or tissue which hâve been engineered to express MAdCAM.

Inhibition of lymphocyte recruitment to gpt-associated lymphoid tissue by anti-MAdCAM antibodies

In another embodiment, the disclosure provides an anti-MAdCAM antibody that binds natively expressed MAdCAM and inhibits the binding of lymphocytes to specialised gastrointestinal lymphoid tissue. In a preferred embodiment, the natively-expressed MAdCAM is human or primate MAdCAM and is either a soluble form, or expressed on the surface of a cell. In another

-64preferred embodiment, the anti-MAdCAM antibody is a human antibody. In another embodiment, the antibody or portion thereof inhibits the recruitment of gut-trophic Ο4β?⁺ lymphocytes to tissues expressing MAdCAM with an IC50 value of no more than 5 mg/kg. In a preferred embodiment, the IC50 value is no more than 1 mg/kg. In a more preferred embodiment, the IC50 value is less than 0.1 mg/kg. In one embodiment, the IC50 value can be determined by measuring the dose effect relationship of recruitment of technetium-labeled peripheral blood lymphocytes to the gastrointestinal tract using gamma scintigraphy or single photon émission computed tomography. In an another embodiment, the IC50 value can be determined by measuring the increase in gut-trophic α4β?⁺ lymphocytes, such as, but not limited to, CD4⁺ «4β7⁺ memory T-cells, in the peripheral circulation using flow cytometry as a function of the dose of anti-MAdCAM antibody.

In order that this disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the disclosure in any manner.

EXAMPLE 1:

Génération of anti-MAdCAM producing hybridomas

Antibodies of the disclosure were prepared, assayed and selected in accordance with the présent Example

Primary Immunogen Préparation:

Two immunogens were prepared for immunisation of the XenoMouse™ mice: (i) a MAdCAMIgGi Fc fusion protein and (ii) cell membranes prepared from cells stably transfected with MAdCAM.

(i) MAdCAM-IgGi Fc Fusion Protein

Expression vector construction:

An EcoRI/BglII cDNA fragment encoding the mature extracellular, immunoglobulin-like domain of MAdCAM was excised from a pINCY Incyte clone (3279276) and cloned into EcoRI/BamHI sites of the pIGl vector (Simmons, D. L. (1993) in Cellular Interactions in Development: A Practical Approach, ed. Hartley, D. A. (Oxford Univ. Press, Oxford), pp. 93127.)) to generate an in frame IgGi Fc fusion. The resulting insert was excised with EcoRI/Notl and cloned into pCDNA3.1+ (Invitrogen). The MAdCAM-IgGi Fc cDNA in the vector was sequence confirmed. The amino acid sequence of the MAdCAM-IgGi Fc fusion protein is shownbelow:

-65 MAdCAM-IgGi Fc Fusion Protein:

MDFGLALLLAGLLGLLLGQSLQVKPLQVEPPEPWAVALGASRQLTCRLACADRGASVQWRG LDTSLGAVQSDTGRSVLTVRNASLSAAGTRVCVGSCGGRTFQHTVQLLVYAFPDQLTVSPAA LVPGDPEVACTAHKVTPVDPNALSFSLLVGGQELEGAQALGPEVQEEEEEPQGDEDVLFRVT ERWRLPPLGTPVPPALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDTTSPES PDTTSQEPPDTTSQEPPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRSPGSTRTRRPE

IQPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 107)

Underlined: signal peptide

Bold: MAdCAM extracellular domain

Recombinant Protein Expression/Purification:

CHO-DHFR cells were transfected with pCDNA3.1+ vector containing MAdCAM- IgGi Fc fusion protein cDNA and stable clones expressing MAdCAM-IgGi Fc fusion protein selected in Iscove’s media containing 600 pg/mL G418 and 100 ng/mL methotrexate. For protein expression, a hollow fibre bioreactor was seeded with stably expressing MAdCAM- IgGi Fc CHO cells in Iscove’s media containing 10% low IgG fêtai bovine sérum (Gibco), non essential amino acids (Gibco), 2 mM glutamine (Gibco), sodium pyruvate (Gibco), 100 pg/mL G418 and 100 ng/mL methotrexate, and used to generate concentrated media supematant. The MAdCAMIgGi Fc fusion protein was purified from the harvested supematant by affinity chromatography. Briefly, supematant was applied to a HiTrap Protein G Sepharose (5 mL, Pharmacia) column (2 mL/min), washed with 25 mM Tris pH 8, 150 mM NaCl (5 column volumes) and eluted with 100 mM glycine pH 2.5 (1 mL/min), immediately neutralising fractions to pH 7.5 with IM Tris pH 8. Fractions containing MAdCAM-IgGi Fc fusion protein were identified by SDS-PAGE, pooled together and applied to a Sephacryl S100 column (Pharmacia), pre-equilibrated with 35 mM BisTris pH 6.5, 150 mM NaCl. The gel filtration was performed at 0.35 mL/min, collecting a peak of MAdCAM-IgGi Fc fusion protein in ca. 3 x 5 mL fractions. These samples were pooled and applied to a Resource Q (6 mL, Pharmacia) column, pre-equilibrated in 35 mM BisTris pH6.5. The column was washed with 5 column volumes of 35 mM Bis Tris pH 6.5, 150 mM NaCl (6 mL/min) and MAdCAM-IgGi Fc fusion protein eluted into a 4-6 mL fraction with 35 mM Bis Tris pH 6.5, 400 mM NaCl. At this stage the protein was 90% pure and migrating as a single band at approximately 68 kD by SDS-PAGE. For use as an immunogen and ail

- 66 subséquent assays, the material was buffer exchanged into 25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 50% glycerol and stored as aliquots at -80°C.

(ii) Cell membranes stably expressing MAdCAM

A Sacl/Notl fragment comprising nucléotides 645-1222 of the published MAdCAM sequence (Shyjan AM, et al., JImmunol., 156, 2851-7 (1996)) was PCR amplified from a colon cDNA library and cloned into Sacl/Notl sites of pIND-Hygro vector (Invitrogen). A Sacl fragment, comprising the additionai 5’ coding sequence was sub-cloned into this construct from pCDNA3.1 MAdCAM-IgGi Fc, to generate the full length MAdCAM cDNA. A KpnI/Notl fragment containing the MAdCAM cDNA was then cloned into corresponding sites in a pEF5FRTV5GWCAT vector (Invitrogen) and replacing the CAT coding sequence. The cDNA insert was sequence verified and used in transfections to generate single stably expressing clones in Flpln NIH 3T3 cells (Invitrogen) by Flp recombinase technology, according to the manufacturer’s instructions. Stably expressing clones were selected by their ability to support the binding of a α^β?' JY human B lymphoblastoid cell line (Chan BM, et al, J. Biol. Chem., 267:8366-70 (1992)), outlined below. Stable clones of CHO cells expressing MAdCAM were prepared in the same way, using Flpln CHO cells (Invitrogen).

MAdCAM-expressing Flpln NIH-3T3 cells were grown in Dulbecco’s modified Eagles Medium (Gibco), containing 2 mM L-glutamine, 10% Donor calf sérum (Gibco) and 200 pg/mL Hygromycin B (Invitrogen) and expanded in roller bottles. MAdCAM-expressing Flpln CHO cells were grown in Ham’s F12/Dulbecco’s modified Eagles Medium (Gibco), containing 2 mM L-glutamine, 10% Donor calf sérum (Gibco) and 350 pg/mL Hygromycin B (Invitrogen) and expanded in roller bottles. Cells were harvested by use of a non- enzymatic cell dissociation solution (Sigma) and scraping, washing in phosphate buffered saline by centrifùgation. Cell membranes were prepared from the cell pellet by two rounds of polytron homogenization in 25 mM Bis Tris pH 8, 10 mM MgCb, 0.015% (w/v) aprotinin, 100 U/mL bacitracin and centrifugation. The final pellet was resuspended in the same buffer, and 50x10⁶ cell équivalents aliquoted into thick-walled eppendorfs and spun at >100,000g to generate cell membrane pellets for XenoMouse mice immunisations. Supematant was decanted and membranes were stored in eppendorfs at -80°C until required. Confirmation of protein expression in the cell membranes was determined by SDS-PAGE and Western blotting with a rabbit anti-peptide antibody raised against the N-terminal residues of MAdCAM ([C]-KPLQVEPPEP).

-67Immunization and hybridoma génération:

Eight to ten week old XENOMOUSE™mice were immunized intraperitoneally or in their hind footpads with either the purified recombinant MAdCAM-IgGi Fc fusion protein (10 pg/dose/mouse), or cell membranes prepared from either stably expressing MAdCAM- CHO or NIH 3T3 cells (10x10⁶ cells/dose/mouse). This dose was repeated five to seven times over a three to eight week period. Four days before fusion, the mice received a final injection of the extracellular domain of human MAdCAM in PB S. Spleen and lymph node lymphocytes from immunized mice were fused with the non-secretory myeloma P3-X63- Ag8.653 cell line and were subjected to HAT sélection as previously described (Galfre and Milstein, Methods Enzymol. 73:3-46 (1981)). A panel of hybridomas ail secreting MAdCAM spécifie human IgG2K and IgG4K antibodies were recovered and sub-cloned. Twelve hybridoma sub-clones, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6,, 7.26.4 and 9.8.2, producing monoclonal antibodies spécifie for MAdCAM were recovered and detected with assays described below. The parental fines 1.7, 1.8, 6.14, 6.22, 6.34, 6.67, 6.73, 6.77, 7.16, 7.20, 7.26 and 9.8, from which the sub-clone hybridoma fines, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2, were derived ail had anti-MAdCAM activity.

X481.2

A selected clone was further engineered to remove an inadvertent splicing event in the final protein. The inadvertent splicing event resulted in the production of an extended protein. It was discovered that the extension was the resuit of a read-through that led to the inadvertent splicing event. Without wishing to be bound by theory, it is believed that the inadvertent splicing event brought together the end of the heavy chain with a région 4244bp downstream (in the SV40 Derived Sequence Région). A new vector was designed to eliminate this observed extension. The heavy chain région was engineered to replace a nucléotide at the 3’end of the heavy chain (a change from T to A) resulting in the removal of the splice donor sight. The resulting MAdCAM antibody from this reengineered clone is X481.2.

ELISA assays:

Détection of antigen-specific antibodies in mouse sérum and hybridoma supematant was determined by ELISA as described (Coligan et al., Unit 2.1 “Enzyme-linked immunosorbent assays,” in Current Protocolslinlmmunology (1994)) using MAdCAM-IgGi Fc fusion protein to capture the antibodies. For animais that were immunised with MAdCAM-IgGi Fc fusion protein, antibodies were screened for non-specific reactivity against human IgGi and for the ability to bind to Flpln CHO MAdCAM cells by flow cytometry.

-68In a preferred ELISA assay, the following techniques are used:

ELISA plates were coated ovemight at 4°C with 100 pL/well of MAdCAM-IgG, Fc fusion (4.5 pg/mL) in plate containing buffer (100 mM sodium carbonate/bicarbonate buffer pH 9.6). After incubation, coating buffer was removed and the plate blocked with 200 pL/well blocking buffer (5% BSA, 0.1% Tween 20, in phosphate buffered saline) and incubated at room température for 1 hour. Blocking buffer was removed and 50 pL/well of hybridoma supernatant or other sérum or supernatant (e.g., positive control) added for 2 hours at room température. After incubation the plate was washed with PBS (3 x 100 pL/well) and the binding of the hybridoma mAb detected with HRP-conjugated secondary antibodies (i.e. 1:1000 mouse anti-human IgG2-HRP (SB Cat. No. 9060-05) for IgG2 antibodies or 1:1000 mouse anti-human IgG4-HRP (Zymed Cat. No. 3840) for IgG4 antibodies) diluted in PBS. The plates were incubated at room température for 1 hour, washed in PBS (3 x 100 pL/well) and finally developed with 100 pL OPD (ophenylenediamine (DAKO S2405) + 5 pL 30% H2O2/I2 mL). The plates were allowed to develop 10-20 mins, stopping the reaction with 100 pL 2M H2SO4. The plates were read at 490 nm.

Adhesion assays:

Antibodies that demonstrated binding to MAdCAM-IgG 1 Fc fusion protein by ELISA, were assessed for antagonist activity in an adhesion assays with oc β ⁺ JY cells and either (i) MAdCAM-IgG 1 Fc fusion protein or (ii) MAdCAM-CHO cells.

(i) MAdCAM-IgG 1 Fc fusion assay lOOpL of a 4.5pg/mL solution of purified MAdCAM-IgGi Fc fùsion protein in Dulbecco’s PBS was adsorbed to 96 well Black Micro fluor “B” u-bottom (Dynex #7805) plates ovemight at 4°C. The MAdCAM coated plates were then inverted and excess liquid blotted off, prior to blocking at 37°C for at least 1 hour in 10% BSA/ PBS. During this time cultured JY cells were counted using tryptan blue exclusion (should be approximately 8x10⁵ cells/mL) and 20x10⁶ cells/assay plate pipetted into a 50 mL centrifuge tube. JY cells were cultured in RPMI1640 media (Gibco), containing 2 mM L-glutamine and 10% heat-inactivated fêtai bovine sérum (Life Technologies #10108-165) and seeded at l-2xlO⁵/mL every 2-3 days to prevent the culture from differentiating. The cells were washed twice with RPMI 1640 media (Gibco) containing 2 mM L-glutamine (Gibco) by centrifugation (240g), resuspending the final cell pellet at 2x10⁶ cells/mL in RPMI 1640 for Calcein AM loading. Calcein AM (Molecular Probes #C-3099) was added to the cells as a 1:200 dilution in DMSO (ca. final concentration 5 pM) and the cells

-69protected from light during the course of the incubation (37°C for 30 min). During this cell incubation step the antibodies to be tested, were diluted as follows: for single dose testing, the antibodies were made up to 3 pg/mL (1 pg/mL final) in 0.1 mg/mL BSA (Sigma#A3059) in PBS; for full IC50 curves, the antibodies were diluted in 0.1 mg/mL BSA/ PBS, with 3 pg/mL (1 pg/mL final) being the top concentration, then doubling dilutions (1:2 ratio) across the plate. The final well of the row was used for determining total binding, so O.lmg/ml BSA in PBS was used.

After blocking, the plate contents were flicked out and 50 pL of antibodies/Controls were added to each well and the plate incubated at 37°C for 20 min. During this time, Calcein-loaded JY cells were washed once with RP MI 1640 media containing 10% fêtai bovine sérum and once with 1 mg/mL BSA/PBS by centrifugation, resuspending the final cell pellet to lxlO⁶/mL in 1 mg/mL BSA/PBS. 100 pL of cells were added to each well of the U bottomed plate, the plate sealed, briefly centrifuged (1000 rpm for 2 min) and the plate then incubated at 37°C for 45 min. At the end of this time, the plates were washed with a Skatron plate washer and fluorescence measured using a Wallac Victor² 1420 Multilabel Reader (excitation λ 485nm, émission λ 535nm count from top, 8 mm from bottom of plate, for 0.1 sec with normal émission aperture). For each antibody concentration, percent adhesion was expressed as a percentage of maximal fluorescence response in the absence of any antibody minus fluorescence associated with nonspecific binding. The IC50 value is defined as the anti-MAdCAM antibody concentration at which the adhesion response is decreased to 50% of the response in the absence of antiMAdCAM antibody. Antibodies that were able to inhibit the binding of JY cells to MAdCAMIgGi Fc fusion with an IC50 value <0.1 pg/mL, were considered to hâve potent antagonist activity and were progressed to the MAdCAM- CHO adhesion assay. Ail twelve of the tested Abs showed potent antagonist activity (Table 3). Monoclonal antibodies 1.7.2, 1.8.2, 7.16.6, 7.20.5 and 7.26.4 were derived from IgGiK lineages, and monoclonal antibodies 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1 and 9.8.2 were derived from IgG4K lineages.

(ii) MAdCAM-CHO cell adhesion assay.

JY cells were cultured as above. MAdCAM-expressing CHO cells were generated with the pEF5FRT MAdCAM cDNA construct and using the Flp recombinase technology (Invitrogen) as described above. Single stable clones of MAdCAM-expressing CHO cells were selected based on their ability to support the adhesion of JY cells and the binding, by flow cytometry, of the rabbit anti-peptide antibody, raised against the N-terminus of MAdCAM and described above. MAdCAM-expressing CHO cells were cultured in a DMEM/F12 media (Gibco # 21331-020)

- 70 containing 2 mM L-glutamine, 10% fêtai bovine sérum (Gibco) and 350 pg/mL Hygromycin B (Invitrogen), splitting 1:5 every 2/3 days. For the adhesion assay, MAdCAM-expressing CHO cells were seeded at 4x10⁴ cells/well in 96 well black plates-clear bottom (Costar # 3904) in 200 pL culture medium and cultured ovemight at 37°C/5% CO₂.

The following day, hybridoma supematant or purified monoclonal antibody was diluted from a starting concentration of 30 pg/mL (équivalent to a final concentration of 10 pg/mL) in 1 mg/mL BSA/PBS, as described above. For the MAdCAM CHO plates, the plate contents were flicked out and 50 pL of antibodies/controls were added to each well and the plate incubated at 37°C for 20 min. The final well of the row was used for determining total binding, so 0.1 mg/mL BSA in PBS was used. Calcein AM-loaded JY cells, to a final concentration of lxlO⁶/mL in 1 mg/mL BSA/PBS, were prepared as above, then 100 pL added to the plate after the 20 min incubation period with the antibody. The plate was then incubated at 37°C for 45 min, then washed on a Tecan plate washer (PW 384) and fluorescence measured using the Wallac plate reader as described above. For each antibody concentration, percent adhesion was expressed as a percentage of maximal fluorescence response in the absence of any antibody minus fluorescence associated with non-specific binding. Antibodies that were able to inhibit the binding of JY cells to MAdCAM CHO cells with an IC50 value <1 pg/mL were considered to hâve potent antagonist activity. As before, the IC50 value is defined as the anti-MAdCAM antibody concentration at which the adhesion response had decreased to 50% of the response in the absence of antiMAdCAM antibody.

The IC50potencies for 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 in this assay are described below in Table 3.

Table 3. IC50 values of exemplified anti-MAdCAM antibodies

Clone	MAdCAM IgG t Fc fusion	MAdCAM Flpln CHO Assay Mean IC50 (pg/mL)	n
Mean IC50 (pg/mL)	n
1.7.2	0.030 ± 0.011	6	0.502 ± 0.280	9
1.8.2	0.027 ± 0.011	4	0.424 ± 0.107	8
7.16.6	0.019 ± 0.009	7	0.389 ± 0.093	16
7.20.5	0.025 ± 0.027	7	0.387 ± 0.202	9
7.26.4	0.021 ± 0.040	4	0.574 ± 0.099	15
6.14.2	0.011 ± 0.005	4	0.291 ± 0.096	6
6.22.2	0.018 ± 0.011	4	0.573 ± 0.168	7
6.34.2	0.013 ± 0.008	4	0.285 ± 0.073	7

Clone	MAdCAM IgG 1 Fc fusion	MAdCAM Flpln CHO Assay Mean IC50 (pg/mL)	n
Mean IC50 (pg/mL)	n
6.67.1	0.013 ± 0.070	4	0.298 ± 0.115	8
6.73.2	0.020 ± 0.010	4	0.369 ± 0.103	8
6.77.1	0.022 ± 0.004	4	0.520 ± 0.100	4
9.8.2	0.020 ± 0.050	4	0.440 ± 0.342	8
lgG2
lgG4

To measure the antagonist potency of anti-MAdCAM mAbs in flow-based assays, under sheer stress conditions that are designed to mimic the microvascular environment on the high endothélial venules which serve the gut associated lymphoid tissue, CHO cells expressing MAdCAM were plated in glass microslides (50 x 4 mm) and allowed to adhéré to form a confluent monolayer (ca. 2.5 x 10⁵ cells). The cells were then incubated with affmity- purified mAb over a range of concentrations (0.1-10 pg/mL) for 20 mins at 37°C, before being connected to the flow assay System. An isotype matched IgG2 or IgG4 mAb (10 pg/mL) was used as a négative control. Normal donor peripheral blood lymphocytes (PBLs) were perfused over the cell monolayer at a constant shear stress of 0.05 Pa. Experiments were videoed and total adhesion of lymphocytes (rolling + firm adhesion) was calculated.

Ail of the tested monoclonal antibodies were shown to be potent antagonists under the conditions described.

(iii) Stamper-Woodruff assays

To visualise MAdCAM⁺ vessels, biotinylated anti-MAdCAM mAb was generated on 1 -2 mg of affinity-purified protein, using a 20 molar excess of biotin-NHS (Pierce) in phosphate buffer saline, according to manufacturer’s instructions. The reaction was allowed to sit at room température (30 min), and desalted with a PD-10 (Pharmacia) column and the protein concentration determined.

Normal liver lymph node was removed from a donor organ, snap-frozen in liquid nitrogen and stored at -70°C until use. 10 pm cryostat sections were eut, air-dried on poly-L lysine coated slides, and fixed in acetone prior to the assay. Sections were blocked using an avidin-biotin blocking System (DAKO), and then incubated with biotinylated anti-MAdCAM mAb over a range of concentrations (1-50 pg/mL) at room température (2 hrs). An isotype matched IgG2 or IgG4 mAb (50 pg/mL) was used as a négative control and a blocking anti-β? antibody (50 pg/mL) as a positive control.

- 72 Peripheral blood lymphocytes, taken from normal donors, were labeled with a mouse anti-human CD2 mAb (DAKO) to allow subséquent visualisation of adhèrent cells. 5x10⁵ PBLs were added to each lymph node section and incubated for 30 mins before being gently rinsed off to avoid detachment of adhèrent cells. Sections were then re-fixed in acetone, and re-incubated with biotinylated anti-MAdCAM mAb (10 pg/mL), followed by biotinylated goat-anti-mouse mAb (to recognise CD2 labeled PBLs and unstained MAdCAM⁴ vessels) and then streptABcomplex/HRP (DAKO). Finally MAdCAM¹ vessels & CD2 labeled PBLs were visualised by addition of DAB substrate (DAKO) to the sections, with a brown reaction product showing areas of positive staining. Lymphocyte adhesion was quantified by counting the number of lymphocytes adhering to 50 MAdCAM-1 ⁺ vessels of portai tracts, veins or sinusoids. Data, expressed as mean values, were then normalised to percent adhesion, using the adhesion of PBLs in the absence of any antibody taken as 100%. The data were compiled on the basis of n=3 different PBL donors and for different liver lymph node donors. Représentative data for biotinylated purified monoclonal antibodies

1.7.2 and 7.16.6 are depicted in Figure 4 compared to a blocking anti-β? antibody control.

Selectivity assays:

VCAM and fibronectin are close structural and sequence homologues to MAdCAM. Affinitypurified anti-MAdCAM mAbs were assessed for MAdCAM-specificity by detennining their ability to block the binding of α4βι⁺/α5βι⁺Jurkat T-cells (ATCC) to their cognate cell adhesion molécule. lOOpL of a 4.5pg/mL solution of Fibronectin cell binding fragment (110 Kd, Europa Bioproducts Ltd, Cat. No. UBF4215-18) or VCAM (Panvera) in Dulbecco’s PBS was adsorbed to 96 well Black Microfluor “B” u-bottom (Dynex #7805) plates ovemight at 4°C. The coated plates were then inverted and excess liquid blotted off, prior to blocking at 37°C for at least 1 hour in 10% BSA/ PBS. During this time cultured Jurkat T cells were counted using tryptan blue exclusion and loaded with Calcein AM dye as previously described for JY cells above. The antibodies to be tested, were diluted from a top concentration of 10 pg/mL in 0.1 mg/ml BSA in PBS. The final well of the row was used for detennining total binding, so O.lmg/ml BSA in PBS was used. Echistatin (Bachem, Cat. No. H-9010) prepared in PBS was used at a top concentration of 100 nM to block the o^i/Fibronectin interaction. An anti-CD 106 mAb (Clone 51-10C9, BD Pharmingen Cat. No. 555645) at a top concentration of 1 pg/mL was used to block the α4βl/VCAM interaction.

After blocking, the plate contents were flicked out and 50 pL of antibodies/controls were added to each well and the plate incubated at 37°C for 20 min. Calcein-loaded Jurkat T cells were

- 73 washed once as before, resuspending the final cell pellet to lxlO⁶/mL in 1 mg/mL BSA/PBS.

100 pL of cells were added to each well of the U bottomed plate, the plate sealed, briefly centrifuged (1000 rpm for 2 min) and the plate then incubated at 37°C for 45 min. At the end of this time, the plates were washed with a Skatron plate washer and fluorescence measured using a

Wallac Victor² 1420 Multilabel Reader (excitation À485nm, émission

X535nm count from top, 8 mm from bottom of plate, for 0.1 sec with normal émission aperture).

For each antibody, the degree of inhibition is expressed below pictorially, in Table 4 (- negligible inhibition of adhesion, *** complété inhibition of adhesion). Ail mAbs exemplified are potent and sélective anti-MAdCAM antagonists, demonstrating substantially greater than 100 fold selectivity for MAdCAM over VCAM and fibronectin.

Table 4. Comparative selectivity of anti-MAdCAM antibody for MAdCAM over other cell adhesion molécules, Fibronectin and VCAM

Clone	Inhibition in a 531/Fibronectin assay (10 pg/mL)	Inhibition in a4pi/VCAM assay (10 pg/mL)	Inhibition in α 4p7/MAdCAM assay (0.1 pg/mL)
1.7.2	-	-
1.8.2	-	-	* * *
7.16.6	-	-	* * *
7.20.5	-	-	* * *
7.26.4	-	-	***
6.14.2	-	-
6.22.2	-	-
6.34.2	-	-	* * ❖
6.67.1	-	-	***
6.73.2	-	-	***
6.77.1	-	-
9.8.2	-	-	* * *
igG2	-	-	* * *
igG4	-	-

Hybridomas were deposited in the European Collection of Cell Cultures (ECACC), H.P.A at

CAMR, Porton Down, Salisbury, Wiltshire SP4 0JG on 9^th September 2003 with the following deposit numbers:

Hybridoma

1.7.2

1.8.2

6.14.2

Deposit No.

03090901

03090902

03090903

Hybridoma	Deposit No.
6.22.2	03090904
6.34.2	03090905
6.67.1	03090906
6.73.2	03090907
6.77.1	03090908
7.16.6	03090909
7.20.5	03090910
7.26.4	03090911
9.8.2	03090912

EXAMPLE II:

Détermination of Affinity Constants (Kd) of Fully Human Anti-MAdCAM Monoclonal Antibodies by BIAcore

We performed affinity measures of purified antibodies by surface plasmon résonance using the BIAcore 3000 instrument, following the manufacturer’s protocols.

Protocol 1

To perform kinetic analyses, a high density mouse anti-human (IgG2 and IgG4) antibody surface over a CM5 BIAcore sensor chip was prepared using routine amine coupling. Hybridoma supematants were diluted 10, 5, 2-fold in HBS-P (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Surfactant P20) running buffer containing 100 pg/mL BSA and 10 mg/mL carboxymethyldextran or used neat. Each mAb was captured onto a separate surface using a 1 min contact time and a 5 min wash for stabilization of the mAb baseline. MAdCAM-IgGi Fc (141 nM) fusion protein was then injected at over ail surfaces for one minute, followed by a 3 min dissociation. The data were normalized for the amount of antibody captured on each surface and evaluated with global fit Langmuir 1:1, using baseline drift models available on the BIAevaluation software provided by BIAcore.

Protocol 2

Affinity-purified mAb were immobilized onto the dextran layer of a CM5 biosensor chip using amine coupling. Chips were prepared using pH 4.5 acetate buffer as the immobilization buffer and protein densifies of 2.5-5.5 kRU were achieved. Samples of MAdCAM-IgGi Fc fusion protein in running buffer were prepared at concentrationsranging from 0.2-55 nM (a 0 nM solution comprising running buffer alone was included as a zéro reference). Samples were randomized and injected in duplicate for 3 min each across 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%

- 75 Surfactant P20) as running buffer. A flow rate of 100 pL/min was used to minimize mass transport limitations. Dissociation of MAdCAM-IgGi Fc fusion protein was monitored for 180 mins, the surface regenerated by a 6 sec injection of 25 mM H3PO4(50 pL/min), or 10 mM (6.22.2), 20 mM (6.67.1, 6.73.2, 6.77.1) to 25 mM (6.34.2) and 45 mM NaOH (6.14.2) and the data analysed using the BIAévaluation (v3.1) software package.

Table 5 lists affinity measurements for représentative anti-MAdCAM antibodies of the présent disclosure:

Table 5. Détermination of affinity constant, Kd, by surface plasmon résonance (BIAcore)

CLONE	Protocol 1	Protocol 2
kon (1/Ms)	koff(l/s)	Kd (pM)	kon(l/Ms)	koff(l/s)	Kd (Pm)
1.7.2	2.4 x 105	1x10	42	5.5 x 103	1.3 x 10’7	23.6
1.8.2	2.9 x 105	1 x 10’5	35	1.8 x 105	2.3 x 10’5	128
7.16.6	1.5 x 106	2.2 x 10'6	1.5	2.9 x 105	1.4 x 10'6	4.8
7.20.5	4.5 x 105	1.9 x 10’5	42.2	1.6 x 105	1.2 x 10’5	75
7.26.4	9.6 x 105	2.6 x 10'4	271	1.5 x 105	1.2 x 10'5	80
6.14.2	1.3 x 105	1 x 10'5	7.7	5 x 105	< 5 x 10‘6	< 10
6.22.2	1.5 x 106	1.4 x 10’5	9.3	2.3 x 105	8.7 x 10'7	3.8
6.34.2	1.2 x 106	1,9x 10'5	15.8	3.3 x 105	< 5 x 10'6	<15
6.67.1	5.9 x 105	1 x 10'5	17	2.4 x 105	< 5 x 10’6	<20
6.73.2	1.4 x 105	1.3x10’4	93
6.77.1	1.5 x 105	1 x 10'5	6.7
9.8.2	2.3 x 106	2.3 x 10’4	100	4.4 x 105	1.4 x 10'5	32.5
lgG2
lgG4

The kinetic analyses indicate that the antibodies prepared in accordance with the disclosure possess high affinities and strong binding constants for the extracellular domain of MAdCAM.

EXAMPLE III:

Identification of epitope selectivity and species cross-reactivity of anti-MAdCAM mAbs

Antibodies recognize surface-exposed epitopes on antigens as régions of linear (primary) sequence or structural (secondary) sequence. Luminex epitope binning, BIAcore binning and species immunohistochemical analysis were used in concert, in order to define the functional epitope landscape of the anti-MAdCAM antibodies.

- 76 Luminex-based Epitope Binning:

MxhlgG 2,3.4-conjugated beads (Calbiochem Ml 1427) were coupled to the primary unknown anti-MAdCAM antibody. We added 150 pL of primary unknown antibody dilution (0.1 pg/mL diluted in hybridoma medium) to the well of a 96-well tissue culture plate. The bead stock was gently vortexed and diluted in supematant to a concentration of 0.5 x 10⁵ beads/mL. The beads were incubated in the supematant on a shaker overnight in the dark at 4°C.

Each well of a 96-well microtiter filter plate (Millipore # MABVN1250) was pre- wetted by adding 200 pL wash buffer (PBS containing 0.05% Tween20) and removed by aspiration. Next, 50 pL/well of the 0.5 x 10⁵ beads/mL stock was added to the filter plate, and the wells washed with wash buffer (2 xlOO pL/well). 60 pL/well of MAdCAM-IgGiFc antigen diluted in hybridoma medium (0.1 pg/mL) was added. The plates were covered and incubated at room température with gentle shaking for one hour. The wells were washed twice by addition of 100 pL/well wash buffer followed by aspiration. Next, we added 60 pL/well of secondary unknown anti-MAdCAM antibody diluted in hybridoma medium (0.1 pg/mL). The plates were shaken at room température in the dark for two hours. Next, the wells were washed twice by addition of 100 pL/well wash buffer followed by aspiration. Next, 60 pL/well of biotinylated MxhlgG 2,3,4 (0.5 pg/mL) was added. The plates were shaken at room température in the dark for one hour.

The wells were washed twice by addition of 100 pL/well wash buffer followed by aspiration. To each well, 60 pL of 1 pg/mL MxhlgG 2,3,4 Streptavidin-PE (Pharmacia #554061) diluted in hydridoma medium was added. The plates were shaken at room température in the dark for twenty minutes. The wells were washed twice by addition of 100 pL/well wash buffer followed by aspiration.

Next, each well was resuspended in 80 pL blocking buffer (PBS with 0.5% bovine sérum albumin, 0.1% TWEEN and 0.01% Thimerosal) carefully pipetted up and down to resuspend the beads.

Using Luminex 100 and its accompanying software (Luminex® Corporation) the plates were read to détermine luminescence readings. Based on the luminescence data obtained for the various anti-MAdCAM antibodies tested, the anti-MAdCAM antibodies were grouped according to their binding specificities. The anti-MAdCAM antibodies that were tested fall into a sériés of epitope bins, represented in Table 8.

BIAcore binning:

- 77 In a similar method to that described above, BIAcore can also be used to déterminé the epitope exclusivity of the anti-MAdCAM antibodies exemplified by this disclosure. Nine antiMAdCAM antibody clones, 6.22.2, 6.34.2, 6.67.1, 6.77.1, 7.20.5, 9.8.2, 1.7.2, 7.26.4 and 7.16.6, were immobilized onto the dextran layer of separate flow cells of a CM5 biosensor chip using amine coupling. The immobilization buffer was either 10 mM acetate buffer pH 4.5 (clones 6.22.2, 6.34.2, 7.20.5, 9.8.2, 1.7.2, 7.26.4 and 7.16.6) or 10 mM acetate buffer pH 5.5 (clones 6.67.1 and 6.77.1). A protein density of approximately 3750 RU was achieved in ail cases. Deactivation of unreacted N-hydroxysuccinimide esters was performed using 1 M ethanolamine hydrochloride, pH 8.5.

MAdCAM-IgGi Fc fùsion protein was diluted to a concentration of 1.5 pg/mL (approximately 25 nM) in HBS-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate 20). It was then injected across the first flow cell, in a volume of 50 pL at a rate of 5 pL/min. After the injection was complété, the first antibody probe was added to the same flow cell. Ail test antibodies were diluted to a concentration of approximately 20 pg/mL in HBS-EP, and also injected in a volume of 50 pL at a flow rate of 5 pL/min. When no binding of the test antibody was observed, the next test clone was injected immediately afterwards. When binding did occur, the sensor surface was regenerated to remove both the MAdCAM-IgGi Fc fusion protein and the test antibody. A variety of régénération solutions were useddepending upon the immobilized antibody and the test antibody présent. A summary of the régénération conditions used is depicted in Table 6.

Table 6. Summary of régénération conditions used to perform BIAcore epiope mapping

Immobilisée! antibody	Antibody probe to be removed	Régénération solution	Injection volume
7.16.6	6.22.2	40 mM Phosphoric Acid	20 pL
6.34.2	40 mM Phosphoric Acid	40 pL
7.20.5	40 mM Phosphoric Acid	20 pL
6.77.1	9.8.2	40 mM Phosphoric Acid	10 pL
1.7.2	40 mM Phosphoric Acid	5 pL
7.16.6	40 mM Phosphoric Acid	10 pL
1.7.2	6.77.1	25 mM Phosphoric Acid	5 pL
9.8.2	25 mM Phosphoric Acid	5 pL
7.20.5	25 mM Phosphoric Acid	5 pL
6.22.2	25 mM Phosphoric Acid	5 pL
6.34.2	25 mM Sodium Hydroxide	5 pL
6.67.1	25 mM Sodium Hydroxide	5 pL
6.22.2	9.8.2	25 mM Sodium Hydroxide	20 pL
7.26.4	25 mM Sodium Hydroxide	5 pL
6.34.2	9.8.2	25 mM Sodium Hydroxide	70 pL
1.7.2	40 mM Sodium Hydroxide	5 pL

	7.26.4	40 mM Sodium Hydroxide	5 pL
6.67.1	9.8.2	40 mM Sodium Hydroxide	5 pL
1.7.2	40 mM Sodium Hydroxide	5 pL
7.20.5	9.8.2	25 mM Phosphoric Acid	5 pL
1.7.2	25 mM Phosphoric Acid	5 pL
7.26.4	25 mM Phosphoric Acid	5 pL
7.26.4	9.8.2	40 mM Sodium Hydroxide	20 pL
6.22.2	75 mM Phosphoric Acid	20 pL
7.20.5	75 mM Phosphoric Acid	20 pL
7.16.6	75 mM Phosphoric Acid	20 pL
9.8.2	9.8.2	25 mM Phosphoric Acid	15 pL
6.22.2	25 mM Phosphoric Acid	10 pL
7.20.5	25 mM Phosphoric Acid	20 pL
7.16.6	25 mM Phosphoric Acid	10 pL

(Flow rate was 50 pL/min during ail régénération procedures)

After régénération, MAdCAM-IgGi Fc fusion protein was bound again and further test antibodies were injected. These procedures were carried out until the entire panel of clones had been injected over the surface of the immobilised antibody, with bound MAdCAM-IgGi Fc fusion protein. A new flow cell with a different immobilised antibody and bound MAdCAM was then used for probing with the nine test clones. Anti-MAdCAM antibodies 1.7.2 and 1.8.2 were expected to recognise the same MAdCAM epitope, based on the close primary amino acid sequence homology of their heavy and kappa light chains, SEQ ID NOS: 2, 4, 6, 8 respectively. Accordingly, only 1.7.2 was assessed though the BIAcore response matrix. Antibodies 6.14.2 and 6.73.2 were omitted from this analysis, but ail other combinations of anti-MAdCAM antibody pairs were tested in this way. An arbitrary level of 100 RU was chosen as the threshold between binding/non-binding and a response matrix, (Table 7), was created based on whether binding was observed.

Table 7.BIAcore epitope binning response matrix

Immobilised antibody	Secondary antibody
6.22.2	6.34.2	6.67.1	6.77.1	7.20.5	9.8.2	1.7.2	7.26.4	7.16.6
6.22.2	-	-	-	-	-	X	X	X	X
6.34.2	-	-	-	-	-	X	X	X	X
6.67.1	-	-	-	-	-	X	X	-	-
6.77.1	-	-	-	-	-	X	X	-	X
7.20.5	-	-	-	-	-	X	X	X	X
9.8.2	X	X	X	X	X	X	-	-	X
1.7.2	X	X	X	X	X	X	-	-	X
7.26.4	X	X	-	-	X	X	-	-	X
7.16.6	X	X	-	-	X	-	-	-	X

-79Response matrix for ail combinations of antibody pairs. - indicates no binding of the antibody probe, x indicates binding was observed (above a chosen threshold level of 100 RU).

The matrix diagonal in Table 7 (shaded grey) holds the binding data for identical probe pairs. In ail instances, except for the two clones 7.16.6 and 9.8.2, the antibodies were self-blocking. Antibodies 7.16.6 and 9.8.2 do not cross compete. The lack of self-blocking could be due to a mAb-induced conformational change in the fusion protein that permits additional binding ofthe mAb to a second site on MAdCAM-IgFc. Grouping the clones that show the same reactivity pattern gives rise to at least six different epitope bins, as shown in the graphical représentation, Figure 5).

Further précisé identification of the MAdCAM epitope sequences with which an anti-MAdCAM antibody interacts can be determined by any of a number of methods, including, but not limited to, Western analysis of spotted peptide library arrays (Reineke et al., Curr. Topics in Microbiol. andImmunol 243: 23-36 (1999), M. Famulok, E-L Winnacker, C-H Wong eds., Springer-Verlag , Berlin), phage or bacterial flagellin//7zC expression library display, or simple MALDI-TOF analysis of bound protein fragments following limited proteolysis.

Immunohistochemical assays:

OCT or sucrose-embedded frozen tissue specimens of ileum (Peyer’s patches), mesenteric lymph node, spleen, stomach, duodénum, jéjunum and colon were used as a positive staining Controls for the anti-MAdCAM mAbs. For staining human sections with human IgGi mAbs, biotinylated dérivatives of the anti-MAdCAM mAbs were generated. 10 pm frozen tissue sections were eut onto poly L-lysine coated slides, placed directly into 100% acetone 4°C (10 min), then 3% hydrogen peroxide in methanol (10 min), washing between steps with PBS. The slides were blocked with Biotin Blocking System (DAKO Cat. No. X0590), prior to incubation with the primary antibody ( 1:100 - 1:1000) in PBS (1 hr), washed with PBS-Tween 20 (0.05%) and then binding developed with HRP-Streptavidin (BD Bioscience Cat. No.550946, 30 min) and DAB substrate (Sigma Cat. No. D5905). For IgG4mAbs, an HRP-conjugated, mouse anti-human IgG4 (Zymed Cat. No. 3840) secondary was used. The slides were counterstained with Mayer’s Haemalum (1 min), washed and then mounted in DPX.

Binding affinity was compared for a number of species (mouse, rat, rabbit, dog, pig, cynomolgus and human tissue). There was no reactivity for rat, rabbit and pig tissue by immunohistochemistry and no cross-reactivity of the anti-MAdCAM antibodies for recombinant mouse MAdCAM, when analyzed by ELISA. The data for human, cynomolgus and dog tissue are presented in table form, Table 8 below:

- 80 Table 8. Pattern of cross reactivity of anti-MAdCAM antibodies to MAdCAM species orthologues n.d: not determined

	IHC cross-reactivitity
	Luminex	human	cyno	marmoset	dog
CLONE	BIN	ileum	ileum	ileum	ileum
1.7.2	3a
1.8.2	3a
7.16.6	3b
7.20.5	2b		n.d
7.26.4	3b		n.d
6.14.2	2		n.d
6.22.2	2		n.d
6.34.2	6		n.d
6.67.1	5		n.d
6.73.2	3		n.d
6.77.1	1		n.d
9.8.2	3a

lgG2 lgG4

No Binding

Binding

Anti-MAdCAM binding to specialised endothélial structures and lymphoid tissue is indicated by the shading, according to the key. The epitope bin based on Luminex epitope analysis and the pattern of MAdCAM cross-reactivity are indicated for each antibody.

Luminex epitope binning data for anti-MAdCAM antibodies 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.3 and 6.77.1 (italics) were derived from separate experiments than that for 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 (bold type), as indicated by the différence in font character.

Ail anti-MAdCAM antibodies tested had the ability to recognize a human MAdCAM epitope expressed on vascular endothélial compartments of the gastrointestinal tract. Apart from 1.7.2 and 1.8.2, ail other anti-MAdCAM antibodies tested were able to specifically bind the vascular endothélial compartments of the cynomolgus gastrointestinal tract Certain other anti-MAdCAM antibodies, namely 6.14.2 and 6.67.1 also had the ability to specifically recognize the dog MAdCAM orthologue as well as cynomolgus MAdCAM.

Génération of a functionally active chimeric cynomolgus/human MAdCAM-expressing CHO cell line:

- 81 The différences in binding affmity of certain anti-MAdCAM antibodies for human and cynomolgus MAdCAM led us to déterminé whether a structural basis for this observation could be made.

Based on the published amino acid sequence for Macaque MAdCAM (Shyjan AM, et al., J Immunol., 156, 2851-7 (1996)), primers were designed to PCR amplify the cynomolgus MAdCAM ΟΜβ? binding domain sequence. Total RNA was prepared from frozen excised cynomolgus mesenteric lymph node (ca. 200 mg) using the Trizol method (Invitrogen) according to the manufacturer’s instructions. 1-2 pg was oligo-dT primed and reverse transcribed with AMV reverse transcriptase (Promega). A proportion of the reverse transcribed product was subjected to PCR with forward 5’-AGC ATG GAT CGG GGC CTG GCC-3’ (SEQ ID NO: 67) and reverse 5’-GTG CAG GAC CGG GAT GGC CTG-3’ (SEQ ID NO: 68) primers with GC-2 polymerase in IM GC melt (Clontech) and at an annealing température of 62°C. An RT-PCR product of the appropriate size was excised and purified from a 1 % agarose gel after electrophoresis, then TOPO-TA cloned (Invitrogen) between EcoRI sites of pCR2.1. The insert was sequence confirmed. The nucléotide and predicted translated amino acid sequences are shown in SEQ ID NOS 49 and 50, respectively.

The predicted human and cynomolgus MAdCAM amino acid sequences for the οωβ? binding domain show a high degree of sequence identity (90.8%) when aligned (Figure 3 provides this sequence alignment). To generate a functionally active cynomolgus

MAdCAM-expressing cell line, which mimicked the anti-MAdCAM binding pattern represented by Table 8, a Sacl fragment corresponding to the cynomolgus οωβ? binding domain sequence in pCR2.1, was subcloned directly into the C-terminal human MAdCAM pIND-Hygro construct containing carboxyl-terminal mucin stalk and transmembrane domain, described above. The sequence and orientation was verified, then a KpnI/Notl fragment was cloned into pEF5FRTV5GWCAT vector (Invitrogen), replacing the CAT coding sequence and used in transfections to generate single stably expressing clones in Flp In CHO cells (Invitrogen), according to the manufacturer’s instructions.

The binding of anti-MAdCAM antibody clones to the CHO cells expressing cynomolgus/human MAdCAM chimera was assessed by flow cytometry and the functional activity of antiMAdCAM antibodies was determined using a very similar JY cell adhesion assay as that described above. The binding and functional activity of anti-MAdCAM antibodies areexpressed in Table 9.

- 82 Table 9. Corrélation between the functional activity in the cynomolgus/human MAdCAMCHO/JY adhesion assay and human and cynomolgus/human MAdCAM CHO cell binding, as measured by FACS, for a range of anti-MAdCAM antibodies.

	Functional	FACS binding
CLONE	IC50 (pg/mL)	human	cyno/human
1.7.2
1.8.2
7.16.6
7.20.5
7.26.4
6.14.2
6.22.2
6.34.2
6.67.1
6.73.2
6.77.1
9.8.2

lgG2	No Binding
lgG4	Binding

Taken together, there is a good corrélation between the ability of a given anti- MAdCAM antibody to bind human or cynomolgus MAdCAM, as detected by immunohistochemistry (Table 8), with recombinant cell-based binding and functional activity (Table 9). Anti-MAdCAM antibodies 1.7.2, 1.8.2 and 6.73.2, for instance, demonstrated a consistent lack of binding to cynomolgus tissue and cells expressing a chimeric cynomolgus/human MAdCAM protein. AntiMAdCAM antibodies 1.7.2, 1.8.2 and 6.73.2 also did not hâve the ability to detect functional blocking activity in the cynomolgus/human MAdCAM/JY adhesion assay.

Similar approaches could be used to define the epitope of the anti-MAdCAM antibodies 6.14.2 and 6.67.1 that recognise dog MAdCAM.

EXAMPLE IV:

Use of anti-MAdCAM mAbs in the détection of circulating soluble MAdCAM as a method of disease diagnosis

Anti-MAdCAM antibodies can be used for the détection of circulating soluble MAdCAM (sMAdCAM). Détection of sMAdCAM in clinical plasma, sérum samples or other biofluid, such as, but not limited to, stool, urine, sputum, is likely to be a useful surrogate disease biomarker for underlying disease, including, but not limited to, inflammatory bowel disease.

- 83 Based on the epitope binning data (Tables 7 and 8), anti-MAdCAM antibodies 1.7.2 and 7.16.6 appear to recognise different epitopes on human MAdCAM. ELISA plates were coated ovemight at 4°C with 100 pL/well of a 50 pg/mL solution of 1.7.2 in phosphate buffered saline (PBS). After incubation the plate was blocked for 1.5 hours with a PBS blocking buffer containing 10% milk (200 pL/well). After incubation the plate was washed with PBS (2 x 100 pL/well) and serial dilutions of MAdCAM-IgGl-Fc fusion protein, from a top concentration of 50 pg/mL down to approximately 5 ng/mL in PBS, to a final volume of 100 pL, were added to the plate for incubation of 2 hours at room température. In a similar approach the MAdCAMIgGl-Fc protein can be diluted in plasma or sérum, or some other such relevant biofluid and used to détermine the expression of soluble MAdCAM in a clinical sample, as described below. As a négative control, only buffer was added to the wells containing the primary anti-MAdCAM antibody. After this time, the plate was washed with PBS (3 x 100 pL/well) and the plate then incubated in the dark with an Alexa488-labelled 7.16.6 (100 pL, 5 pg/mL). The Alexa488labelled 7.16.6 was generated using a commercially available kit (Molecular Probes, A-20181), following Manufacturer’s protocols.

The plate was washed with PBS containing 0.05% Tween-20, and binding of labeled 7.16.6 to captured soluble MAdCAM determined by measuring the fluorescence (Wallac Victor² 1420 Multilabel Reader, excitation À485nm, émission λ535ηιη count from top, 3 mm from bottom of plate, for 0.1 sec with normal émission aperture). When fluorescence is plotted as a function of the concentration of MAdCAM-IgGl-Fc fusion protein, Figure 6, it indicates that 1.7.2 and a labeled 7.16.6 can be used for diagnostic purposes to détermine the level of circulating soluble MAdCAM expressed in a biofluid or clinical sample. This sandwich ELISA approach is not restricted to the use of 1.7.2 and 7.16.6, but any combination of anti-MAdCAM antibodies that recognise different epitopes on MAdCAM, as outlined by the data and interprétation of table 7 and Figure 5. Similar strategies could be applied to the development of similar assays, such as immunohistochemistry and Western Blot, with the other anti-MAdCAM antibodies described, using different partners, variants, labels, etc.

EXAMPLE V:

Amino acid structure of anti-MAdCAM mAbs prepared in accordance to the disclosure

In the following discussion, structural information related to the anti-MAdCAM mAbs prepared in accordance with the disclosure is provided.

- 84 To analyze structures of mAbs produced in accordance with the disclosure, we cloned the genes encoding the heavy and light chain fragments out of the spécifie hybridoma clone. Gene cloning and sequencing was accomplished as follows:

Poly(A)+ mRNA was isolated from approximately 2x10⁵ hybridoma cells derived from immunized XenoMouse mice using Fast-Track kit (Invitrogen). The génération of random primed cDNA was followed by PCR. Human VH or Vk family spécifie primers (Marks et al., Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genese and design of family-specific oligonucleotide probes’; Eur. J. Immunol., 21, 985991 (1991)) or a universal human VH primer, MG-30 (5’-CAG GTG CAG CTG GAG CAG TCI GG-3 (SEQ ID NO: 108) was used in conjunction with primers spécifie for the human Cy2, MG40-d (5’-GCT GAG GGA GTA GAG TCC TGA GGA-3 (SEQ ID NO: 109) or Cy4 constant région, MG-40d (5’GCT GAG GGA GTA GAG TCC TGA GGA CTG T -3 (SEQ ID NO: 110), or Ck constant région (1ικΡ2; as previously described in Green et al., 1994). Sequences of the human mAb-derived heavy and kappa chain transcripts from hybridomas were obtained by direct sequencing of PCR products generated from poly (A+) RNA using the primers described above. PCR products were cloned into pCR2.1 using a TOPO-TA cloning kit (Invitrogen) and both strands were sequenced using Prism dye terminator sequencing kits and an ABI 377 sequencing machine. Ail sequences were analysed by alignments to the ‘V BASE sequence directory’ (Tomlinson, et al, J. Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet., 3, 853-860 (1994);

EMBOJ., 14, 4628-4638 (1995).)

Further each of the antibodies, 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4, 9.8.2, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, were subjected to full length DNA sequencing. For such, total RNA was isolated from approximately 3-6x10⁶ hybridoma cells using an RNeasy kit (Qiagen). The mRNA was reverse transcribed using oligo-dT and an AMV-based reverse transcriptase System (Promega). V BASE was used to design 5’ spécifie amplification primers, containing an optimal Kozak sequence and ATG start codon (underlined) and 3 ’ reverse primers for the spécifie heavy and kappa chains as depicted in Table 10.

Table 10: PCR primer pairs for cDNA amplification from anti-MAdCAM mAb-expressing hybridomas and primers used in the construction of modified versions of anti-MAdCAM antibodies.

Oligo sequence

VH1-18

5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGACTGGACCTGGAGCATCCTT 3’ (SEQ ID NO: 70)

VH3-15 VH3-21 VH3-23 VH3-30 VH3-33 VH4-4	5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGATT 3’ (SEQ ID NO:
71)
5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAACTGGGGCTCCGCTGGGTT 3’ (SEQ ID NO:
72)
5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGCTT 3’ (SEQ ID NO:
73)
5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGGTT 3’ (SEQ ID NO:
74)
5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGGAGTTTGGGCTGAGCTGGGTT 3’ (SEQ ID NO:
75)
5’ TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACACCTGTGGTTCTTCCTC 3’ (SEQ ID NO:
76)
A2/A3	5’ TATCTAAGCTTCTAGACCCGGGCGCCACCATGAGGCTCCCTGCTCAGCTCCTG 3’ (SEQ ID NO: 77)
A2 6	5’ TATCTAAGCTTCTAGACCCGGGCGCCACCATGTTGCCATCACAACTCATTGGG 3’ (SEQ ID NO: 78)
B3	5’ TATCTAAGCTTCTAGACCCGGGCGCCACCATGGTGTTGCAGACCCAGGTCTTC 3’ (SEQ ID NO: 79)
012	5’ TATCTAAGCTTCTAGACCCGGGCGCCACCATGGACATGAGGGTCCCCGCTCAG 3’ (SEQ ID NO: 80)
018	5’ TATCTAAGCTTCTAGACCCGGGCGCCACCATGGACATGAGGGTCCCTGCTCAG 3’ (SEQ ID NO: 81)
RevIgG2	5’ TTCTCTGATCAGAATTCCTATCATTTACCCGGAGACAGGGAGAG 3’ (SEQ ID NO: 82)
RevKappa	5’ TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC 3’ (SEQ ID NO: 83) 5’ TTCTCTGATCAGAATTCCTATCATTTACCCAGAGACAGGGAGAG 3’ (SEQ ID NO: 84)
6.22.2VK_F1	5’ -GGA TCT GGG ACA GAT TTC ACC CTC ACC ATC AAT AGC CTG GAA GC-3’ (SEQ ID	NO: 85)
	5’ -GCT TCC AGG CTA TTG ATG GTG AGG GTG AAA TCT GTC CCA GAT CC-3’ (SEQ ID	NO: 86)
6.22.2VK RI	5’ -GCA GCG TCT GGA TTC ACC TTC AGT AGC-3’ (SEQ ID NO: 87) 5’ -GCT ACT GAA GGT GAA TCC AGA CGC TGC-3’ (SEQ ID NO: 88)
6.22.2VH Fl
6.22.2VH_R1 6.22.2VH CS*	5 -CGG AGG TGC TTC TAG AGC AGG GCG-3’ (SEQ ID NO: 89)
6.34.2VK_F1	5’ -GCA AGT CAG AGT ATT AGT AGC TAT TTA AAT TGG TAT CAG CAG AAA CC3’ (SEQ ID NO: 90)
6.34.2VK_R1 6.34.2VK F2	5’ -GGT TTC TGC TGA TAC CAA TTT AAA TAG CTA CTA ATA CTC TGA CTT GC3’ (SEQ ID NO: 91)
	5 -CCA TCA GTT CTC TGC AAC CTG AGG ATT TTG CAA CTT ACT ACT GTC ACC-
6.34.2VK_R3	3’ (SEQ ID NO: 92) 5’ -GGT GAC AGT AGT AAG TTG CAA AAT CCT CAG GTT GCA GAG AAC TGA TGG-
6.34.2VH_F16	3’ (SEQ ID NO: 93)
. 34	5’ -GCA AAT GAA CAG CCT GCG CGC TGA GGA CAC G-3’ (SEQ ID NO: 94)
. 2VH_R1	5’ -CGT GTC CTC AGC GCG CAG GCT GTT CAT TTG C-3’ (SEQ ID NO: 95)
6.67.1VK_F1	5’ -CAA TAA GAA CTA CTT AGC TTG GTA CCA ACA GAA ACC AGG ACA GCC3’ (SEQ ID NO: 96)
6.67.1VK_R1	5’ -GGC TGT CCT GGT TTC TGT TGG TAC CAA GCT AAG TAG TTC TTA TTG-3’ (SEQ ID	NO: 97)
6.67.1VH Fl	5’ -CGC TCA GGG GTC GAG TCA CCA TGT CAG TAG ACA CGT CCA AGAACC-3’ (SEQ ID	NO: 98)
	5’ -GGT TCT TGG ACG TGT CTA CTG ACA TGG TGA CTC GAC CGC TGA GGG-3’ (SEQ ID	NO: 99)
6.67.1VH_R1 6.67.1VH CS*	5’ -ATT CTA GAG CAG GGC GCC AGG-3’ (SEQ ID NO: 100)
6.77 .1VK_F1	5’ -CCA TCT CCT GCA AGT CTA GTC AGA GCC TCC-3’ (SEQ ID NO: 101)
6.77.1VK_R1	5’ -GGA GGC TCT GAC TAG ACT TGC AGG AGA TGG-3’ (SEQ ID NO: 102)
6.77.1VK_F2	5’ -GGT TTA TTA CTG CAT GCA AAG TAT ACA GCT TAT GTC CAG TTT TGG CC -

	Oligo sequence
	3’ (SEQ ID NO: 103)

6.77.1VK_R2

5’ -GGC CAA AAC 3’ (SEQ ID NO: 1

TGG ACA 04)

TAA

GCT GTA TAC TTT GCA TGC AGT

AAT AAA CC -

7.26.4K Fl

5’ -CCT

GCA

AGT

CTA

GTC

AGA

GCC

TCC-3’

(SEQ

ID

NO:

105)

7.26.4K RI

5’ -GGA

GGC

TCT

GAC

TAG

ACT

TGC

AGG-3’

(SEQ

ID

NO:

106)

The primers pairs were used to amplify the cDNAs using Expand High Fidelity Taq polymerase (Roche), and the PCR products cloned into pCR2.1 TOPO-TA (Invitrogen) for subséquent sequencing. Heavy and kappa light chain sequence verified clones were then cloned into pEE6.1 5 and pEE12.1 vectors (LONZA) using Xbal/EcoRI and HindlII/EcoRI sites respectively.

Gene Utilization Analysis

Table 11 displays the heavy and kappa light chain gene utilization for each hybridoma outlined in the disclosure.

CLONE	Heavy Chain	Kappa light Chain
VH	I d I	JH	Vk	Jk
1.7.2	VH3-15	D6-19	JH4b	A3	JK5
1.8.2	VH3-15	D6-19	JH4b	A3	JK5
7.16.6	VH1-18	D6-6	JH6b	A2	JK1
7.20.5	VH4-4	D3-10	JH6b	A3	JK4
7.26.4	VH1-18	D6-6	JH6b	A2	JK1
6.14.2	VH3-23	D5-5	JH4b	012	JK5
6.22.2	VH3-33	D5-12	JH6b	A26	JK4
6.34.2	VH3-30	D4-23	JH6b	012	JK3
6.67.1	VH4-4	D3-10	JH4b	B3	JK4
6.73.2	VH3-23	D6-19	JH6b	012	JK2
6.77.1	VH3-21	D6-19	JH6b	A2	JK2
9.8.2	VH3-33	D3-10or D3-16	JH4b	018	JK5

lgG2 lgG4

Table 11 : Heavy and Kappa light chain Gene Utilization

Sequence Analysis

To further examine antibody structure predicted amino acid sequences of the antibodies were obtained from the cDNAs obtained from the clones.

Sequence identifier numbers (SEQ ID NO:) 1-48 and 51-68 provide the nucléotide and amino acid sequences of the heavy and kappa light chains of the anti-MAdCAM antibodies 1.7.2 (SEQ

IDNOS 1-4), 1.8.2 (SEQ ID NOS 5-8), 6.14.2 (SEQ ID NOS 9-12), 6.22.2 (SEQ ID NOS 1316), 6.34.2 (SEQ ID NOS 17-20), 6.67.1 (SEQ ID NOS 21-24), 6.73.2 (SEQ ID NOS 25-28), 6.77.1 (SEQ ID NOS 29-32), 7.16.6 (SEQ ID NOS 33-36), 7.20.5 (SEQ ID NOS 37-40), 7.26.4 (SEQ ID NOS 41-44), 9.8.2 (SEQ ID NOS 45-48) and the modified anti-MAdCAM antibodies

6.22.2-mod (SEQ ID NOS 51-54), 6.34.2-mod (SEQ ID NOS 55-58), 6.67.1-mod (SEQ ID NOS 59-62) and 6.77.1-mod (SEQ ID NOS 63-66) and 7.26.4-mod (SEQ ID NOS 41-42, 67-68). For each anti-MAdCAM antibody sequence cloned, the sequences of the signal peptide sequence (or the bases encoding the same) are indicated in lower case and underlined.

Figures 1A-1J provide sequence alignments between the predicted heavy chain amino acid sequences of antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 and the amino acid sequence of the respective germline gene products. The positions of the CDR1, CDR2 and CDR3 sequences of the antibodies are underlined, différences between the expressed sequence the corresponding germline sequence are indicated in bold and where there are additions in the expressed sequence compared to the germline these are indicated as a (-) in the germline sequence.

Figures 1K-1T provide sequence alignments between the predicted kappa light chain amino acid sequences ofthe antibodies 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.16.6, 7.20.5, 7.26.4 and 9.8.2 and the amino acid sequence of the respective germline gene products. The positions of the CDR1, CDR2 and CDR3 sequences of the antibodies are underlined, différences between the expressed sequence the corresponding germline they are indicated in bold and where there are additions in the expressed sequence compared to the germline these are indicated as a (-) in the germline sequence.

Presence of post-translational modification: glycosylation and deamidation:

The effect of some of the changes in the expressed anti-MAdCAM antibody sequence, compared with the derived germline sequence, is to introduce residues that potentially could be subject to N-linked glycosylation (Asn-X-Ser/Thr) and/or deamidation (Asn-Gly) (see Table 12). The nucleic acid sequences encoding the kappa light chain variable domain amino acid sequences of the anti-MAdCAM antibodies 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.26.4 and 9.8.2, (SEQ ID NOS: 16, 20, 24, 28, 32, 44 and 48) and the heavy chain variable domain of antibody 6.14.2, (SEQ ID NO: 10), predict the presence of N- linked glycosylation. The presence of this posttranslational modification was investigated using a combination of SDS-PAGE and Pro-Q® Emerald 488 Glycoprotein (Molecular Probes) staining with mAbs 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.26.4 and 9.8.2.

Briefly, approximately 2 pg of reduced anti-MAdCAM antibody was loaded onto a 4-12% SDSpolyacrylamide gel using a MOPS buffer. Following electrophoresis, the gel was fixed in 50% MeOH, 5% acetic acid and washed in 3% acetic acid. Any carbohydrates on the gel were then oxidised with periodic acid and stained using Pro-Q® Emerald 488 Glycoprotein Stain Kit

- 88 (Molecular Probes). After a final wash step, glycoprotein staining was visualised using a fluorescence scanner set at a wavelength of 473 nm.

After glycoprotein staining, the gel was stained for total protein using SYPRO Ruby protein gel stain and analysed using a fluorescence scanner set at a wavelength of 473 nm.

The kappa light chains of anti-MAdCAM antibodies, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, 7.26.4 and 9.8.2, ail stained positively for the presence of glycosylation. As an additional confirmation, anti-MAdCAM antibody 7.26.4, was subjected to tryptic/chymotrypic digestion, the LC-MS/MS analysis confirmed the presence of a modified tryptic peptide and provided additional confirmation of kappa light chain glycosylation.

Spécifie Asn-Gly sequences in the CDR1 régions of anti-MAdCAM antibodies, 1.7.2, 1.8.2, 6.22.2 and 7.20.5, render these régions sensitive to deamidation. Deamidation at neutral pH introduces a négative charge and can also lead to β-isomerisation, which could affect the properties of an antibody. For anti-MAdCAM antibodies 1.7.2, 1.8.2 and 7.20.5, the presence of deamidated Asn-isoaspartate residues was assessed by mass spectroscopy foilowing trapping the isoaspartate side chain with MeOH.

In brief, for the anti-MAdCAM antibody 1.7.2, the status of the tryptic/Asp-N peptide SSQSLLQSNGYNYL (SEQ ID NO: 69) (1573.7 Da) was selected for monitoring by LCMS/MS. Anti-MAdCAM antibody 1.7.2 was reduced in 10 mM DTT, alkylated in 5 mM Na iodoacetate and subsequently buffer exchanged into trypsin digestion buffer (50mM Tris- HCl, ImM CaCb, pH 7.6). The antibody was then mixed with sequencing grade modified trypsin (Promega) in a protease:protein ratio of 1:20. Protein was digested in trypsin for 15 hours at 30°C, and the resulting peptides separated by HPLC using a C-18 RPC on an Ettan LC System. The ³³Asn-containing peptide (4032 Da) was collected from the column and diluted in Asp-N digestion buffer (50 mM sodium phosphate buffer, pH 8.0). Endoproteinase Asp-N (Roche) was then added at an approximate peptide:enzyme ratio of 10:1.

Acetyl chloride (100 pL) was added to a sample of methanol (1 mL, -20°C), the mixture warmed to room température. The tryptic+Asp-N digest was dried in a Speed-Vac and then 5 pL of the methanol/acetyl chloride was added (45 min, room temp), then dried again in a Speed-Vac. The resulting residue was re-constituted in 0.1% TFA and peptides were analysed initially on the Voyager-DE STR MALDI-TOF mass spectrometer using either the nitrocellulose thin layer sample préparation method or reverse phase purification using Cl8 ZipTips (Millipore) followed by droplet mixing with α-cyano matrix. Themethylated peptide mixture was also analysed using LC-MS/MS on a Deçà XP Plus Ion Trap Mass Spectrometer as above. The elution was plumbed

- 89 straight into the Ion Trap MS and peptides were subsequently analysed by MS and MS/MS. The MS was set to analyse ail ions between 300 and 2000 Da. The strongest ion in any particular scan was then subjected to MS/MS analysis.

Table 12. Post-translational modification of anti-MAdCAM antibodies

CLONE	Heavy Chain	Kappa light chain
Glycosylation (NXS/T)	Confirmed	Glycosylation (NXS/T)	Confirmed	Deamidation (NG)	Confirmed
1.7.2					LQSNGYN	MS
1.8.2					LQSNGYN	MS
7.16.6
7.20.5					HGNGYNY	MS
7.26.4			CKSNQSLLY	MS/PAGE
6.14.2	TFNNSAMT	N . D
6.22.2			SGTNFTLTI	PAGE	LTINGLEA	N . D
6.34.2			ASQNISSYL	PAGE
6.67.1			SSNNKTYLA	PAGE
6.73.2			RASQNITN	PAGE
6.77.1			SCNSSQSL	PAGE
9.8.2			HSDNLSIT	PAGE

lgG4 lgG2

Mutagenesis studies:

The primary amino acid sequence of the anti-MAdCAM antibodies exemplified in this disclosure can be modified, by site-directed mutagenesis, to remove potential sites of post-translational modification (e.g., glycosylation, de-amidation) or to alter the isotype background, or to engineer other changes which may improve the therapeutic utility. As an example, PCR was used to engineer changes to the anti-MAdCAM antibodies 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 7.26.4, to revert certain framework sequences to germline, to remove potential glycosylation sites and/or to change the isotype background to a human IgG2. pCR2.1 TOPO-TA cloned cDNAs (100 ng), corresponding to heavy chain nucléotide SEQ ID NOS: 13, 17, 21 and 29, and kappa light nucléotide SEQ ID NOS: 15, 19, 23, 31 and 43, were used as a template in a sériés of PCRs using overlap-extension and a panel of primer sets described in Table 10.

6.22.2 Heavy chain: PCR primer sets 6.22.2_VH_F1 and 6.22.2VH_CS* (1) and VH3-33 and 6.22.2_VH_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 13 . Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with VH3-33 and VK6.22.2_CS* primers, to generate the modified 6.22.2

-90heavy chain V-domain. This modified version contains a His/Phe mutation in FRI and introduces an Xbal restriction site to enable in frame cloning into a pEE6.1 derived vector, termed pEEô.ICH, which contains the corresponding human IgGs constant domain. The final PCR fragment was cloned into the Xbaf site of pEEô.ICH, checked for orientation and the insert full sequence verified. The nucléotide sequence for the modified 6.22.2 heavy chain is found in SEQ ID NO: 51 and the corresponding amino acid sequence in SEQ ID NO: 52. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.22.2 kappa light chain: PCR primer sets 6.22.2_VK_F1 and revKappa (1), and A26 and 6.22.2_VK_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 15. Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with A26 and revKappa primers, to generate the modified 6.22.2 kappa light chain V-domain. This modified version contains Asn/Asp and Gly/Ser changes to the FR3 sequence. The résultant PCR product was cloned into pEE12.1 using HindlII/EcoRl sites and fully sequence verified. The nucléotide sequence for the modified 6.22.2 kappa light chain is found in SEQ ID NO: 53 and the corresponding amino acid sequence in SEQ ID NO: 54. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.34.2 Heavy chain: PCR primer sets 6.34.2_VH_F1 and 6.22.2VH_CS* (1) and VH3-30 and 6.34.2_VH_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 17. Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with VH3-30 and VK6.22.2_CS* primers, to generate the modified 6.34.2 heavy chain V-domain. This modified version contains a Ser/Arg mutation in FR3 and introduces an Xbal restriction site to enable in frame cloning into a pEE6.1 derived vector, termed pEEô.ICH, which contains the corresponding human IgG2 constant domain. The final PCR fragment was cloned into the Xbal site of pEEô.ICH, checked for orientation and the insert full sequence verified. The nucléotide sequence for the modified 6.34.2 heavy chain is found in SEQ ID NO: 55 and the corresponding amino acid sequence in SEQ ID NO: 56. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.34.2 kappa light chain: PCR primer sets 012 and 6.34.2_VK_R1 (1), 6.34.2_VK_F1 and 6.34.2_VK_R2 (2), as well as 6.34.2_VK_F2 and revKappa (3) were used to generate separate PCR products (1), (2) and (3), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 19. Products (1), (2) and (3) were purified and (1) and (2) were combined in a third PCR step (ca. 50 ng each), along with

- 91 012 and 6.34.2_VK_R2 primers, to generate the PCR product (4). PCR products (2) and (3) were combined in a fourth PCR step (ca. 50 ng each), along with 6.34.2_VK_F1 and revKappa, to generate the PCR product (5). PCR products (4) and (5) were purified and combined together (ca.50 ng each) with primers 012 and revKappa to generate the modified 6.34.2 kappa light chain V-domain. This modified version contains an Asn/Ser change in CDR1, a Phe/Tyr change in FR2 and Arg-Thr/Ser-Ser, Asp/Glu and Ser/Tyr changes to the FR3 sequence. The résultant PCR product was cloned into pEE12.1 using HindlII/EcoRl sites and fully sequence verified. The nucléotide sequence for the modified 6.34.2 kappa light chain is found in SEQ ID NO: 57 and the corresponding amino acid sequence in SEQ ID NO: 58. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.67.1 Heavy chain: PCR primer sets 6.67.1_VH_F1 and 6.67.1 VH CS* (1) and VH4-4 and 6.67.1_VH_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 21. Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with VH4-4 and VK6.67.1_CS* primers, to generate the modified 6.67.1 heavy chain V-domain. This modified version contains an Ile-Leu-Ala/Met-Ser-Val conversion in FR3 and introduces an Xbal restriction site to enable in frame cloning into a pEE6.1 derived vector, termed pEE6.1CH, which contains the corresponding human IgG2 constant domain. The final PCR fragment was cloned into the Xbal site of pEE6.1CH, checked for orientation and the insert full sequence verified. The nucléotide sequence for the modified 6.67.1 heavy chain is found in SEQ ID NO: 59 and the corresponding amino acid sequence in SEQ ID NO: 60. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.67.1 kappa light chain: PCR primer sets 6.67.1_VK_F1 and revKappa (1), and B3 and 6.67.1_VK_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 23. Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with B3 and revKappa primers, to generate the modified 6.67.1 kappa light chain V-domain. This modified version contains a Thr/Asn change in CDR1 and an Arg/Gly change in FR2. The résultant PCR product was cloned into pEE12.1 using HindlII/EcoRl sites and fully sequence verified. The nucléotide sequence for the modified 6.67.1 kappa light chain is found in SEQ ID NO: 61 and the corresponding amino acid sequence in SEQ ID NO: 62. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.77.1 Heavy chain: PCR primer sets VH 3-21 and 6.22.2VH_CS* were used to generate a single PCR product using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 29. The PCR products were digested with Xbal, gel purified and cloned into the Xbal site of pEE6.1CH, checking for orientation. The insert was fullly sequence verified. The nucléotide sequence for the modified 6.77.1 heavy chain is found in SEQ ID NO: 63 and the corresponding amino acid sequence in SEQ ID NO: 64. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

6.77.1 kappa light chain: PCR primer sets A2 and 6.77.1_VK_R1 (1), 6.77.1_VK_VK_F1 and 6.77.1_R2 (2), as well as 6.77.1_VK_F2 and revKappa (3) were used to generate separate PCR products (1), (2) and (3), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 31. Products (1), (2) and (3) were purified and, (1) and (2) were combined in a third PCR step (ca. 50 ng each) along with A2 and 6.77.1_VK_R2 primers, to generate PCR product (4). PCR product (2) and (3) were combined in a fourth PCR step (ca. 50 ng each) along with 6.77.1_VK_F1 and revKappa primers, to generate PCR product (5). PCR products (4) and (5) were purified and combined together (ca.5O ng each) with primers A2 and JK2 to generate the modified 6.77.1 kappa light chain V-domain. This modified version contains an Asn/Lys change in CDR1, a Ser/Tyr change in FR3 and a Cys/Ser residue change in CDR3 sequence. The résultant PCR product was cloned into pEE12.1 using HindlII/EcoRl sites and fully sequence verified. The nucléotide sequence for the modified

6.77.1 kappa light chain is found in SEQ ID NO: 65 and the corresponding amino acid sequence in SEQ ID NO: 66. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

7.26.4 kappa light chain: PCR primer sets 7.26.4_VK_F1 and revKappa (1), and A2 and 7.26.4_VK_R1 (2) were used to generate separate PCR products (1) and (2), using an Expand Taq polymerase and a pCR2.1 TOPO-TA cDNA template (100 ng) represented by nucléotide sequence SEQ ID NO: 43. Products (1) and (2) were purified and combined in a third PCR step (ca. 50 ng each) along with A2 and revKappa primers, to generate the modified 7.26.4 kappa light chain V-domain. This modified version contains an Asn/Ser change in CDR1. The résultant PCR product was cloned into pEE12.1 using HindlII/EcoRl sites and fully sequence verified. The nucléotide sequence for the modified 7.26.4 kappa light chain is found in SEQ ID NO: 67 and the corresponding amino acid sequence in SEQ ID NO: 68. The changes in the nucléotide and amino acid sequences compared with the parent are indicated.

- 93 A functional eukaryotic expression vector for each of the modified versions of 6.22.2, 6.34.2, 6.67.1, 6.77.1 and 7.26.4, referred to as 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod, and representing respectively the heavy chain nucléotide sequences SEQ ID NOS: 51, 55, 59, 63 and 41, and corresponding amino acid sequences SEQ ID NOS: 52, 56, 60, 64 and 42, as well as the kappa light chain nucléotide sequences SEQ ID NOS: 53, 57, 61, 65 and 67, and the corresponding amino acid sequences SEQ ID NOS: 54, 58, 62, 66 and 68 were assembled as follows: The heavy chain cDNA inserts corresponding to 6.22.2-mod, 6.34.2-mod, 6.67.1-mod and 6.77.1-mod were excised from the pEE6.1CH vector with Notl/Sall, the parental version of the heavy chains of 7.26.4 was excised from the pEE6.1 vector with Notl/Sall, and the purified fragments were cloned into identical sites into the corresponding pEE12.1 vector containing the modified versions of the kappa light chain sequences 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4- mod. The sequences of the vectors were confirmed, and purified amounts used in transient transfections with HEK 293T cells. Briefly, 9x10⁶ HEK 293T cells, seeded in a Tl65 flask the day before transfection and washed into Optimem, were transiently transfected with vector cDNAs corresponding to 6.22.2-mod, 6.34.2-mod, 6.67.1mod, 6.77.1-mod and 7.26.4-mod (40 pg) using Lipofectamine PLUS (Invitrogen) according to manufacturer’s instructions. The cells were incubated for 3 hrs, then the transfection media replaced with DMEM (Invitrogen 21969-035) media containing 10% ultra-low IgG fêtai calf sérum (Invitrogen 16250-078) and L-Glutamine (50 mL). The media supematant was harvested 5 days later, filter sterilised and the anti-MAdCAM antibody purified using protein G sepharose affinity chromatography, in a similar manner as to that described above. The amount of antibody recovered (20-100 pg) was quantified by a Bradford assay.

The anti-MAdCAM activity of affinity purified antibody corresponding to 6.22.2-mod, 6.34.2mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod was assessed in the MAdCAM- IgGl-Fc fusion assay as described previously. The IC50 values of these anti-MADCAM antibodies compared with the parental anti-MAdCAM antibodies from which they were derived are presented in Table 13. There was minimal effect of the amino acid substitutions described above on the activity of the modified anti-MAdCAM antibodies compared with their parents was minimal. The antibodies also maintained their binding to CHO cells expressing recombinant human MAdCAM or the cynomolgus/human MAdCAM chimera.

Table 13. Activity of modified versions of anti-MAdCAM antibodies, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod compared with their parents.

CLONE	MAdCAM lgG1 Fc fusion Assay Mean 1C50 (pg/mL)
Parent	Modifîed
6.22.2	0.018	0.058
6.34.2	0.013	0.049
6.67.1	0.013	0.037
6.77.1	0.022	0.077
7.26.4	0.021	0.033

EXAMPLE VI

Increase in β?⁺ lymphocytes in the peripheral circulation by blocking anti-MAdCAM antibodies

An assay was developed to identify and correlate a mechanistic effect of an anti- MAdCAM antibody and its circulating level in blood. An inhibitory anti-MAdCAM antibody should hâve the effect of inhibiting the recruitment of leukocytes expressing the ο^ιβγ integrin to the gastrointestinal tract. Classes of α.4β? integrin-bearing leukocytes should, therefore, be restricted to the peripheral circulation.

This was demonstrated with a fully human anti-human MAdCAM mAb 7.16.6, in cynomolgus.

Purified anti-human MAdCAM mAb 7.16.6 (1 mg/kg) or vehicle (20 mM NaAcetate, 0.2 mg/mL polysorbate 80, 45 mg/mL mannitol, and 0.02 mg/mL EDTA atpH 5.5 ) were assessed in a similar manner by intravenous administration via the saphenous vein to two groups of cynomolgus monkeys (n=4/group). At day 3 post-dosing blood samples were collected in EDTA tubes by fémoral venipuncture. LP AM specifc antibodies, which crossreact with the cynomolgus α4βγ integrin, are not commercially available, so an anti-βγ antibody (recognising α4βγ and αεβν integrin) was used instead. Antibodies (30 pL), according to the following table, table 15, were added to tubes containing 100 pL of cynomolgus blood, mixed by gentle vortexing and incubated for 20-30 mins at 4°C.

Table 15. Antibodies (BD Pharmingen) used in immunophenotyping of cynomologus blood

Catalogue Number	Antibody or Isotype
555748	mIgGl,k-FITC
555844	mIgG2a, k-PE
559425	mlgGl - PerCP
555751	mIgGl,k-APC
555728	CD 28-FITC
555945	β7-ΡΕ
558814	CD 95-APC
550631	CD 4-PerCP

- 95 To each tube, 1 mL of 1:10 FACSlyse solution (BD # 349202) was added, mixed by gentle vortex and incubated at room température for approximately 12 minutes in the dark until red blood cell lysis was complété. Then 2 mL of BD stain buffer (# 554656) was added to each tube, mixed and centrifuged at 250 x g for 6-7 mins at room température. The supematant was decanted and the pellet resuspended in 3 mL of stain buffer, mixed again and centrifuged at 250 x g for 6-7 mins at room température. Cytofix buffer (BD # 554655), containing w/v paraformaldéhyde (100 pL) was added to the cell pellets from monkey peripheral blood and mixed thoroughly by low/moderate speed of vortexer. The samples were kept at 4°C in the dark until they acquired on the FACSCalibur. Just prior to acquisition, PBS (100 pL) was added to ail tubes immediately before acquisition. The absolute cell numbers of CD4⁺P ⁺CD951oCD28⁺ (naïve), CD4⁺p ⁺CD95hiCD28⁺ (central memory), CD4⁺p₇-CD95hiCD28⁺ (central memory), CD4 P7⁺CD95hiCD28 (effector memory) were acquired by appropriate gating and quandrant analyses. Other T cell subsets for example, CD8⁺ T central memory cell (^7CD8CD28A'D95Q and any other leukocytes bearing a MAdCAM ligand, may also be analyzed by this method with the appropriate antibodies. Compared with the vehicle control, anti-MAdCAM mAb 7.16.6 caused an approximate 3 fold increase in the levels of circulating CD4⁺P ⁺CD95hiCD28⁺ central memory T cells, as shown in Figure 7. There were no effects on the population of circulating CD4 p7-CD95hiCD28⁺ central memory T cells, indicating that the effect of anti-MAdCAM mAb 7.16.6 is spécifie for gut homing T cells. The effects of anti-MAdCAM mAb 7.16.6, in cynomolgus, on populations of circulating (aQfV lymphocytes indicates that this is a robust surrogate proof of mechanism biomarker, particularly in the context of practical application in a clinicâl setting.

Sequences

SEQ ID NO: 1-48,51-68 and 148-150 provide nucléotide and amino acid sequences of the heavy and kappa light chains for thirteen human anti-MAdCAM antibodies, nucléotide and amino acid sequences of cynomolgus MAdCAM οπβ? binding domain sequences and nucléotide and amino acid sequences of five modified human anti-MAdCAM antibodies.

SEQ ID NO: 1-48 and 148-150 provide the heavy and kappa light chain nucléotide and amino acid sequences of thirteen human monoclonal anti-MAdCAM antibodies: 1.7.2 (SEQ ID NO: 14), 1.8.2 (SEQ ID NO: 5-8), 6.14.2 (SEQ ID NO: 9-12), 6.22.2 (SEQ IDNO: 13-16), 6.34.2 (SEQ ID NO: 17-20), 6.67.1 (SEQ ID NO: 21-24), 6.73.2 (SEQ ID NO: 25-28), 6.77.1 (SEQ ID NO: 29-32), 7.16.6 (SEQ ID NO: 33-36), 7.20.5 (SEQ ID NO: 37-40), 7.26.4 (SEQ ID NO: 4144), 9.8.2 (SEQ ID NO: 45-48), X481.2 (SEQ ID NO: 35, 148-150).

-96SEQ ID NO: 49-50 provide the nucléotide and amino acid sequences of a cynomolgus MAdCAM α4βγ binding domain.

SEQ ID NO: 51-68 provide the heavy and kappa light chain nucléotide and amino acid sequences for the modified monoclonal anti-MAdCAM antibodies: 6.22.2 (SEQ ID NO: 51-54), 5 modified 6.34.2 (SEQ ID NO: 55-58), modified 6.67.1 (SEQ ID NO: 59-62), modified 6.77.1 (SEQ ID NO: 63-66) and the kappa light chain nucléotide and amino acid sequences of modified monoclonal anti-MAdCAM antibody: modified 7.26.4 (SEQ ID NO: 67-68).

SEQ ID NOS: 70-106 and 108-110 provide various primer sequences.

Key:

Signal sequence: underlined lower case

Amino acid changes in modified anti-MAdCAM antibodies sequence compared to parent: underlined upper case

SEQ IDNO. 1

1.7.2 Heavy Chain Nucléotide Sequence i

101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951

1001 1051 1101 1151 1201 1251 1301 1351 atggagtttg ggctgagctg gattttcctt gctgctattt taaaaggtgt ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCTTG GTGAAGCCTG GGGGGTCCCT TAGACTCTCC TGTGTAGCCT CTGGATTCAC TTTCACTAAC GCCTGGATGA TCTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT TGGCCGTATT AAAAGGAAAA CTGATGGTGG GACAACAGAC TACGCTGCAC CCGTGAAAGG CAGATTCACC ATCTCAAGAG ATGATTCAAA AAACACGCTG TATCTGCAAA TGAACAGCCT GAAAACCGAG GACACAGCCG TGTATTACTG TACCACAGGG GGAGTGGCTG AGGACTACTG GGGCCAGGGA ACCCTGGTCA CCGTCTCCTC AGCCTCCACC AAGGGCCCAT CGGTCTTCCC CCTGGCGCCC TGCTCCAGGA GCACCTCCGA GAGCACAGCG GCCCTGGGCT GCCTGGTCAA GGACTACTTC CCCGAACCGG TGACGGTGTC GTGGAACTCA GGCGCTCTGA CCAGCGGCGT GCACACCTTC CCAGCTGTCC TACAGTCCTC AGGACTCTAC TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAACTTCG GCACCCAGAC CTACACCTGC AACGTAGATC ACAAGCCCAG CAACACCAAG GTGGACAAGA CAGTTGAGCG CAAATGTTGT GTCGAGTGCC CACCGTGCCC AGCACCACCT GTGGCAGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACGTG CGTGGTGGTG GACGTGAGCC ACGAAGACCC CGAGGTCCAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC ACGGGAGGAG CAGTTCAACA GCACGTTCCG TGTGGTCAGC GTCCTCACCG TTGTGCACCA GGAGTGGCTG AACGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGGCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAACCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATCC CGGGAGGAGA TGACCAAGAA CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTACCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG AGAACAACTA CAAGACCACA CCTCCCATGC TGGACTCCGA CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA

SEQ IDNO. 2

1.7.2 Predicted Heavy Chain Protein Sequence mefglswifl aailkgvqcE VQLVESGGGL VKPGGSLRLS CVASGFTFTN

- 97 51 AWMIWVRQAP GKGLEWVGRI KRKTDGGTTD YAAPVKGRFT ISRDDSKNTL 101 YLQMNSLKTE DTAVYYCTTG GVAEDYWGQG TLVTVSSAST KGPSVFPLAP 151 CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY 201 SLSSVVTVPS SNFGTQTYTC NVDHKPSNTK VDKTVERKCC VECPPCPAPP 251 VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ FNWYVDGVEV 301 HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS NKGLPAPIEK 351 TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN 401 GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 451 HYTQKSLSLS PGK

SEQ ID NO. 3

1.7.2 Kappa Light Chain Nucléotide Sequence atgaggctcc ctgctcagct cctggggctg ctaatgctct Gggtctctgg 51 atccagtggg GATATTGTGA TGACTCAGTC TCCACTCTCC CTGCCCGTCA

101 CCCCTGGAGA GCCGGCCTCC ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG 151 CAAAGTAATG GATACAACTA TTTGGATTGG TACCTGCAGA AGCCAGGGCA 201 GTCTCCACAG CTCCTGATCT ATTTGGGTTC TAATCGGGCC TCCGGGGTCC 251 CTGACAGGTT CAGTGGCAGT GGATCAGGCA CAGATTTTAC ACTGAAAATC 301 AGCAGAGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAGCTCT 351 ACAAACTATC ACCTTCGGCC AAGGGACACG ACTGGAGATT AAACGAACTG 401 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 451 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA 501 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC 551 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC 601 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC 651 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA 701 ACAGGGGAGA GTGTTAGTGA

SEQ ID NO. 4

1.7.2 Predicted Kappa Light Chain Protein Sequence mrlpagllgl Imlwvsgssg DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL

QSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI

101 SRVEAEDVGV YYCMQALQTI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK

151 SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS

202 STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO. 5

1.8.2 Heavy Chain Nucléotide Sequence

Atggagtttg Ggctgagctg ccagtgtGAG GTGCAGCTGG 101 GGGGGTCCCT TAGACTCTCC 151 GCCTGGATGA TCTGGGTCCG 2 01 TGGCCGTATT AAAAGGAAAA 251 CCGTGAAAGG CAGATTCACC 301 TATCTGCAAA TGAACAGCCT 351 TACCACAGGG GGAGTGGCTG 401 CCGTCTCCTC AGCCTCCACC 451 TGCTCCAGGA GCACCTCCGA 501 GGACTACTTC CCCGAACCGG 551 CCAGCGGCGT GCACACCTTC 601 TCCCTCAGCA GCGTGGTGAC 651 CTACACCTGC AACGTAGATC 701 CAGTTGAGCG CAAATGTTGT 751 GTGGCAGGAC CGTCAGTCTT 801 CATGATCTCC CGGACCCCTG 851 ACGAAGACCC CGAGGTCCAG 901 CATAATGCCA AGACAAAGCC

Gattttcctt Gctgctattt Taaaaggtgt TGGAGTCTGG GGGAGGCTTG GTGAAGCCTG TGTGTAGTCT CTGGATTCAC TTTCACTAAC CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT CTGATGGTGG GACAACAGAC TACGCTGCAC ATCTCAAGAG ATGATTCAAA AAACACGCTG GAAAACCGAG GACACAGCCG TGTATTACTG AGGACTACTG GGGCCAGGGA ACCCTGGTCA AAGGGCCCAT CGGTCTTCCC CCTGGCGCCC GAGCACAGCG GCCCTGGGCT GCCTGGTCAA TGACGGTGTC GTGGAACTCA GGCGCTCTGA CCAGCTGTCC TACAGTCCTC AGGACTCTAC CGTGCCCTCC AGCAACTTCG GCACCCAGAC ACAAGCCCAG CAACACCAAG GTGGACAAGA GTCGAGTGCC CACCGTGCCC AGCACCACCT CCTCTTCCCC CCAAAACCCA AGGACACCCT AGGTCACGTG CGTGGTGGTG GACGTGAGCC TTCAACTGGT ACGTGGACGG CGTGGAGGTG ACGGGAGGAG CAGTTCAACA GCACGTTCCG

951 TGTGGTCAGC GTCCTCACCG

1001 AGTACAAGTG CAAGGTCTCC

1051 ACCATCTCCA AAACCAAAGG

1101 GCCCCCATCC CGGGAGGAGA

1151 TGGTCAAAGG CTTCTACCCC

1201 GGGCAGCCGG AGAACAACTA

1251 CGGCTCCTTC TTCCTCTACA

1301 AGCAGGGGAA CGTCTTCTCA

1351 CACTACACGC AGAAGAGCCT

- 98 TTGTGCACCA GGACTGGCTG AACGGCAAGG AACAAAGGCC TCCCAGCCCC CATCGAGAAA GCAGCCCCGA GAACCACAGG TGTACACCCT TGACCAAGAA CCAGGTCAGC CTGACCTGCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT CAAGACCACA CCTCCCATGC TGGACTCCGA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC TGCTCCGTGA TGCATGAGGC TCTGCACAAC CTCCCTGTCT CCGGGTAAAT GA

SEQ ID NO. 6

1.8.2 Predicted Heavy Chain Protein Sequence mefglswifl aailkgvqcE VQLVESGGGL VKPGGSLRLS CVVSGFTFTN

AWMIWVRQAP GKGLEWVGRI KRKTDGGTTD YAAPVKGRFT ISRDDSKNTL

101 YLQMNSLKTE DTAVYYCTTG GVAEDYWGQG TLVTVSSAST KGPSVFPLAP

151 CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY

201 SLSSVVTVPS SNFGTQTYTC NVDHKPSNTK VDKTVERKCC VECPPCPAPP

251 VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ FNWYVDGVEV

301 HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS NKGLPAPIEK

351 TISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN

401 GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

451 HYTQKSLSLS PGK

SEQ ID NO. 7

1.8.2 Kappa Light Chain Nucléotide Sequence atgaggctcc ctgctcagct atccagtggg GATATTGTGA

101 CCCCTGGAGA GCCGGCCTCC

151 CAAAGTAATG GATTCAACTA

201 GTCTCCACAG CTCCTGATCT

251 CTGACAGGTT CAGTGGCAGT

301 AGCAGAGTGG AGGCTGAGGA

351 ACAAACTATC ACCTTCGGCC

401 TGGCTGCACC ATCTGTCTTC

451 TCTGGAACTG CCTCTGTTGT

501 GGCCAAAGTA CAGTGGAAGG

551 AGGAGAGTGT CACAGAGCAG

601 AGCACCCTGA CGCTGAGCAA

651 CTGCGAAGTC ACCCATCAGG

701 ACAGGGGAGA GTGTTAGTGA cctggggctg ctaatgctct gggtctctgg TGACTCAGTC TCCACTCTCC CTGCCCGTCA ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG TTTGGATTGG TACCTGCAGA AGCCAGGGCA ATTTGGGTTC TAATCGGGCC TCCGGGGTCC GGGTCAGGCA CAGATTTTAC ACTGAAAA.TC TGTTGGGGTT TATTACTGCA TGCAAGCTCT AAGGGACACG ACTGGAGATT AAACGAACTG ATCTTCCCGC CATCTGATGA GCAGTTGAAA GTGCCTGCTG AATAACTTCT ATCCCAGAGA TGGATAACGC CCTCCAATCG GGTAACTCCC GACAGCAAGG ACAGCACCTA CAGCCTCAGC AGCAGACTAC GAGAAACACA AAGTCTACGC GCCTGAGCTC GCCCGTCACA AAGAGCTTCA

SEQ ID NO. 8

1.8.2 Predicted Kappa Light Chain Protein Sequence mrlpaqllgl

QSNGFNYLDW

101 SRVEAEDVGV

151 SGTASVVCLL

202 STLTLSKADY

SEQ ID NO. 9

Imlwvsgssg DIVMTQSPLS YLQKPGQSPQ LLIYLGSNRA YYCMQALQTI TFGQGTRLEI NNFYPREAKV QWKVDNALQS EKHKVYACEV THQGLSSPVT

LPVTPGEPAS ISCRSSQSLL SGVPDRFSGS GSGTDFTLKI KRTVAAPSVF IFPPSDEQLK GNSQESVTEQ DSKDSTYSLS KSFNRGEC

6.14.2 Heavy Chain Nucléotide Sequence atggagtttg ggctgagctg gctttttctt gtqgctattt taaaaggtgt ccagtgtGAG GTGCAGCTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG

-99101 GGGGGTCCCT GAGACTCTCC

151 TCTGCCATGA CCTGGGTCCG

201 CTCAACTACT AGTGGAAGTG

251 AGGGCCGGTT CACCATCTCC

301 CAAATGAACA GCCTGAGAGC

351 CCGTGGATAC AGCTATGGTA

401 CCCTGGTCAC CGTCTCCTCA

451 CTGGCGCCCT GTTCCAGGAG

501 CCTGGTCAAG GACTACTTCC

551 GCGCCCTGAC CAGCGGCGTG

601 GGACTCTACT CCCTCAGCAG

651 CACGAAGACC TACACCTGCA

701 TGGACAAGAG AGTTGAGTCC

751 GCACCTGAGT TCCTGGGGGG

801 CAAGGACACT CTCATGATCT

851 TGGACGTGAG CCAGGAAGAC

901 GGCGTGGAGG TGCATAATGC

951 CAGCACGTAC CGTGTGGTCA

1001 TGAACGGCAA GGAGTACAAG

1051 TCCATCGAGA AAACCATCTC

1101 GGTGTACACC CTGCCCCCAT

1151 GCCTGACCTG CCTGGTCAAA

1201 TGGGAGAGCA ATGGGCAGCC

1251 GCTGGACTCC GACGGCTCCT

1301 AGAGCAGGTG GCAGGAGGGG

1351 GCTCTGCACA ACCACTACAC

1401 ATGA

TGTGCAGCCT CTGGACTCAC CTTTAACAAT CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT GTGGTACCAC ATACTACGCA GACTCCGTGA AGAGACTCTC CCAAGAACAC GCTCTATCTG CGAGGACACG GCCGTATATT ACTGTGCGGC CGACCCCCTA TGAGTACTGG GGCCAGGGAA GCTTCCACCA AGGGCCCATC CGTCTTCCCC CACCTCCGAG AGCACAGCCG CCCTGGGCTG CCGAACCGGT GACGGTGTCG TGGAACTCAG CACACCTTCC CGGCTGTCCT ACAGTCCTCA CGTGGTGACC GTGCCCTCCA GCAGCTTGGG ACGTAGATCA CAAGCCCAGC AACACCAAGG AAATATGGTC CCCCATGCCC ATCATGCCCA ACCATCAGTC TTCCTGTTCC CCCCAAAACC CCCGGACCCC TGAGGTCACG TGCGTGGTGG CCCGAGGTCC AGTTCAACTG GTACGTGGAT CAAGACAAAG CCGCGGGAGG AGCAGTTCAA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGCAAGGTCT CCAACAAAGG CCTCCCGTCC CAAAGCCAAA GGGCAGCCCC GAGAGCCACA CCCAGGAGGA GATGACCAAG AACCAGGTCA GGCTTCTACC CCAGCGACAT CGCCGTGGAG GGAGAACAAC TACAAGACCA CGCCTCCCGT TCTTCCTCTA CAGCAGGCTA ACCGTGGACA AATGTCTTCT CATGCTCCGT GATGCATGAG ACAGAAGAGC CTCTCCCTGT CTCTGGGTAA

SEQ ID NO. 10

6.14.2 Predicted Heavy Chain Protein Sequence mefglswlfl vailkgvgcE VQLLESGGGL VQPGGSLRLS CAASGLTFNN

SAMTWVRQAP GKGLEWVSTT SGSGGTTYYA DSVKGRFTIS RDSPKNTLYL

101 QMNSLRAEDT AVYYCAARGY SYGTTPYEYW GQGTLVTVSS ASTKGPSVFP

151 LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS

201 GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP

251 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD

301 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS

351 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE

401 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE

451 ALHNHYTQKS LSLSLGK

SEQ ID NO. 11

6.14.2 Kappa Light Chain Nucléotide Sequence atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggggcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT

101 CTGCATCTGT AGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCGGAGC

151 ATTAGCAGCT ATTTAAATTG GTATCAGCAG AAACCAGGGA AAGCCCCTAA

201 AGTCCTGATC TTTTTTGTGT CCAGTTTGCA AAGTGGGGTC CCATCAAGGT

251 TCAGTGGCAG TGGCTCTGGG ACAGATTTCA CTCTCACCAT CAGCAGTCTG

301 CAACCTGAAG ATTTTGCAAC TTACTACTGT CAACAGAATT ACATTCCCCC

351 TATTACCTTC GGCCAGGGGA CACGACTGGA GATCAGACGA ACTGTGGCTG

401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA

451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA

501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA

551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC

601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA

651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG

701 GAGAGTGTTA G

- 100SEQ IDNO. 12

6.14.2 Predicted Kappa Light Chain Protein Sequence mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASRS

ISSYLNWYQQ KPGKAPKVLI FFVSSLQSGV PSRFSGSGSG TDFTLTISSL

101 QPEDFATYYC QQNYIPPITF GQGTRLEIRR TVAAPSVFIF PPSDEQLKSG

151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST

202 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO. 13

6.22.2 Heavy Chain Nucléotide Sequence

10	1	atggagtttg	ggctgagctg	ggttttcctc	gttgctcttt	taagaqqtqt
	51	ccagtgtCAG	GTGCAGCTGG	TGGAGTCTGG	GGGAGGCGTG	GTCCAGCCTG
	101	GGAGGTCCCT	GAGACTCTCC	TGTGCAGCGT	CTGGACACAC	CTTCAGTAGC
	151	GATGGCATGC	ACTGGGTCCG	CCAGGCTCCA	GGCAAGGGGC	TGGAGTGGGT
	201	GGCAATTATA	TGGTATGATG	GAAGTAATAA	ATATTATGCA	GACTCCGTGA
	251	AGGGCCGATT	CACCATCTCC	AGAGACAATT	CCAAGAACAC	GCTGTATCTG
	301	CAAATGAACA	GCCTGAGAGC	CGAGGACACG	GCTGTATATT	ACTGTGCGAG
	351	AGATCCCGGC	TACTATTACG	GTATGGACGT	CTGGGGCCAA	GGGACCACGG
	401	TCACCGTCTC	CTCAGCTTCC	ACCAAGGGCC	CATCCGTCTT	CCCCCTGGCG
	451	CCCTGCTCCA	GGAGCACCTC	CGAGAGCACA	GCCGCCCTGG	GCTGCCTGGT
	501	CAAGGACTAC	TTCCCCGAAC	CGGTGACGGT	GTCGTGGAAC	TCAGGCGCCC
	551	TGACCAGCGG	CGTGCACACC	TTCCCGGCTG	TCCTACAGTC	CTCAGGACTC
	601	TACTCCCTCA	GCAGCGTGGT	GACCGTGCCC	TCCAGCAGCT	TGGGCACGAA
	651	GACCTACACC	TGCAACGTAG	ATCACAAGCC	CAGCAACACC	AAGGTGGACA
	701	AGAGAGTTGA	GTCCAAATAT	GGTCCCCCAT	GCCCATCATG	CCCAGCACCT
	751	GAGTTCCTGG	GGGGACCATC	AGTCTTCCTG	TTCCCCCCAA	AACCCAAGGA
	801	CACTCTCATG	ATCTCCCGGA	CCCCTGAGGT	CACGTGCGTG	GTGGTGGACG
	851	TGAGCCAGGA	AGACCCCGAG	GTCCAGTTCA	ACTGGTACGT	GGATGGCGTG
	901	GAGGTGCATA	ATGCCAAGAC	AAAGCCGCGG	GAGGAGCAGT	TCAACAGCAC
	951	GTACCGTGTG	GTCAGCGTCC	TCACCGTCCT	GCACCAGGAC	TGGCTGAACG
	1001	GCAAGGAGTA	CAAGTGCAAG	GTCTCCAACA	AAGGCCTCCC	GTCCTCCATC
	1051	GAGAAAACCA	TCTCCAAAGC	CAAAGGGCAG	CCCCGAGAGC	CACAGGTGTA
	1101	CACCCTGCCC	CCATCCCAGG	AGGAGATGAC	CAAGAACCAG	GTCAGCCTGA
	1151	CCTGCCTGGT	CAAAGGCTTC	TACCCCAGCG	ACATCGCCGT	GGAGTGGGAG
	1201	AGCAATGGGC	AGCCGGAGAA	CAACTACAAG	ACCGCGCCTC	CCGTGCTGGA
	1251	CTCCGACGGC	TCCTTCTTCC	TCTACAGCAG	GCTAACCGTG	GACAAGAGCA
	1301	GGTGGCAGGA	GGGGAATGTC	TTCTCATGCT	CCGTGATGCA	TGAGGCTCTG
	1351	CACAACCACT	ACACACAGAA	GAGCCTCTCC	CTGTCTCTGG	GTAAATGA

SEQ ID NO. 14

6.22.2 Predicted Heavy Chain Protein Sequence mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGHTFSS

DGMHWVRQAP GKGLEWVAII WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL

101 QMNSLRAEDT AVYYCARDPG YYYGMDVWGQ GTTVTVSSAS TKGPSVFPLA

151 PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL

201 YSLSSVVTVP SSSLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPSCPAP

251 EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV

301 EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI

351 EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE

401 SNGQPENNYK TAPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL

451 HNHYTQKSLS LSLGK

SEQ ID NO. 15

6.22.2 Kappa Light Chain Nucléotide Sequence

- 101 - atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagcttc caggggtGAA ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC

101 CAAAAGAGAA AGTCACCATC ACCTGCCGGG CCAGTCAGAG AATTGGTAGT

151 AGCTTACACT GGTACCAGCA GAAACCAGAT CAGTCTCCAA AACTCCTCAT

201 CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG TTCAGTGGCA

251 GTGGATCTGG GACAAATTTC ACCCTCACCA TCAATGGCCT GGAAGCTGAA

301 GATGCTGCAA CTTATTACTG TCATCAGAGT GGTCGTTTAC CGCTCACTTT

351 CGGCGGAGGG ACCAAGGTGG AGATCAAACG AACTGTGGCT GCACCATCTG

401 TCTTCATCTT CCCGCCATCT GATGAGCAGT TGAAATCTGG AACTGCCTCT

451 GTTGTGTGCC TGCTGAATAA CTTCTATCCC AGAGAGGCCA AAGTACAGTG

501 GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG AGTGTCACAG

551 AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG

601 AGCAAAGCAG ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA

651 TCAGGGCCTG AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT

701 AGTGA

SEQ ID NO. 16

6.22.2 Predicted Kappa Light Chain Protein Sequence mlpsqligfl llwvpasrgE IVLTQSPDFQ SVTPKEKVTI TCRASQRIGS 5 51 SLHWYQQKPD QSPKLLIKYA SQSFSGVPSR FSGSGSGTNF TLTINGLEAE

101 DAATYYCHQS GRLPLTFGGG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS

151 VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL

201 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC

SEQ ID NO. 17

6.34.2 Heavy Chain Nucléotide Sequence atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtCAG GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG

101 GGAGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC

151 TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT

201 GGCAGTTATA TCAAATGATG GAAATAATAA ATACTATGCA GACTCCGTGA

251 AGGGCCGATT CACCATCTCC AGAGACAATT CCAAAAACAC GCTGTATCTG

301 CAAATGAACA GCCTGAGCGC TGAGGACACG GCTGTGTATT ACTGTGCGAG

351 AGATAGTACG GCGATAACCT ACTACTACTA CGGAATGGAC GTCTGGGGCC

401 AAGGGACCAC GGTCACCGTC TCCTCAGCTT CCACCAAGGG CCCATCCGTC

451 TTCCCCCTGG CGCCCTGCTC CAGGAGCACC TCCGAGAGCA CAGCCGCCCT

501 GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA

551 ACTCAGGCGC CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG

601 TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAG

651 CTTGGGCACG AAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA

701 CCAAGGTGGA CAAGAGAGTT GAGTCCAAAT ATGGTCCCCC ATGCCCATCA

751 TGCCCAGCAC CTGAGTTCCT GGGGGGACCA TCAGTCTTCC TGTTCCCCCC

801 AAAACCCAAG GACACTCTCA TGATCTCCCG GACCCCTGAG GTCACGTGCG

851 TGGTGGTGGA CGTGAGCCAG GAAGACCCCG AGGTCCAGTT CAACTGGTAC

901 GTGGATGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA

951 GTTCAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG

1001 ACTGGCTGAA CGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC

1051 CCGTCCTCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA

1101 GCCACAGGTG TACACCCTGC CCCCATCCCA GGAGGAGATG ACCAAGAACC

1151 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC

1201 GTGGAGTGGG AGAGCAATGG ACAGCCGGAG AACAACTACA AGACCACGCC

1251 TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AGGCTAACCG

1301 TGGACAAGAG CAGGTGGCAG GAGGGGAATG TCTTCTCATG CTCCGTGATG

1351 CATGAGGCTC TGCACAACCA CTACACACAG AAGAGCCTCT CCCTGTCTCT

1401 GGGTAAATGA

SEQ ID NO. 18

- 102 -

6.34.2 Predicted Heavy Chain Protein Sequence mef^lswyfj^jzajLlrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS

YGMHWVRQAP GKGLEWVAVI SNDGNNKYYA DSVKGRFTIS RDNSKNTLYL

101 QMNSLSAEDT AVYYCARDST AITYYYYGMD VWGQGTTVTV SSASTKGPSV

151 FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ

201 SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS

251 CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY

301 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL

351 PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA

401 VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

451 HEALHNHYTQ KSLSLSLGK

SEQ ID NO. 19

6.34.2 Kappa Light Chain Nucléotide Sequence atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT

101 CTGCATCTGT CGGAGACAGA GTCACCATCA CTTGCCGGGC AAGTCAGAAT

151 ATTAGTAGCT ATTTAAATTG GTTTCAGCAG AAACCAGGGA AAGCCCCTAA

201 GCTCCTGATC TATGCTGCAT CCGGTTTGAA GCGTGGGGTC CCATCACGGT

251 TCAGTGGTAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGGACTCTG

301 CAACCTGATG ATTTTGCAAC TTACTCCTGT CACCAGAGTT ACAGTCTCCC

351 ATTCACTTTC GGCCCTGGGA CCAAAGTGGA TATCAAACGA ACTGTGGCTG

401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA

451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA

501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA

551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC

601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA

651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG

701 GAGAGTGTTA GTGA

SEQ ID NO. 20

6.34.2 Predicted Kappa Light Chain Protein Sequence mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQN

ISSYLNWFQQ KPGKAPKLLI YAASGLKRGV PSRFSGSGSG TDFTLTIRTL

101 QPDDFATYSC HQSYSLPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG

151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST

201 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO. 21

6.67.1 Heavy Chain Nucléotide Sequence atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtccCAG GTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT

101 CGGAGACCCT GTCCCTCACC TGCACTGTCT CTGGTGACTC CATCAGTAGT

151 AACTATTGGA GCTGGATCCG GCAGCCCGCC GGGAAGGGAC TGGAGTGGAT

201 TGGGCGTATC TATACCAGTG GGGGCACCAA CTCCAACCCC TCCCTCAGGG

251 GTCGAGTCAC CATTTTAGCA GACACGTCCA AGAACCAGTT CTCTCTGAAA

301 CTGAGTTCTG TGACCGCCGC GGACACGGCC GTGTATTACT GTGCGAGAGA

351 TCGTATTACT ATAATTCGGG GACTTATTCC ATCCTTCTTT GACTACTGGG

401 GCCAGGGAAC CCTGGTCACC GTCTCCTCAG CTTCCACCAA GGGCCCATCC

451 GTCTTCCCCC TGGCGCCCTG CTCCAGGAGC ACCTCCGAGA GCACAGCCGC

501 CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT

551 GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA

601 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG

651 CAGCTTGGGC ACGAAGACCT ACACCTGCAA CGTAGATCAC AAGCCCAGCA

701 ACACCAAGGT GGACAAGAGA GTTGAGTCCA AATATGGTCC CCCATGCCCA

751 TCATGCCCAG CACCTGAGTT CCTGGGGGGA CCATCAGTCT TCCTGTTCCC

- 103 -

801	CCCAAAACCC	AAGGACACTC
851	GCGTGGTGGT	GGACGTGAGC
901	TACGTGGATG	GCGTGGAGGT
951	GCAGTTCAAC	AGCACGTACC
1001	AGGACTGGCT	GAACGGCAAG
1051	CTCCCGTCCT	CCATCGAGAA
1101	AGAGCCACAG	GTGTACACCC
1151	ACCAGGTCAG	CCTGACCTGC
1201	GCCGTGGAGT	GGGAGAGCAA
1251	GCCTCCCGTG	CTGGACTCCG
1301	CCGTGGACAA	GAGCAGGTGG
1351	ATGCATGAGG	CTCTGCACAA
1401	TCTGGGTAAA	T GA

TCATGATCTC CCGGACCCCT GAGGTCACGT CAGGAAGACC CCGAGGTCCA GTTCAACTGG GCATAATGCC AAGACAAAGC CGCGGGAGGA GTGTGGTCAG CGTCCTCACC GTCCTGCACC GAGTACAAGT GCAAGGTCTC CAACAAAGGC AACCATCTCC AAAGCCAAAG GGCAGCCCCG TGCCCCCATC CCAGGAGGAG ATGACCAAGA CTGGTCAAAG GCTTCTACCC CAGCGACATC TGGGCAGCCG GAGAACAACT ACAAGACCAC ACGGCTCCTT CTTCCTCTAC AGCAGGCTAA CAGGAGGGGA ATGTCTTCTC ATGCTCCGTG CCACTACACA CAGAAGAGCC TCTCCCTGTC

SEQ ID NO. 22

6.67.1 Predicted Heavy Chain Protein Sequence mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGDSISS

NYWSWIRQPA GKGLEWIGRI YTSGGTNSNP SLRGRVTILA DTSKNQFSLK

101 LSSVTAADTA VYYCARDRIT IIRGLIPSFF DYWGQGTLVT VSSASTKGPS

151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL

201 QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP

251 SCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW

301 YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG

351 LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI

401 AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV

451 MHEALHNHYT QKSLSLSLGK

SEQ ID NO. 23

6.67.1 Kappa Light Chain Nucléotide Sequence atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT

101 CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA

151 TACAGCTCCA ACAATAAGAC CTACTTAGCT TGGTACCAAC AGAAACCAAG

201 ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG

251 TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC

301 ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTTCT GTCAACAATA

351 TTATAGTATT CCTCCCCTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA

401 AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG

451 CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA

501 TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG

551 GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC

601 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA

651 AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA

701 AGAGCTTCAA CAGGGGAGAG TGTTAGTGA

SEQ ID NO. 24

6.67.1 Predicted Kappa Light Chain Protein Sequence mvlqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL

YSSNNKTYLA WYQQKPRQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT

101 ISSLQAEDVA VYFCQQYYSI PPLTFGGGTK VEIKRTVAAP SVFIFPPSDE

151 QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY 201 SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

SEQ ID NO. 25

6.73.2 Heavy Chain Nucléotide Sequence

- 104 1

101

151

201

251

301

351

401

451

501

551

601

651

701

751

801

851

901

951

1001

1051

1101

1151

1201

1251

1301

1351

1401 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtGAG GTGCAGCTGT TGGAGTCTGG GGGAGACTTG GTCCAGCCTG GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTTAGAAGT TATGCCATGA ACTGGGTCCG ACAGGCTCCA GGGAAGGGGC TGGAGTGGGT CTCAGTTATT AGTGGTCGTG GTGGTACTAC ATACTACGCA GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG CAAATGAACA GCCTGAGAGC CGAGGACGCG GCCGTATATT ACTGTGCGAA GATAGCAGTG GCTGGAGAGG GGCTCTACTA CTACTACGGT ATGGACGTCT GGGGCCAAGG GACCACGGTC ACCGTCTCCT CAGCTTCCAC CAAGGGCCCA TCCGTCTTCC CCCTGGCGCC CTGCTCCAGG AGCACCTCCG AGAACACAGC CGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG GTGACGGTGT CGTGGAACTC AGGCGCCCTG ACCAGCGGCG TGCACACCTT CCCGGCTGTC CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC TAGCAGCTTG GGCACGAAGA CCTACACCTG CAACGTAGAT CACAAGCCCA GCAACACCAA GGTGGACAAG AGAGTTGAGT CCAAATATGG TCCCCCATGC CCATCATGCC CAGCACCTGA GTTCCTGGGG GGACCATCAG TCTTCCTGTT CCCCCCAAAA CCCAAGGACA CTCTCATGAT CTCCCGGACC CCTGAGGTCA CGTGCGTGGT GGTGGACGTG AGCCAGGAAG ACCCCGAGGT CCAGTTCAAC TGGTACGTGG ATGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTTC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GGCCTCCCGT CCTCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAGCCA CAGGTGTACA CCCTGCCCCC ATCCCAGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAGGC TAACCGTGGA CAAGAGCAGG TGGCAGGAGG GGAATGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACACAGAAGA GCCTCTCCCT GTCTCTGGGT AAATGATAG

SEQ ID NO. 26

6.73.2 Predicted Heavy Chain Protein Sequence mefglswlfl vailkgvqcE VQLLESGGDL VQPGGSLRLS CAASGFTFRS

YAMNWVRQAP GKGLEWVSVI SGRGGTTYYA DSVKGRFTIS RDNSKNTLYL

101 QMNSLRAEDA AVYYCAKIAV AGEGLYYYYG MDVWGQGTTV TVSSASTKGP

151 SVFPLAPCSR STSENTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV

201 LQSSGLYSLS SVVTVPSSSL GTKTYTCNVD HKPSNTKVDK RVESKYGPPC

251 PSCPAPEFLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN

301 WYVDGVEVHN AKTKPREEQF NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK

351 GLPSSIEKTI SKAKGQPREP QVYTLPPSQE EMTKNQVSLT CLVKGFYPSD

401 IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSRLTVDKSR WQEGNVFSCS

451 VMHEALHNHY TQKSLSLSLG K

SEQ ID NO. 27

6.73.2 Kappa Light Chain Nucléotide Sequence atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT

101 CTGCATCTGT AGGTGACAGA GTCACCTTCA CTTGCCGGGC AAGTCAGAAC

151 ATTACCAACT ATTTAAATTG GTATCAGCAG AAACCAGGGA AGGCCCCTAA

201 GCTCCTGATC TATGCTGCGT CCAGTTTGCC AAGAGGGGTC CCATCAAGGT

251 TCCGTGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGTCTG

301 CAACCTGAAG ATTTTGCAAC TTACTACTGT CAACAGAGTT ACAGTAATCC

351 TCCGGAGTGC GGTTTTGGCC AGGGGACCAC GCTGGATATC AAACGAACTG

401 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA

451 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA

501 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC

551 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC

601 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC

651 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA

- 105 701 ACAGGGGAGA GTGTTAGTGA

SEQ ID NO. 28

6.73.2 Predicted Kappa Light Chain Protein Sequence

101 151 201 mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTFTCRASQN ITNYLNWYQQ KPGKAPKLLI YAASSLPRGV PSRFRGSGSG TDFTLTISSL QPEDFATYYC QQSYSNPPEC GFGQGTTLDI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO. 29

6.77.1 Heavy Chain Nucléotide Sequence atggaactgg ggctccgctg ggttttcctt gttgctattt tagaaggtgt ccagtgtGAG GTGCAGCTGG TGGAGTCTGG GGGAGGCCTG GTCAAGCCTG

101 GGGGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTCAGTAGC

151 TATAGCATGA ACTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT

201 CTCATCCATT AGTAGTAGTA GTAGTTACAT ATACTACGCA GACTCAGTGA

251 AGGGCCGATT CACCATCTCC AGAGACAACG CCAAGAACTC ACTGTATCTG

301 CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG

351 AGATGGGTAT AGCAGTGGCT GGTCCTACTA CTACTACTAC GGTATGGACG

401 TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGCTTC CACCAAGGGC

451 CCATCCGTCT TCCCCCTGGC GCCCTGCTCC AGGAGCACCT CCGAGAGCAC

501 AGCCGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG

551 TGTCGTGGAA CTCAGGCGCC CTGACCAGCG GCGTGCACAC CTTCCCGGCT

601 GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC

651 CTCCAGCAGC TTGGGCACGA AGACCTACAC CTGCAACGTA GATCACAAGC

701 CCAGCAACAC CAAGGTGGAC AAGAGAGTTG AGTCCAAATA TGGTCCCCCA

751 TGCCCATCAT GCCCAGCACC TGAGTTCCTG GGGGGACCAT CAGTCTTCCT

801 GTTCCCCCCA AAACCCAAGG ACACTCTCAT GATCTCCCGG ACCCCTGAGG

851 TCACGTGCGT GGTGGTGGAC GTGAGCCAGG AAGACCCCGA GGTCCAGTTC

901 AACTGGTACG TGGATGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG

951 GGAGGAGCAG TTCAACAGCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC

1001 TGCACCAGGA CTGGCTGAAC GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC

1051 AAAGGCCTCC CGTCCTCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA

1101 GCCCCGAGAG CCACAGGTGT ACACCCTGCC CCCATCCCAG GAGGAGATGA

1151 CCAAGAACCA GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTACCCCAGC

1201 GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA

1251 GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA

1301 GGCTAACCGT GGACAAGAGC AGGTGGCAGG AGGGGAATGT CTTTTCACGC

1351 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACACAGA AGAGCCTCTC

1401 CCTGTCTCTG GGTAAATGAT AGGAATTCTG ATGA

SEQ ID NO. 30

6.77.1 Predicted Heavy Chain Protein Sequence melglrwvfl vailegvqcE VQLVESGGGL VKPGGSLRLS CAASGFTFSS

YSMNWVRQAP GKGLEWVSSI SSSSSYIYYA DSVKGRFTIS RDNAKNSLYL 101 QMNSLRAEDT AVYYCARDGY SSGWSYYYYY GMDVWGQGTT VTVSSASTKG 151 PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 201 VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP 251 CPSCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF 301 NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 351 KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS 401 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSR 451 SVMHEALHNH YTQKSLSLSL GK

SEQ ID NO. 31

- 106 -

6.77.1 Kappa Light Chain Nucléotide Sequence atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg atccagtgca GATATTGTGA

101 CTCCTGGACA GCCGGCCTCC

151 CTTAGTGATG GAAAGACCTA

201 GCCTCCACAG CTCCTGATCT

251 CAGACAGGTT CAGTGGCAGC

301 AGCCGGGTGG AGGCTGAGGA

351 ACAGCTTATG TGCAGTTTTG

401 CTGTGGCTGC ACCATCTGTC

451 AAATCTGGAA CTGCCTCTGT

501 AGAGGCCAAA GTACAGTGGA

551 CCCAGGAGAG TGTCACAGAG

601 AGCAGCACCC TGACGCTGAG

651 CGCCTGCGAA GTCACCCATC

701 TCAACAGGGG AGAGTGTTAG

TGACCCAGAC TCCACTCTCT CTGTCCGTCA ATCTCCTGCA ACTCTAGTCA GAGCCTCCTG TTTGAATTGG TACCTGCAGA AGCCCGGCCA ATGAAGTTTC CAACCGGTTC TCTGGAGTGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC TGTTGGGGTT TATTCCTGCA TGCAAAGTAT GCCAGGGGAC CAAGCTGGAG ATCAAACGAA TTCATCTTCC CGCCATCTGA TGAGCAGTTG TGTGTGCCTG CTGAATAACT TCTATCCCAG AGGTGGATAA CGCCCTCCAA TCGGGTAACT CAGGACAGCA AGGACAGCAC CTACAGCCTC CAAAGCAGAC TACGAGAAAC ACAAAGTCTA AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT T GA

SEQ ID NO. 32

6.77.1 Predicted Kappa Light Chain Protein Sequence

101

151

201 mrlpaqllgl Imlwipgssa

LSDGKTYLNW SRVEAEDVGV KSGTASVVCL SSTLTLSKAD

YLQKPGQPPQ YSCMQSIQLM LNNFYPREAK YEKHKVYACE

DIVMTQTPLS LLIYEVSNRF CSFGQGTKLE VQWKVDNALQ VTHQGLSSPV

LSVTPGQPAS SGVPDRFSGS IKRTVAAPSV SGNSQESVTE TKSFNRGEC

ISCNSSQSLL GSGTDFTLKI FIFPPSDEQL QDSKDSTYSL

SEQ ID NO. 33

7.16.6 Heavy Chain Nucléotide Sequence atggactgga cctggagcat ccactccCAG GTTCAGCTGG

101 GGGCCTCAGT GAAGGTCTCC

151 TATGGTATCA ACTGGGTGCG

201 GGGATGGATC AGCGTTTACA

251 AGGGCAGAGT CACCATGACC

301 GACCTGAGGA GCCTGAGATC

351 AGAGGGTAGC AGCTCGTCCG

401 GCCAAGGGAC CACGGTCACC

451 GTCTTCCCCC TGGCGCCCTG

501 CCTGGGCTGC CTGGTCAAGG

551 GGAACTCAGG CGCTCTGACC

601 CAGTCCTCAG GACTCTACTC

651 CAACTTCGGC ACCCAGACCT

701 ACACCAAGGT GGACAAGACA

751 CCGTGCCCAG CACCACCTGT

801 AAAACCCAAG GACACCCTCA

851 TGGTGGTGGA CGTGAGCCAC

901 GTGGACGGCG TGGAGGTGCA

951 GTTCAACAGC ACGTTCCGTG

1001 ACTGGCTGAA CGGCAAGGAG

1051 CCAGCCCCCA TCGAGAAAAC

1101 ACCACAGGTG TACACCCTGC

1151 AGGTCAGCCT GACCTGCCTG

1201 GTGGAGTGGG AGAGCAATGG

1251 TCCCATGCTG GACTCCGACG

1301 TGGACAAGAG CAGGTGGCAG

1351 CATGAGGCTC TGCACAACCA

1401 GGGTAAATGA

SEQ ID NO. 34 ccttttcttg gtggcagcag caacaggtgc TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG TGCAAGGCTT CTGGTTACAC CTTTACCAGC ACAGGCCCCT GGACAAGGGC TTGAGTGGAT GTGGTAACAC AAACTATGCA CAGAAGGTCC GCAGACACAT CCACGAGCAC AGCCTACATG TGACGACACG GCCGTGTATT ACTGTGCGAG GAGACTACTA TTACGGTATG GACGTCTGGG GTCTCCTCAG CCTCCACCAA GGGCCCATCG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC ACTACTTCCC CGAACCGGTG ACGGTGTCGT AGCGGCGTGC ACACCTTCCC AGCTGTCCTA CCTCAGCAGC GTGGTGACCG TGCCCTCCAG ACACCTGCAA CGTAGATCAC AAGCCCAGCA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA GGCAGGACCG TCAGTCTTCC TCTTCCCCCC TGATCTCCCG GACCCCTGAG GTCACGTGCG GAAGACCCCG AGGTCCAGTT CAACTGGTAC TAATGCCAAG ACAAAGCCAC GGGAGGAGCA TGGTCAGCGT CCTCACCGTT GTGCACCAGG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA CCCCATCCCG GGAGGAGATG ACCAAGAACC GTCAAAGGCT TCTACCCCAG CGACATCGCC GCAGCCGGAG AACAACTACA AGACCACACC GCTCCTTCTT CCTCTACAGC AAGCTCACCG CAGGGGAACG TCTTCTCATG CTCCGTGATG CTACACGCAG AAGAGCCTCT CCCTGTCTCC

- 107 -

7.16.6 Predicted Heavy Chain Protein Sequence mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS

YGINWVRQAP GQGLEWMGWI SVYSGNTNYA QKVQGRVTMT ADTSTSTAYM

101 DLRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS

151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL

201 QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP

251 PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY

301 VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL

351 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA

401 VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM

451 HEALHNHYTQ KSLSLSPGK

SEQ ID NO. 35

7.16.6 Kappa Light Chain Nucléotide Sequence and X481.2 Kappa Light Chain Nucléotide Sequence

1 atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg atccagtgca GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA

101 CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG

151 CATACTGATG GAACGACCTA TTTGTATTGG TACCTGCAGA AGCCAGGCCA

201 GCCTCCACAG CTCCTGATCT ATGAAGTTTC CAACCGGTTC TCTGGAGTGC

251 CAGATAGGTT CAGTGGCAGC GGGTCAGGGA CAGATTTCAC ACTGAAAATC

301 AGCCGGGTGG AGGCTGAGGA TGTTGGGATT TATTACTGCA TGCAAAATAT

351 ACAGCTTCCG TGGACGTTCG GCCAAGGGAC CAAGGTGGAA ATCAAACGAA

401 CTGTGGCTGC ACCATCTGTC TTCATCTTCC CGCCATCTGA TGAGCAGTTG

451 AAATCTGGAA CTGCCTCTGT TGTGTGCCTG CTGAATAACT TCTATCCCAG

501 AGAGGCCAAA GTACAGTGGA AGGTGGATAA CGCCCTCCAA TCGGGTAACT

551 CCCAGGAGAG TGTCACAGAG CAGGACAGCA AGGACAGCAC CTACAGCCTC

601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA

651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT

01 TCAACAGGGG AGAGTGTTAG TGA

SEQ ID NO. 36

7.16.6 Kappa Light Chain Protein Sequence mrlpaqllgl Imlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL

HTDGTTYLYW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI

101 SRVEAEDVGI YYCMQNIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL

151 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL

201 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

SEQ ID NO. 37

7.20.5 Heavy Chain Nucléotide Sequence

1	atgaaacacc	tgtggttctt	cctcctgctg	gtggcagctc	ccagatgggt
51	cctgtccCAG	GTGCAGCTGG	AGGAGTCGGG	CCCAGGACTG	GTGAAGCCTT
101	CGGAGACCCT	GTCCCTCACC	TGCACTGTCT	CTGGTAGCTC	CATCAGTAGT
151	TACCACTGGA	ACTGGATCCG	GCAGCCCGCC	GGGAAGGGAC	TGGAGTGGAT
201	TGGGCGTATC	TATACCAGTG	GGAGCACCAA	CTACAACCCC	TCCCTCAAGA
251	GTCGAGTCAC	CATGTCACTA	GACACGTCCA	AGAACCAGTT	CTCCCTGAAG
301	CTGAGCTCTG	TGACCGCCGC	GGACACGGCC	GTGTATTACT	GTGCGAGAGA
351	GGGGGTCAGG	TATTACTATG	CTTCGGGGAG	TTATTACTAC	GGTCTGGACG
401	TCTGGGGCCA	AGGGACCACG	GTCACCGTCT	CCTCAGCCTC	CACCAAGGGC
451	CCATCGGTCT	TCCCCCTGGC	GCCCTGCTCC	AGGAGCACCT	CCGAGAGCAC
501	AGCGGCCCTG	GGCTGCCTGG	TCAAGGACTA	CTTCCCCGAA	CCGGTGACGG
551	TGTCGTGGAA	CTCAGGCGCT	CTGACCAGCG	GCGTGCACAC	CTTCCCAGCT
601	GTCCTACAGT	CCTCAGGACT	CTACTCCCTC	AGCAGCGTGG	TGACCGTGCC
651	CTCCAGCAAC	TTCGGCACCC	AGACCTACAC	CTGCAACGTA	GATCACAAGC

- 108 701 CCAGCAACAC CAAGGTGGAC AAGACAGTTG AGCGCAAATG TTGTGTCGAG

751 TGCCCACCGT GCCCAGCACC ACCTGTGGCA GGACCGTCAG TCTTCCTCTT

801 CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA

851 CGTGCGTGGT GGTGGACGTG AGCCACGAAG ACCCCGAGGT CCAGTTCAAC

901 TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCACGGGA

951 GGAGCAGTTC AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC

1001 ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA

1051 GGCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC

1101 CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA

1151 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC

1201 ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC

1251 CACACCTCCC ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC

1301 TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC

1351 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT

1401 GTCTCCGGGT AAATGA

SEQ ID NO. 38

7.20.5 Predicted Heavy Chain Protein Sequence mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGSSISS

YHWNWIRQPA GKGLEWIGRI YTSGSTNYNP SLKSRVTMSL DTSKNQFSLK

101 LSSVTAADTA VYYCAREGVR YYYASGSYYY GLDVWGQGTT VTVSSASTKG

151 PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA

201 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE

251 CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN

301 WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK

351 GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD

401 IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS

451 VMHEALHNHY TQKSLSLSPG K

SEQ ID NO. 39

7.20.5 Kappa Light Chain Nucléotide Sequence

1 atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtctctgg atccagtggg GATATTGTGA TGACTCAGTC TCCACTCTCC CTGCCCGTCA

101 CCCCTGGAGA GCCGGCCTCC ATCTCCTGCA GGTCTAGTCA GAGCCTCCTG

151 CATGGTAATG GATACAACTA TTTGGATTGG TACCTGCAGA AGCCAGGGCA

201 GTCTCCACAG CTCCTGATCT ATTTGGGTTC TAATCGGGCC TCCGGGGTCC

251 CTGACAGGTT CAGTGGCAGT GGATCAGGCA CAGATTTTAC ACTGAAAATC

301 AGCAGAGTGG AGGCTGAGGA TGTTGGGGTT TATTACTGCA TGCAAGCTCT

351 ACAAACTCTC ACTTTCGGCG GAGGGACCAA GGTGGAGATC AAACGAACTG

401 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA

451 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA

501 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC

551 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC

601 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC

651 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA

701 ACAGGGGAGA GTGTTAGTGA

SEQ ID NO. 40

7.20.5 Predicted Kappa Light Chain Protein Sequence mrlpagllgl Imlwvsgssg DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL

HGNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI

101 SRVEAEDVGV YYCMQALQTL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK

151

201

SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO. 41

- 109 -

7.26.4 Heavy Chain Nucléotide Sequence atggactgga cctggagcat ccactccCAG GTTCAGCTGG

101 GGGCCTCAGT GAAGGTCTCC

151 TATGGTATCG ACTGGGTGCG

201 GGGATGGATC AGCGTTTACA

251 AGGGCAGAGT CACCATGTCC

301 GAGCTGAGGA GCCTGAGATC

351 AGAGGGTAGC AGCTCGTCCG

401 GCCAAGGGAC CACGGTCACC

451 GTCTTCCCCC TGGCGCCCTG

501 CCTGGGCTGC CTGGTCAAGG

551 GGAACTCAGG CGCTCTGACC

601 CAGTCCTCAG GACTCTACTC

651 CAACTTCGGC ACCCAGACCT

701 ACACCAAGGT GGACAAGACA

751 CCGTGCCCAG CACCACCTGT

801 AAAACCCAAG GACACCCTCA

851 TGGTGGTGGA CGTGAGCCAC

901 GTGGACGGCG TGGAGGTGCA

951 GTTCAACAGC ACGTTCCGTG

1001 ACTGGCTGAA CGGCAAGGAG

1051 CCAGCCCCCA TTGAGAAAAC

1101 ACCACAGGTG TACACCCTGC

1151 AGGTCAGCCT GACCTGCCTG

1201 GTGGAGTGGG AGAGCAATGG

1251 TCCCATGCTG GACTCCGACG

1301 TGGACAAGAG CAGGTGGCAG

1351 CATGAGGCTC TGCACAACCA

1402 GGGTAAATGA

SEQ ID NO. 42 ccttttcttg gtggcagcag caacaggtgc TGCAGTCTGG AGCTGAGGTG AAGAAGCCTG TGCGAGGCTT CTGGTTACAC CTTTACCAGC ACAGGCCCCT GGACAAGGGC TTGAGTGGAT GTGGTAACAC AAACTATGCA CAGAAGCTCC ACAGACACAT CCACGAGCAC AGCCTACATG TGACGACACG GCCGTGTATT ACTGTGCGAG GAGACTACTA CTACGGTATG GACGTCTGGG GTCTCCTCAG CCTCCACCAA GGGCCCATCG CTCCAGGAGC ACCTCCGAGA GCACAGCGGC ACTACTTCCC CGAACCGGTG ACGGTGTCGT AGCGGCGTGC ACACCTTCCC AGCTGTCCTA CCTCAGCAGC GTGGTGACCG TGCCCTCCAG ACACCTGCAA CGTAGATCAC AAGCCCAGCA GTTGAGCGCA AATGTTGTGT CGAGTGCCCA GGCAGGACCG TCAGTCTTCC TCTTCCCCCC TGATCTCCCG GACCCCTGAG GTCACGTGCG GAAGACCCCG AGGTCCAGTT CAACTGGTAC TAATGCCAAG ACAAAGCCAC GGGAGGAGCA TGGTCAGCGT CCTCACCGTT GTGCACCAGG TACAAGTGCA AGGTCTCCAA CAAAGGCCTC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA CCCCATCCCG GGAGGAGATG ACCAAGAACC GTCAAAGGCT TCTACCCCAG CGACATCGCC GCAGCCGGAG AACAACTACA AGACCACACC GCTCCTTCTT CCTCTACAGC AAGCTCACCG CAGGGGAACG TCTTCTCATG CTCCGTGATG CTACACGCAG AAGAGCCTCT CCCTGTCTCC

7.26.4 Predicted Heavy Chain Protein Sequence mdwtwsilfl vaaatgahsQ VQLVQSGAEV KKPGASVKVS CEASGYTFTS

YGIDWVRQAP GQGLEWMGWI SVYSGNTNYA QKLQGRVTMS TDTSTSTAYM 101 ELRSLRSDDT AVYYCAREGS SSSGDYYYGM DVWGQGTTVT VSSASTKGPS 151 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL 201 QSSGLYSLSS VVTVPSSNFG TQTYTCNVDH KPSNTKVDKT VERKCCVECP 251 PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY 301 VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL 351 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA 401 VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM 451 HEALHNHYTQ KSLSLSPGK

SEQ ID NO. 43

7.26.4 Kappa Light Chain Nucléotide Sequence

1	atgaggctcc	ctgctcagct	cctggggctg	ctaatgctct	ggatacctgg
51	atccagtgcg	GATAT T GT GA	TGAGCCAGAC	TCCACTCTCT	CTGTCCGTCA
101	CCCCTGGACA	GCCGGCCTCC	ATCTCCTGCA	AGTCTAATCA	GAGCCTCCTG
151	TATAGTGATG	GAAAGACCTA	TTTGTTTTGG	TACCTGCAGA	AGCCAGGCCA
201	GCCTCCACAG	CTCCTGATCT	ATGAAGTTTC	CAACCGATTC	TCTGGAGTGC
251	CAGATAGGTT	CAGTGGCAGC	GGGTCAGGGA	CAGATTTCAC	ACTGAAAATC
301	AGCCGGGTGG	AGGCTGAGGA	TGTTGGGGTT	TATTACTGCA	TGCAAAGTAT
351	ACAGCTTCCG	TGGACGTTCG	GCCAAGGGAC	CAAGGTGGAA	ATCAAACGAA
401	CTGTGGCTGC	ACCATCTGTC	TTCATCTTCC	CGCCATCTGA	TGAGCAGTTG
451	AAATCTGGAA	CTGCCTCTGT	TGTGTGCCTG	CTGAATAACT	TCTATCCCAG
501	AGAGGCCAAA	GTACAGTGGA	AGGTGGATAA	CGCCCTCCAA	TCGGGTAACT
551	CCCAGGAGAG	TGTCACAGAG	CAGGACAGCA	AGGACAGCAC	CTACAGCCTC

- 110601 AGCAGCACCC TGACGCTGAG CAAAGCAGAC TACGAGAAAC ACAAAGTCTA

651 CGCCTGCGAA GTCACCCATC AGGGCCTGAG CTCGCCCGTC ACAAAGAGCT

701 TCAACAGGGG AGAGTGTTAG TGA

SEQ ID NO. 44

7.26.4 Predicted Kappa Light Chain Protein Sequence

101 151 201 mrlpaqllgl Imlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSNQSLL YSDGKTYLFW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

SEQ ID NO. 45

9.8.2 Heavy Chain Nucléotide Sequence

1	atggagtttg	ggctgagctg	ggttttcctc	gttgctcttt	taagaggtgt
51	ccagtgtCAG	GTGCAGCTGG	TGGAGTCTGG	GGGAGGCGTG	GTCCAGCCTG
101	GGAGGTCCCT	GAGACTCTCC	TGTGCAGCGT	CTGGATTCAC	CTTCAGTAGC
151	TATGGCATGC	ACTGGGTCCG	CCAGGCTCCA	GGCAAGGGGC	TGGAGTGGGT
201	GGCAGTTATA	TGGTATGATG	GAAGTAATGA	ATACTATGCA	GACTCCGTGA
251	AGGGCCGATT	CACCATCTCC	AGAGACAATT	CCAAGAACAC	GCTGTATCTG
301	CAAATGAACA	GCCTGAGAGC	CGAGGACACG	GCTGTGTATT	ACTGTGCGAG
351	GGGGGCGTAC	CACTTTGCCT	ACTGGGGCCA	GGGAACCCTG	GTCACCGTCT
401	CCTCAGCTTC	CACCAAGGGC	CCATCCGTCT	TCCCCCTGGC	GCCCTGCTCC
451	AGGAGCACCT	CCGAGAGCAC	AGCCGCCCTG	GGCTGCCTGG	TCAAGGACTA
501	CTTCCCCGAA	CCGGTGACGG	TGTCGTGGAA	CTCAGGCGCC	CTGACCAGCG
551	GCGTGCACAC	CTTCCCGGCT	GTCCTACAGT	CCTCAGGACT	CTACTCCCTC
601	AGCAGCGTGG	TGACCGTGCC	CTCCAGCAGC	TTGGGCACGA	AGACCTACAC
651	CTGCAACGTA	GATCACAAGC	CCAGCAACAC	CAAGGTGGAC	AAGAGAGTTG
701	AGTCCAAATA	TGGTCCCCCA	TGCCCATCAT	GCCCAGCACC	TGAGTTCCTG
751	GGGGGACCAT	CAGTCTTCCT	GTTCCCCCCA	AAACCCAAGG	ACACTCTCAT
801	GATCTCCCGG	ACCCCTGAGG	TCACGTGCGT	GGTGGTGGAC	GTGAGCCAGG
851	AAGACCCCGA	GGTCCAGTTC	AACTGGTACG	TGGATGGCGT	GGAGGTGCAT
901	AATGCCAAGA	CAAAGCCGCG	GGAGGAGCAG	TTCAACAGCA	CGTACCGTGT
951	GGTCAGCGTC	CTCACCGTCC	TGCACCAGGA	CTGGCTGAAC	GGCAAGGAGT
1001	ACAAGTGCAA	GGTCTCCAAC	AAAGGCCTCC	CGTCCTCCAT	CGAGAAAACC
1051	ATCTCCAAAG	CCAAAGGGCA	GCCCCGAGAG	CCACAGGTGT	ACACCCTGCC
1101	CCCATCCCAG	GAGGAGATGA	CCAAGAACCA	GGTCAGCCTG	ACCTGCCTGG
1151	TCAAAGGCTT	CTACCCCAGC	GACATCGCCG	TGGAGTGGGA	GAGCAATGGG
1201	CAGCCGGAGA	ACAACTACAA	GACCACGCCT	CCCGTGCTGG	ACTCCGACGG
1251	CTCCTTCTTC	CTCTACAGCA	GGCTAACCGT	GGACAAGAGC	AGGTGGCAGG
1301	AGGGGAATGT	CTTCTCATGC	TCCGTGATGC	ATGAGGCTCT	GCACAACCAC
1351	TACACACAGA	AGAGCCTCTC	CCTGTCTCTG	GGTAAATGA

SEQ ID NO. 46

9.8.2 Predicted Heavy Chain Chain Protein Sequence meJjgjLswyfJ^yaJJ^rgyqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS

YGMHWVRQAP GKGLEWVAVI WYDGSNEYYA DSVKGRFTIS RDNSKNTLYL 101 QMNSLRAEDT AVYYCARGAY HFAYWGQGTL VTVSSASTKG PSVFPLAPCS

151 RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL

201 SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPSCPAPEFL

251 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH

301 NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT

351 ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG

401 QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH

451 YTQKSLSLSL GK

SEQ ID NO. 47

- 111 -

9.8.2 Kappa Light Chain Nucléotide Sequence atggacatga gggtccctgc tcagctcctg gggctcctgc tgctctggct ctcagtcgca ggtgccagat gtGACATCCA GATGACCCAG TCTCCATCCT

101 CCCTGTCTGC ATCTGTAGGA GACAGAGTCA CCATCACTTG CCAGGCGAGT

151 CAGGACATTA GCAACTATTT AAATTGGTAT CAGCAGAAAC CAGGGAAAGC

201 CCCTAAGCTC CTGATCTACG ATGCATCCAA TTTGGAAACA GGGGTCCCAT

251 CAAGGTTCAG TGGAAGTGGA TCTGGGACAG ATTTTACTTT CACCATCAGC

301 AGCCTGCAGC CTGAAGATAT TGCAACATAT TCCTGTCAAC ACTCTGATAA

351 TCTCTCGATC ACCTTCGGCC AGGGGACACG ACTGGAGATT AAACGAACTG

401 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA

451 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ACCCCAGAGA

501 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC

551 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC

601 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC

651 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA

701 ACAGGGGAGA GTGTTAGTGA

SEQ ID NO. 48

9.8.2 Predicted Kappa Light Chain Protein Sequence mdmrvpaqll gllllwlsva garcDIQMTQ SPSSLSASVG DRVTITCQAS

QDISNYLNWY QQKPGKAPKL LIYDASNLET GVPSRFSGSG SGTDFTFTIS

101 SLQPEDIATY SCQHSDNLSI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK

151 SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS

201 STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO. 49

Nucléotide Sequence of cynomolgus MAdCAM Ο4β7 binding domain

ATGGATCGGG GCCTGGCCCT CCTGCTGGCG GGGCTTCTGG GGCTCCTCCA

GCCGGGCTGC GGCCAGTCCC

101 CGGAGCCGGT GGTGGCCGTG

151 CGCCTGGACT GCGCGGACGG

201 CACCAGCCTG GGCGCGGTGC

251 TGCGCAACGC CTCGCTGTCG

301 TGCGGGGGCC GCACCTTCCA

351 CCCGGACCAG CTGACCATCT

401 AGGTGGCCTG TACGGCTCAC

451 TCCTTCTCCC TGCTCCTGGG

501 GGGCCCGGAG GTGGAGGAGG

551 TGTTCAGGGT GACAGAGCGC

601 CTGCCCGCGC TCTACTGCCA

651 CAGCCACCGC CAGGCCATCC

SEQ ID NO. 50

TCCAGGTGAA GCCCCTGCAG GTGGAGCCCC GCCCTGGGCG CCTCTCGCCA GCTCACCTGC CGGGGCCACG GTGCAGTGGC GGGGCCTGGA AGTCGGACGC GGGCCGCAGC GTCCTCACCG GCGGCCGGGA CCCGTGTGTG CGTGGGCTCC GCACACCGTG CGGCTCCTTG TGTACGCCTT CCCCGGCAGC CCTGGTGCCT GGTGACCCGG AAAGTCACGC CTGTGGACCC CAATGCGCTC GGACCAGGAA CTGGAGGGGG CCCAGGCTCT AGGAGGAGCC CCAGGAGGAG GAGGACGTGC TGGCGGCTGC CGACCCTGGC AACCCCTGTC GGCCACGATG AGGCTGCCTG GCTTGGAGCT CGGTCCTGCA C

Amino acid sequence of cynomolgus MAdCAM ο^β? binding domain

MDRGLALLLA GLLGLLQPGC GQSLQVKPLQ VEPPEPVVAV ALGASRQLTC

RLDCADGGAT VQWRGLDTSL GAVQSDAGRS VLTVRNASLS AAGTRVCVGS

101 CGGRTFQHTV RLLVYAFPDQ LTISPAALVP GDPEVACTAH KVTPVDPNAL

151 SFSLLLGDQE LEGAQALGPE VEEEEEPQEE EDVLFRVTER WRLPTLATPV

201 LPALYCQATM RLPGLELSHR QAIPVLH

SEQ ID NO. 51

Modified 6.22.2 Heavy Chain Nucléotide Sequence atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt

- 112 -

51	ccagtgtCAG	GTGCAGCTGG	TGGAGTCTGG	GGGAGGCGTG	GTCCAGCCTG
101	GGAGGTCCCT	GAGACTCTCC	TGTGCAGCGT	CTGGATTCAC	CTTCAGTAGC
151	GATGGCATGC	ACTGGGTCCG	CCAGGCTCCA	GGCAAGGGGC	TGGAGTGGGT
201	GGCAATTATA	TGGTATGATG	GAAGTAATAA	ATATTATGCA	GACTCCGTGA
251	AGGGCCGATT	CACCATCTCC	AGAGACAATT	CCAAGAACAC	GCTGTATCTG
301	CAAATGAACA	GCCTGAGAGC	CGAGGACACG	GCTGTATATT	ACTGTGCGAG
351	AGATCCCGGC	TACTATTACG	GTATGGACGT	CTGGGGCCAA	GGGACCACGG
401	TCACCGTCTC	CTCAGCTTCC	ACCAAGGGCC	CATCCGTCTT	CCCCCTGGCG
451	CCCTGCTCTA	GAAGCACCTC	CGAGAGCACA	GCGGCCCTGG	GCTGCCTGGT
501	CAAGGACTAC	TTCCCCGAAC	CGGTGACGGT	GTCGTGGAAC	TCAGGCGCTC
551	TGACCAGCGG	CGTGCACACC	TTCCCAGCTG	TCCTACAGTC	CTCAGGACTC
601	TACTCCCTCA	GCAGCGTGGT	GACCGTGCCC	TCCAGCAACT	TCGGCACCCA
651	GACCTACACC	TGCAACGTAG	ATCACAAGCC	CAGCAACACC	AAGGTGGACA
701	AGACAGTTGA	GCGCAAATGT	TGTGTCGAGT	GCCCACCGTG	CCCAGCACCA
751	CCTGTGGCAG	GACCGTCAGT	CTTCCTCTTC	CCCCCAAAAC	CCAAGGACAC
801	CCTCATGATC	TCCCGGACCC	CTGAGGTCAC	GTGCGTGGTG	GTGGACGTGA
851	GCCACGAAGA	CCCCGAGGTC	CAGTTCAACT	GGTACGTGGA	CGGCGTGGAG
901	GTGCATAATG	CCAAGACAAA	GCCACGGGAG	GAGCAGTTCA	ACAGCACGTT
951	CCGTGTGGTC	AGCGTCCTCA	CCGTTGTGCA	CCAGGACTGG	CTGAACGGCA
1001	AGGAGTACAA	GTGCAAGGTC	TCCAACAAAG	GCCTCCCAGC	CCCCATCGAG
1051	AAAACCATCT	CCAAAACCAA	AGGGCAGCCC	CGAGAACCAC	AGGTGTACAC
1101	CCTGCCCCCA	TCCCGGGAGG	AGATGACCAA	GAACCAGGTC	AGCCTGACCT
1151	GCCTGGTCAA	AGGCTTCTAC	CCCAGCGACA	TCGCCGTGGA	GTGGGAGAGC
1201	AATGGGCAGC	CGGAGAACAA	CTACAAGACC	ACACCTCCCA	TGCTGGACTC
1251	CGACGGCTCC	TTCTTCCTCT	ACAGCAAGCT	CACCGTGGAC	AAGAGCAGGT
1301	GGCAGCAGGG	GAACGTCTTC	TCATGCTCCG	TGATGCATGA	GGCTCTGCAC
1351	AACCACTACA	CGCAGAAGAG	CCTCTCCCTG	TCTCCGGGTA	AATGATAG

SEQ ID NO. 52

Modified 6.22.2 Heavy Chain Amino Acid Sequence

	1	mefglswvf1	vallrgvqcQ	VQLVESGGGV	VQPGRSLRLS	CAASGFTFSS
	51	DGMHWVRQAP	GKGLEWVAII	WYDGSNKYYA	DSVKGRFTIS	RDNSKNTLYL
5	101	QMNSLRAEDT	AVYYCARDPG	YYYGMDVWGQ	GTTVTVSSAS	TKGPSVFPLA
	151	PCSRSTSEST	AALGCLVKDY	FPEPVTVSWN	SGALTSGVHT	FPAVLQSSGL
	201	YSLSSVVTVP	SSNFGTQTYT	CNVDHKPSNT	KVDKTVERKC	CVECPPCPAP
	251	PVAGPSVFLF	PPKPKDTLMI	SRTPEVTCVV	VDVSHEDPEV	QFNWYVDGVE
	301	VHNAKTKPRE	EQFNSTFRVV	SVLTVVHQDW	LNGKEYKCKV	SNKGLPAPIE
10	351	KTISKTKGQP	REPQVYTLPP	SREEMTKNQV	SLTCLVKGFY	PSDIAVEWES
	401	NGQPENNYKT	TPPMLDSDGS	FFLYSKLTVD	KSRWQQGNVF	SCSVMHEALH
	451	NHYTQKSLSL	SPGK

SEQ ID NO. 53

Modified 6.22.2 Kappa Light Chain Nucléotide Sequence

1 atqttqccat cacaactcat caqgggtGAA ATTGTGCTGA

101 CAAAAGAGAA AGTCACCATC

151 AGCTTACACT GGTACCAGCA

201 CAAGTATGCT TCCCAGTCCT

251 GTGGATCTGG GACAGATTTC

301 GATGCTGCAA CTTATTACTG

351 CGGCGGAGGG ACCAAGGTGG

401 TCTTCATCTT CCCGCCATCT

451 GTTGTGTGCC TGCTGAATAA

501 GAAGGTGGAT AACGCCCTCC

551 AGCAGGACAG CAAGGACAGC

601 AGCAAAGCAG ACTACGAGAA

651 TCAGGGCCTG AGCTCGCCCG

701 AGTGA tgggtttctg ctgctctggg ttccagcttc CTCAGTCTCC AGACTTTCAG TCTGTGACTC ACCTGCCGGG CCAGTCAGAG AATTGGTAGT GAAACCAGAT CAGTCTCCAA AACTCCTCAT TCTCAGGGGT CCCCTCGAGG TTCAGTGGCA ACCCTCACCA TCAATAGCCT GGAAGCTGAA TCATCAGAGT GGTCGTTTAC CGCTCACTTT AGATCAAACG AACTGTGGCT GCACCATCTG GATGAGCAGT TGAAATCTGG AACTGCCTCT CTTCTATCCC AGAGAGGCCA AAGTACAGTG AATCGGGTAA CTCCCAGGAG AGTGTCACAG ACCTACAGCC TCAGCAGCAC CCTGACGCTG ACACAAAGTC TACGCCTGCG AAGTCACCCA TCACAAAGAG CTTCAACAGG GGAGAGTGTT

SEQ ID NO. 54

- 113 Modifîed 6.22.2 Kappa Light Chain Amino Acid Sequence mlpsqligfl llwvpasrgE

SLHWYQQKPD QSPKLLIKYA

101 DAATYYCHQS GRLPLTFGGG

151 VVCLLNNFYP REAKVQWKVD

201 SKADYEKHKV YACEVTHQGL

SEQ ID NO. 55

IVLTQSPDFQ SVTPKEKVTI TCRASQRIGS SQSFSGVPSR FSGSGSGTDF TLTINSLEAE TKVEIKRTVA APSVFIFPPS DEQLKSGTAS NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SSPVTKSFNR GEC

Modifîed 6.34.2 Heavy Chain Nucléotide Sequence

1	atggagtttg	ggctgagctg	ggttttcctc	gttgctcttt	taagaggtgt
51	ccagtgtCAG	GTGCAGCTGG	TGGAGTCTGG	GGGAGGCGTG	GTCCAGCCTG
101	GGAGGTCCCT	GAGACTCTCC	TGTGCAGCCT	CTGGATTCAC	CTTCAGTAGC
151	TATGGCATGC	ACTGGGTCCG	CCAGGCTCCA	GGCAAGGGGC	TGGAGTGGGT
201	GGCAGTTATA	TCAAATGATG	GAAATAATAA	ATACTATGCA	GACTCCGTGA
251	AGGGCCGATT	CACCATCTCC	AGAGACAATT	CCAAAAACAC	GCTGTATCTG
301	CAAATGAACA	GCCTGCGCGC	TGAGGACACG	GCTGTGTATT	ACTGTGCGAG
351	AGATAGTACG	GCGATAACCT	ACTACTACTA	CGGAATGGAC	GTCTGGGGCC
401	AAGGGACCAC	GGTCACCGTC	TCCTCAGCTT	CCACCAAGGG	CCCATCCGTC
451	TTCCCCCTGG	CGCCCTGCTC	TAGAAGCACC	TCCGAGAGCA	CAGCGGCCCT
501	GGGCTGCCTG	GTCAAGGACT	ACTTCCCCGA	ACCGGTGACG	GTGTCGTGGA
551	ACTCAGGCGC	TCTGACCAGC	GGCGTGCACA	CCTTCCCAGC	TGTCCTACAG
601	TCCTCAGGAC	TCTACTCCCT	CAGCAGCGTG	GTGACCGTGC	CCTCCAGCAA
651	CTTCGGCACC	CAGACCTACA	CCTGCAACGT	AGATCACAAG	CCCAGCAACA
701	CCAAGGTGGA	CAAGACAGTT	GAGCGCAAAT	GTTGTGTCGA	GTGCCCACCG
751	TGCCCAGCAC	CACCTGTGGC	AGGACCGTCA	GTCTTCCTCT	TCCCCCCAAA
801	ACCCAAGGAC	ACCCTCATGA	TCTCCCGGAC	CCCTGAGGTC	ACGTGCGTGG
851	TGGTGGACGT	GAGCCACGAA	GACCCCGAGG	TCCAGTTCAA	CTGGTACGTG
901	GACGGCGTGG	AGGTGCATAA	TGCCAAGACA	AAGCCACGGG	AGGAGCAGTT
951	CAACAGCACG	TTCCGTGTGG	TCAGCGTCCT	CACCGTTGTG	CACCAGGACT
1001	GGCTGAACGG	CAAGGAGTAC	AAGTGCAAGG	TCTCCAACAA	AGGCCTCCCA
1051	GCCCCCATCG	AGAAAACCAT	CTCCAAAACC	AAAGGGCAGC	CCCGAGAACC
1101	ACAGGTGTAC	ACCCTGCCCC	CATCCCGGGA	GGAGATGACC	AAGAACCAGG
1151	TCAGCCTGAC	CTGCCTGGTC	AAAGGCTTCT	ACCCCAGCGA	CATCGCCGTG
1201	GAGTGGGAGA	GCAATGGGCA	GCCGGAGAAC	AACTACAAGA	CCACACCTCC
1251	CATGCTGGAC	TCCGACGGCT	CCTTCTTCCT	CTACAGCAAG	CTCACCGTGG
1301	ACAAGAGCAG	GTGGCAGCAG	GGGAACGTCT	TCTCATGCTC	CGTGATGCAT
1351	GAGGCTCTGC	ACAACCACTA	CACGCAGAAG	AGCCTCTCCC	TGTCTCCGGG
1401	TAAATGATAG

SEQ ID NO. 56

Modifîed 6.34.2 Heavy Chain Amino Acid Sequence mefglswvfl vallrgvqcQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS

YGMHWVRQAP GKGLEWVAVI SNDGNNKYYA DSVKGRFTIS RDNSKNTLYL

101 QMNSLRAEDT AVYYCARDST AITYYYYGMD VWGQGTTVTV SSASTKGPSV

151 FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ

201 SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP

251 CPAPPVAGPS_VRTRPREmÇD_Q?114ISRJTEyjrCVVVp^

301 DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP

351 APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV

401 EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH

451 EALHNHYTQK SLSLSPGK

SEQ ID NO. 57

Modifîed 6.34.2 Kappa Light Chain Nucléotide Sequence atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc agatgtGACA TCCAGATGAC CCAGTCTCCA TCCTCCCTGT

- 114101 CTGCATCTGT CGGAGACAGA

151 ATTAGTAGCT ATTTAAATTG

201 GCTCCTGATC TATGCTGCAT

251 TCAGTGGTAG TGGATCTGGG

301 CAACCTGAGG ATTTTGCAAC

351 ATTCACTTTC GGCCCTGGGA

401 CACCATCTGT CTTCATCTTC

451 ACTGCCTCTG TTGTGTGCCT

501 AGTACAGTGG AAGGTGGATA

551 GTGTCACAGA GCAGGACAGC

601 CTGACGCTGA GCAAAGCAGA

651 AGTCACCCAT CAGGGCCTGA

701 GAGAGTGTTA GTGA

GTCACCATCA CTTGCCGGGC AAGTCAGAGT GTATCAGCAG AAACCAGGGA AAGCCCCTAA CCGGTTTGAA GCGTGGGGTC CCATCACGGT ACAGATTTCA CTCTCACCAT CAGTTCTCTG TTACTACTGT CACCAGAGTT ACAGTCTCCC CCAAAGTGGA TATCAAACGA ACTGTGGCTG CCGCCATCTG ATGAGCAGTT GAAATCTGGA GCTGAATAAC TTCTATCCCA GAGAGGCCAA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA AAGGACAGCA CCTACAGCCT CAGCAGCACC CTACGAGAAA CACAAAGTCT ACGCCTGCGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG

SEQ ID NO. 58

Modified 6.34.2 Kappa Light Chain Amino Acid Sequence mdmrvpaqll gllllwlrga rcDIQMTQSP SSLSASVGDR VTITCRASQS

ISSYLNWYQQ KPGKAPKLLI YAASGLKRGV PSRFSGSGSG TDFTLTISSL

101 QPEDFATYYC HQSYSLPFTF GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG

151 TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST

201 LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO. 59

Modified 6.67.1 Heavy Chain Nucléotide Sequence

1	atgaaacacc	tgtggttctt	cctcctgctg	gtggcagctc	ccagatgggt
51	cctgtccCAG	GTGCAGCTGC	AGGAGTCGGG	CCCAGGACTG	GTGAAGCCTT
101	CGGAGACCCT	GTCCCTCACC	TGCACTGTCT	CTGGTGACTC	CATCAGTAGT
151	AACTATTGGA	GCTGGATCCG	GCAGCCCGCC	GGGAAGGGAC	TGGAGTGGAT
201	TGGGCGTATC	TATACCAGTG	GGGGCACCAA	CTCCAACCCC	TCCCTCAGGG
251	GTCGAGTCAC	CATGTCAGTA	GACACGTCCA	AGAACCAGTT	CTCTCTGAAA
301	CTGAGTTCTG	TGACCGCCGC	GGACACGGCC	GTGTATTACT	GTGCGAGAGA
351	TCGTATTACT	ATAATTCGGG	GACTTATTCC	ATCCTTCTTT	GACTACTGGG
401	GCCAGGGAAC	CCTGGTCACC	GTCTCCTCAG	CTTCCACCAA	GGGCCCATCC
451	GTCTTCCCCC	TGGCGCCCTG	CTCTAGAAGC	ACCTCCGAGA	GCACAGCGGC
501	CCTGGGCTGC	CTGGTCAAGG	ACTACTTCCC	CGAACCGGTG	ACGGTGTCGT
551	GGAACTCAGG	CGCTCTGACC	AGCGGCGTGC	ACACCTTCCC	AGCTGTCCTA
601	CAGTCCTCAG	GACTCTACTC	CCTCAGCAGC	GTGGTGACCG	TGCCCTCCAG
651	CAACTTCGGC	ACCCAGACCT	ACACCTGCAA	CGTAGATCAC	AAGCCCAGCA
701	ACACCAAGGT	GGACAAGACA	GTTGAGCGCA	AATGTTGTGT	CGAGTGCCCA
751	CCGTGCCCAG	CACCACCTGT	GGCAGGACCG	TCAGTCTTCC	TCTTCCCCCC
801	AAAACCCAAG	GACACCCTCA	TGATCTCCCG	GACCCCTGAG	GTCACGTGCG
851	TGGTGGTGGA	CGTGAGCCAC	GAAGACCCCG	AGGTCCAGTT	CAACTGGTAC
901	GTGGACGGCG	TGGAGGTGCA	TAATGCCAAG	ACAAAGCCAC	GGGAGGAGCA
951	GTTCAACAGC	ACGTTCCGTG	TGGTCAGCGT	CCTCACCGTT	GTGCACCAGG
1001	ACTGGCTGAA	CGGCAAGGAG	TACAAGTGCA	AGGTCTCCAA	CAAAGGCCTC
1051	CCAGCCCCCA	TCGAGAAAAC	CATCTCCAAA	ACCAAAGGGC	AGCCCCGAGA
1101	ACCACAGGTG	TACACCCTGC	CCCCATCCCG	GGAGGAGATG	ACCAAGAACC
1151	AGGTCAGCCT	GACCTGCCTG	GTCAAAGGCT	TCTACCCCAG	CGACATCGCC
1201	GTGGAGTGGG	AGAGCAATGG	GCAGCCGGAG	AACAACTACA	AGACCACACC
1251	TCCCATGCTG	GACTCCGACG	GCTCCTTCTT	CCTCTACAGC	AAGCTCACCG
1301	TGGACAAGAG	CAGGTGGCAG	CAGGGGAACG	TCTTCTCATG	CTCCGTGATG
1351	CATGAGGCTC	TGCACAACCA	CTACACGCAG	AAGAGCCTCT	CCCTGTCTCC
1401	GGGTAAATGA	TAG

SEQ ID NO. 60

Modified 6.67.1 Heavy Chain Amino Acid Sequence mkhlwfflll vaaprwvlsQ VQLQESGPGL VKPSETLSLT CTVSGDSISS

- 115 -

51 101 151	NYWSWIRQPA LSSVTAADTA VFPLAPCSRS	GKGLEWIGRI VYYCARDRIT TSESTAALGC	YTSGGTNSNP IIRGLIPSFF LVKDYFPEPV	SLRGRVTMSV DYWGQGTLVT TVSWNSGALT	DTSKNQFSLK VSSASTKGPS SGVHTFPAVL
201	QSSGLYSLSS	VVTVPSSNFG	TQTYTCNVDH	KPSNTKVDKT	VERKCCVECP
251	PCPAPPVAGP	SVFLFPPKPK	DTLMISRTPE	VTCVVVDVSH	EDPEVQFNWY
301	VDGVEVHNAK	TKPREEQFNS	TFRVVSVLTV	VHQDWLNGKE	YKCKVSNKGL
351	PAPIEKTISK	TKGQPREPQV	YTLPPSREEM	TKNQVSLTCL	VKGFYPSDIA
401	VEWESNGQPE	NNYKTTPPML	DSDGSFFLYS	KLTVDKSRWQ	QGNVFSCSVM
451	HEALHNHYTQ	KSLSLSPGK

SEQ ID NO. 61

Modified 6.67.1 Kappa Light Chain Nucléotide Sequence atggtgttgc agacccaggt cttcatttct ctgttgctct ggatctctgg 51 tgcctacggg GACATCGTGA TGACCCAGTC TCCAGACTCC CTGGCTGTGT

101 CTCTGGGCGA GAGGGCCACC ATCAACTGCA AGTCCAGCCA GAGTGTTTTA 151 TACAGCTCCA ACAATAAGAA CTACTTAGCT TGGTACCAAC AGAAACCAGG 201 ACAGCCTCCT AAATTGCTCA TTTACTGGGC ATCTATACGG GAATATGGGG 251 TCCCTGACCG ATTCAGTGGC AGCGGGTCTG GGACAGATTT CACTCTCACC 301 ATCAGCAGCC TGCAGGCTGA AGATGTGGCA GTTTATTTCT GTCAACAATA 351 TTATAGTATT CCTCCCCTCA CTTTCGGCGG AGGGACCAAG GTGGAGATCA 401 AACGAACTGT GGCTGCACCA TCTGTCTTCA TCTTCCCGCC ATCTGATGAG 451 CAGTTGAAAT CTGGAACTGC CTCTGTTGTG TGCCTGCTGA ATAACTTCTA 501 TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC CTCCAATCGG 551 GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC 601 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA 651 AGTCTACGCC TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA 701 AGAGCTTCAA CAGGGGAGAG TGTTAGTGA

SEQ ID NO. 62

Modified 6.67.1 Kappa Light Chain Amino Acid Sequence mvlqtqvfis lllwisgayg DIVMTQSPDS LAVSLGERAT INCKSSQSVL

YSSNNKNYLA WYQQKPGQPP KLLIYWASIR EYGVPDRFSG SGSGTDFTLT

101 ISSLQAEDVA VYFCQQYYSI PPLTFGGGTK VEIKRTVAAP SVFIFPPSDE

151 QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY

201 SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

SEQ ID NO. 63

Modified 6.77.1 Heavy Chain Nucléotide Sequence

1	atggaactgg	qgctccgctg	qqttttcctt	gttgctattt	tagaaggtgt
51	ccagtgtGAG	GTGCAGCTGG	TGGAGTCTGG	GGGAGGCCTG	GTCAAGCCTG
101	GGGGGTCCCT	GAGACTCTCC	TGTGCAGCCT	CTGGATTCAC	CTTCAGTAGC
151	TATAGCATGA	ACTGGGTCCG	CCAGGCTCCA	GGGAAGGGGC	TGGAGTGGGT
201	CTCATCCATT	AGTAGTAGTA	GTAGTTACAT	ATACTACGCA	GACTCAGTGA
251	AGGGCCGATT	CACCATCTCC	AGAGACAAGG	CCAAGAACTC	ACTGTATCTG
301	CAAATGAACA	GCCTGAGAGC	CGAGGACACG	GCTGTGTATT	ACTGTGCGAG
351	AGATGGGTAT	AGCAGTGGCT	GGTCCTACTA	CTACTACTAC	GGTATGGACG
401	TCTGGGGCCA	AGGGACCACG	GTCACCGTCT	CCTCAGCTTC	CACCAAGGGC
451	CCATCCGTCT	TCCCCCTGGC	GCCCTGCTCT	AGAAGCACCT	CCGAGAGCAC
501	AGCGGCCCTG	GGCTGCCTGG	TCAAGGACTA	CTTCCCCGAA	CCGGTGACGG
551	TGTCGTGGAA	CTCAGGCGCT	CTGACCAGCG	GCGTGCACAC	CTTCCCAGCT
601	GTCCTACAGT	CCTCAGGACT	CTACTCCCTC	AGCAGCGTGG	TGACCGTGCC
651	CTCCAGCAAC	TTCGGCACCC	AGACCTACAC	CTGCAACGTA	GATCACAAGC
701	CCAGCAACAC	CAAGGTGGAC	AAGACAGTTG	AGCGCAAATG	TTGTGTCGAG
751	TGCCCACCGT	GCCCAGCACC	ACCTGTGGCA	GGACCGTCAG	TCTTCCTCTT
801	CCCCCCAAAA	CCCAAGGACA	CCCTCATGAT	CTCCCGGACC	CCTGAGGTCA
851	CGTGCGTGGT	GGTGGACGTG	AGCCACGAAG	ACCCCGAGGT	CCAGTTCAAC
901	TGGTACGTGG	ACGGCGTGGA	GGTGCATAAT	GCCAAGACAA	AGCCACGGGA

- 116 -

951 1001 1051 1101 1151 1201 1251 1301 1351 1401

GGAGCAGTTC AACAGCACGT TCCGTGTGGT CAGCGTCCTC ACCGTTGTGC ACCAGGACTG GCTGAACGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA GGCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAACCA AAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCA AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA CCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACACCTCCC ATGCTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGATAG

SEQ ID NO. 64

Modified 6.77.1 Heavy Chain Protein Sequence melglrwvfl vailegvqcE VQLVESGGGL VKPGGSLRLS CAASGFTFSS

YSMNWVRQAP GKGLEWVSSI SSSSSYIYYA DSVKGRFTIS RDNAKNSLYL

101 QMNSLRAEDT AVYYCARDGY SSGWSYYYYY GMDVWGQGTT VTVSSASTKG

151 PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA

201 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE

251 CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN

301 WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK

351 GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD

401 IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS

451 VMHEALHNHY TQKSLSLSPG K

SEQ ID NO. 65

Modified 6.77.1 Kappa Light Chain Nucléotide Sequence

1	atgaggctcc	ctgctcagct	cctggggctg	ctaatgctct	ggatacctgg
51	atccagtgca	GATATTGTGA	TGACCCAGAC	TCCACTCTCT	CTGTCCGTCA
101	CTCCTGGACA	GCCGGCCTCC	ATCTCCTGCA	AGTCTAGTCA	GAGCCTCCTG
151	CTTAGTGATG	GAAAGACCTA	TTTGAATTGG	TACCTGCAGA	AGCCCGGCCA
201	GCCTCCACAG	CTCCTGATCT	ATGAAGTTTC	CAACCGGTTC	TCTGGAGTGC
251	CAGACAGGTT	CAGTGGCAGC	GGGTCAGGGA	CAGATTTCAC	ACTGAAAATC
301	AGCCGGGTGG	AGGCTGAGGA	TGTTGGGGTT	TATTACTGCA	TGCAAAGTAT
351	ACAGCTTATG	TGCAGTTTTG	GCCAGGGGAC	CAAGCTGGAG	ATCAAACGAA
401	CTGTGGCTGC	ACCATCTGTC	TTCATCTTCC	CGCCATCTGA	TGAGCAGTTG
451	AAATCTGGAA	CTGCCTCTGT	TGTGTGCCTG	CTGAATAACT	TCTATCCCAG
501	AGAGGCCAAA	GTACAGTGGA	AGGTGGATAA	CGCCCTCCAA	TCGGGTAACT
551	CCCAGGAGAG	TGTCACAGAG	CAGGACAGCA	AGGACAGCAC	CTACAGCCTC
601	AGCAGCACCC	TGACGCTGAG	CAAAGCAGAC	TACGAGAAAC	ACAAAGTCTA
651	CGCCTGCGAA	GTCACCCATC	AGGGCCTGAG	CTCGCCCGTC	ACAAAGAGCT
701	TCAACAGGGG	AGAGTGTTAG	TGA

SEQ ID NO. 66

Modified 6.77.1 Kappa Light Chain Amino Acid Sequence mrlpaqllgl Imlwipgssa

LSDGKTYLNW YLQKPGQPPQ

101 SRVEAEDVGV YSCMQSIQLM

151 KSGTASVVCL LNNFYPREAK

201 SSTLTLSKAD YEKHKVYACE

SEQ ID NO. 67

DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI SSFGQGTKLE IKRTVAAPSV FIFPPSDEQL VQWKVDNALQ SGNSQESVTE QDSKDSTYSL VTHQGLSSPV TKSFNRGEC

Modified 7.26.4 Kappa Light Chain Nucléotide Sequence atgaggctcc ctgctcagct cctggggctg ctaatgctct ggatacctgg atccagtgcg GATATTGTGA TGACCCAGAC TCCACTCTCT CTGTCCGTCA

101 CCCCTGGACA GCCGGCCTCC ATCTCCTGCA AGTCTAGTCA GAGCCTCCTG

- 117-

151	TATAGTGATG	GAAAGACCTA	TTTGTTTTGG	TACCTGCAGA	AGCCAGGCCA
201	GCCTCCACAG	CTCCTGATCT	ATGAAGTTTC	CAACCGATTC	TCTGGAGTGC
251	CAGATAGGTT	CAGTGGCAGC	GGGTCAGGGA	CAGATTTCAC	ACTGAAAATC
301	AGCCGGGTGG	AGGCTGAGGA	TGTTGGGGTT	TATTACTGCA	TGCAAAGTAT
351	ACAGCTTCCG	TGGACGTTCG	GCCAAGGGAC	CAAGGTGGAA	ATCAAACGAA
401	CTGTGGCTGC	ACCATCTGTC	TTCATCTTCC	CGCCATCTGA	TGAGCAGTTG
451	AAATCTGGAA	CTGCCTCTGT	TGTGTGCCTG	CTGAATAACT	TCTATCCCAG
501	AGAGGCCAAA	GTACAGTGGA	AGGTGGATAA	CGCCCTCCAA	TCGGGTAACT
551	CCCAGGAGAG	TGTCACAGAG	CAGGACAGCA	AGGACAGCAC	CTACAGCCTC
601	AGCAGCACCC	TGACGCTGAG	CAAAGCAGAC	TACGAGAAAC	ACAAAGTCTA
651	CGCCTGCGAA	GTCACCCATC	AGGGCCTGAG	CTCGCCCGTC	ACAAAGAGCT
701	TCAACAGGGG	AGAGTGTTAG	T GA

SEQ ID NO. 68

Modified 7.26.4 Kappa Light Chain Amino Acid Sequence i

101 151 201 mrlpaqllgl Imlwipgssa DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL YSDGKTYLFW YLQKPGQPPQ LLIYEVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQSIQLP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

SEQ ID NO: 148

X481.2 Heavy Chain Amino Acid Sequence

QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYGINWVRQA PGQGLEWMGW 51 ISVYSGNTNY AQKVQGRVTM TADTSTSTAY MDLRSLRSDD TAVYYCAREG 101 SSSSGDYYYG MDVWGQGTTV TVSSASTKGP SVFPLAPCSR STSESTAALG 151 CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSNF 201 GTQTYTCNVD HKPSNTKVDK TVERKCCVEC PPCPAPPVAG PSVFLFPPKP 251 KDTLMISRTP EVTCVVVDVS HEDPEVQFNW YVDGVEVHNA KTKPREEQFN 300 STFRVVSVLT VVHQDWLNGK EYKCKVSNKG LPAPIEKTIS KTKGQPREPQ 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPM 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

SEQ ID NO: 149

X481.2 Heavy Chain Nucléotide Sequence

1	ATGGACTGGA	CCTGGAGCAT	CCTTTTCTTG	GTGGCAGCAG	CAACAGGTGC
51	CCACTCCCAG	GTTCAGCTGG	TGCAGTCTGG	AGCTGAGGTG	AAGAAGCCTG
101	GGGCCTCAGT	GAAGGTCTCC	TGCAAGGCTT	CTGGTTACAC	CTTTACCAGC
151	TATGGTATCA	ACTGGGTGCG	ACAGGCCCCT	GGACAAGGGC	TTGAGTGGAT
201	GGGATGGATC	AGCGTTTACA	GTGGTAACAC	AAACTATGCA	CAGAAGGTCC
251	AGGGCAGAGT	CACCATGACC	GCAGACACAT	CCACGAGCAC	AGCCTACATG
301	GACCTGAGGA	GCCTGAGATC	TGACGACACG	GCCGTGTATT	ACTGTGCGAG
351	AGAGGGTAGC	AGCTCGTCCG	GAGACTACTA	TTACGGTATG	GACGTCTGGG
401	GCCAAGGGAC	CACGGTCACC	GTCTCCTCAG	CCTCCACCAA	GGGCCCATCG
451	GTCTTCCCCC	TGGCGCCCTG	CTCCAGGAGC	ACCTCCGAGA	GCACAGCGGC
501	CCTGGGCTGC	CTGGTCAAGG	ACTACTTCCC	CGAACCGGTG	ACGGTGTCGT
551	GGAACTCAGG	CGCTCTGACC	AGCGGCGTGC	ACACCTTCCC	AGCTGTCCTA
601	CAGTCCTCAG	GACTCTACTC	CCTCAGCAGC	GTGGTGACCG	TGCCCTCCAG
651	CAACTTCGGC	ACCCAGACCT	ACACCTGCAA	CGTAGATCAC	AAGCCCAGCA
701	ACACCAAGGT	GGACAAGACA	GTTGAGCGCA	AATGTTGTGT	CGAGTGCCCA
751	CCGTGCCCAG	CACCACCTGT	GGCAGGACCG	TCAGTCTTCC	TCTTCCCCCC
801	AAAACCCAAG	GACACCCTCA	TGATCTCCCG	GACCCCTGAG	GTCACGTGCG
851	TGGTGGTGGA	CGTGAGCCAC	GAAGACCCCG	AGGTCCAGTT	CAACTGGTAC
901	GTGGACGGCG	TGGAGGTGCA	TAATGCCAAG	ACAAAGCCAC	GGGAGGAGCA
951	GTTCAACAGC	ACGTTCCGTG	TGGTCAGCGT	CCTCACCGTT	GTGCACCAGG
1001	ACTGGCTGAA	CGGCAAGGAG	TACAAGTGCA	AGGTCTCCAA	CAAAGGCCTC

- 118 1051 CCAGCCCCCA TCGAGAAAAC CATCTCCAAA ACCAAAGGGC AGCCCCGAGA 1101 ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC 1151 AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCC 1201 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACACC 1251 TCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG 1301 TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG 1351 CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC 1401 GGGAAAATGA TAG

SEQ IDNO: 150

X481.2 Light Chain Amino Acid Sequence

DIVMTQTPLS LSVTPGQPAS

LLIYEVSNRF SGVPDRFSGS

101 WTFGQGTKVE IKRTVAAPSV

151 VQWKVDNALQ SGNSQESVTE

201 VTHQGLSSPV TKSFNRGEC

ISCKSSQSLL HTDGTTYLYW YLQKPGQPPQ GSGTDFTLKI SRVEAEDVGI YYCMQNIQLP FIFPPSDEQL KSGTASVVCL LNNFYPREAK QDSKDSTYSL SSTLTLSKAD YEKHKVYACE

Claims (27)

1. We Claim:

   1. A human monoclonal antibody or an antigen-binding portion thereof that specifically binds to Mucosal Adressin Cell Adhesion Molécule (MAdCAM), wherein the antibody comprises a heavy chain variable région encoded by a nucléotide sequence comprising SEQ ID NO: 149 and a light chain variable région encoded by a nucléotide sequence comprising SEQ ID NO: 35.
2. 2. The human monoclonal antibody or antigen binding portion of claim 1, wherein nucléotides defined by SEQ ID NO: 149 encodes amino acid sequence SEQ ID NO: 148.
3. 3. The human monoclonal antibody or antigen binding portion of claim 1, wherein the nucléotides defined by SEQ ID NO: 35 without the signal sequence encodes amino acid SEQ ID NO: 150.
4. 4. The human monoclonal antibody or antigen-binding portion according to claim 1, wherein said antibody or portion possesses at least one of the following properties:

   (a) has a selectivity for MAdCAM over VCAM or fibronectin of at least 100 fold;

   (b) binds to human MAdCAM with a Kd of 3 x 10-10 M or less; or (c) inhibits the binding of α4β7 expressing cells to human MAdCAM.
5. 5. A nucleic acid sequence encoding the amino acid sequences of claim 2 or 3.
6. 6. A cell comprising the nucleic acid sequence of any one of daims 1-3 or 5.
7. 7. A hybridoma cell line that produces the human monoclonal antibody according to claim

   1.
8. 8. The human monoclonal antibody or antigen binding portion thereof produced by the hybridoma cell line according to claim 7.
9. 9. A human monoclonal antibody or an antigen-binding portion thereof, wherein the heavy chain of said antibody or portion thereof comprises the heavy chain CDR1, CDR2 and CDR3 or wherein the light chain comprises the light chain CDR1, CDR2 and CDR3 of a monoclonal antibody encoded by the nucleic acid of any one of daims 1-3 or 5.

   - 120-
10. 10. The human monoclonal antibody according to any one of claims 1-4 or 9, wherein the antibody is an immunoglobulin G (IgG), an IgM, an IgE, and IgA or an IgD molécule, a humanized antibody, a chimeric antibody or a bispecific antibody.
11. 11. The antigen-binding portion according to any one of claims 1-4 or 9, wherein the antigenbinding portion is an Fab fragment, an F(ab’)2 fragment, an FV fragment or a single chain antibody.
12. 12. A pharmaceutical composition comprising an effective amount of the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-4 or 9-11.
13. 13. An isolated cell line that produces the monoclonal antibody or antigen-binding portion according to any one of claims 1-4 or 9-11 or the heavy chain or light chain of said antibody or of said portion thereof.
14. 14. An isolated nucleic acid molécule comprising a nucléotide sequence that encodes the heavy chain or an antigen-binding portion thereof or the light chain or an antigen-binding portion thereof of an antibody according to any one of claims 1-4 or 9-11, and wherein the nucleic acid sequence that encodes the heavy chain variable sequence is SEQ ID NO: 149, and wherein the nucleic acid sequence that encodes the light chain sequence is SEQ ID NO: 35.
15. 15. A vector comprising the nucleic acid molécule according to claim 14, wherein the vector comprises an expression control sequence operably linked to the nucleic acid molécule.
16. 16. A host cell comprising the vector according to claim 15 or the nucleic acid molécule according to claim 14.
17. 17. A host cell according to claim 16 comprising a nucleic acid molécule encoding the heavy chain variable sequence or an antigen-binding portion thereof and a nucleic acid molécule encoding the light chain or an antigen-binding portion thereof of an antibody or antigen-binding portion according to any one of claims 1-4 or 9-11.
18. 18. A method for producing a human monoclonal antibody or antigen-binding portion thereof that specifically binds MAdCAM, comprising culturing the host cell according to claim 16 or 17 or the cell line according to claim 18 under suitable conditions and recovering said antibody or

    - 121 antigen-binding portion.
19. 19. A pharmaceutical composition comprising the antibody of any one of daims 1-3.
20. 20. The pharmaceutical composition of claim 19 further comprising one or more additional anti-inflammatory or immunomodulatory agents.
21. 21. The pharmaceutical composition according to claim 20, wherein the one or more additional anti-inflammatory or immunomodulatory agents are selected from the group consisting of: corticosteroids, aminosalicylates, azathioprine, methotrexate, cyclosporin, FK506, IL-10, GM-CSF, rapamycin, anti-TNFa agents and adhesion molécule antagonists.
22. 22. A vaccine comprising an effective amount of the human antibody according to any one of daims 1-4 or 9-11 or antigen-binding portion and a pharmaceutically acceptable carrier.
23. 23. The vaccine according to claim 22, wherein the vaccine is mucosal.
24. 24. A method of manufacturing a human monoclonal antibody or an antigen-binding fragment portion thereof that specifically binds to Mucosal Adressin Cell Adhesion Molécule (MAdCAM), wherein the antibody comprises a heavy chain variable région encoded by a nucléotide sequence comprising SEQ ID NO: 149 and a light chain variable région encoded by a nucléotide sequence comprising SEQ ID NO: 35.
25. 25. The method of claim 24, wherein nucléotides defined by SEQ ID NO: 149 encodes amino acid sequence SEQ ID NO: 148.
26. 26. The method of claim 24 or 25, wherein the nucléotides defined by SEQ ID NO: 35 without the signal sequence encodes amino acid SEQ ID NO: 150.
27. 27. A monoclonal antibody, or antigen-binding portion thereof, that binds MAdCAM comprising the variable région of the light chain of SEQ ID NO: 150 and the variable région of the heavy chain of SEQ ID NO: 148.