HK1099695B - Antibodies to madcam - Google Patents

Description

MAdCAM antibodies

This application claims the benefit of U.S. provisional application 60/535,490 filed on 9/1/2004.

Background

Mucosal addressin cell adhesion molecule (MAdCAM) is a member of the immunoglobulin superfamily of cell adhesion receptors. The selectivity of lymphocyte homing (homing) to specialized lymphoid tissue and mucosal sites of the gastrointestinal tract was determined by endothelial 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 the high endothelial venules of organized intestinal lymphoid tissue, such as Peyer's patches and mesenteric lymph nodes (Streetr et al, Nature, 331: 41-6 (1988); Nakache et al, Nature, 337: 179-81 (1989); Briskin et al, am. J. Pathol.151-97-110(1997)), but also in other lymphoid organs such as the pancreas, gall bladder and spleen venules and the marginal sinuses of the spleen's white marrow (Briin et al (1997), supra; Kraal et al, am. J. Path., 147: 763- (1995)).

Although MAdCAM plays a physiological role in intestinal immune surveillance, it has been shown to promote excessive extravasation of lymphocytes in inflammatory bowel disease under chronic gastrointestinal inflammatory conditions. TNF and other proinflammatory agentsThe cytokine enhances endothelial MAdCAM expression and MAdCAM expression is locally enhanced about 2-3 fold at the site of inflammation in biopsy specimens taken from patients with crohn's disease and ulcerative colitis (Briskin et al (1997), Souza et al, Gut,45: 856-63 (1999); arihiro et al, Pathol int, 52: 367-74(2002)). Increased expression of similar patterns has 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, J Leukoc biol., 65: 349-55 (1999); Kato et al, J Parmacol Exp ther., 295: 183-9 (2000); Hokarri et al, Clin Exp Immunol., 26: 259-65 (2001); Shigematsu et al, Am J Physiol Gastrointet light physiology., 281: G1309-15 (2001)). In inflammatory disorders such as insulin-dependent Diabetes mellitus (Yang et al Diabetes, 46: 1542-7 (1997));etc., J immunol., 160: 6018-25(1998), graft versus host disease (Fujisaki et al, Scand J gastroenterol, 38: 437-42(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, immunological Cell biol., 78: 641-5(2000)) and gastritis (Barrett et al, J Leukoc biol., 67: 169-73 (2000); hatanaka et al, Clin Exp immunol., 130: 183-9(2002)) Re-awakened and activated α expression of fetal MAdCAM in other preclinical models of disease pathogenesis₄β₇ ⁺Involvement of lymphocytes. In these inflammatory models and hapten-mediated (e.g., TNBS, DSS, etc.) or inherited metastasis (CD 4)⁺CD45Rb^high) Rat anti-mouse MAdCAM monoclonal antibody (mAb) MECA-367, which blocks alpha in a mouse colitis model₄β₇ ⁺Binding of lymphocytes to MAdCAM reduces lymphocyte recruitment, tissue extravasation, inflammation, and severity of disease. Mouse monoclonal antibodies (mAbs) against human MAdCAM have also been reported (see, e.g., WO 96/24673 and W)O 99/58573)。

The role of MAdCAM in Inflammatory Bowel Disease (IBD) and other gastrointestinal or other tissue-related inflammatory diseases is known, one for the inhibition of alpha₄β₇Methods of binding and MAdCAM-mediated leukocyte recruitment are desirable. It would be more desirable to have a treatment with advantageous properties including, but not limited to, the absence of undesirable interactions with other drug therapies in patients, and advantageous physicochemical properties such as pK/pD values in humans, solubility, stability, shelf life and in vivo half-life. Therapeutic proteins, such as antibodies, will advantageously be free from unwanted post-translational modifications or aggregate formation. Therefore, therapeutic anti-MAdCAM antibodies are urgently needed.

Summary of The Invention

The invention provides an isolated antibody that specifically binds MAdCAM, wherein at least the CDR sequences of the antibody are human CDR sequences, or an antigen-binding portion of the antibody. In some embodiments the antibody is a human antibody, preferably an antibody that is an antagonist of MAdCAM. Compositions comprising the antibodies or moieties are also provided.

The invention also provides compositions comprising the heavy and/or light chains or variable regions or other antigen-binding portions thereof of the anti-MAdCAM antagonist antibody or a nucleic acid molecule encoding any of the foregoing, and a pharmaceutically acceptable carrier. The compositions of the present invention may further comprise additional ingredients such as therapeutic or diagnostic agents. Diagnostic and therapeutic methods are also provided.

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

The invention also provides nucleic acid molecules encoding the heavy and/or light chains of the anti-MAdCAM antibodies or variable regions thereof or antigen-binding portions thereof.

The invention provides vectors and host cells comprising the nucleic acid molecules, and methods for recombinantly producing a polypeptide encoded by the nucleic acid molecules.

Also provided are non-human transgenic animals or plants expressing the heavy and/or light chains of the anti-MAdCAM antibodies, or antigen-binding portions thereof.

Brief Description of Drawings

FIG. 1 is an alignment of the predicted amino acid sequences of the heavy chain and kappa light chain variable regions of the 12 human anti-MAdCAM monoclonal antibodies with the germline amino acid sequences of the corresponding human genes.

FIG. 1A shows the predicted alignment of the amino acid sequences of the heavy chains of antibodies 1.7.2 and 1.8.2 (residues 20-138 of SEQ ID NOS: 2 and 6, respectively) with the germline human VH 3-15 gene product (SEQ ID NO: 113).

FIG. 1B shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.14.2 (residues 20-141 of SEQ ID NO: 10) with the germline human VH 3-23 gene product (SEQ ID NO: 114).

FIG. 1C shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.22.2 (residues 20-139 of SEQ ID NO: 14) with the germline human VH3-33 gene product (SEQ ID NO: 115).

FIG. 1D shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.34.2 (residues 20-143 of SEQ ID NO: 18) with the germline human VH3-30 gene product (SEQ ID NO: 116).

FIG. 1E shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.67.1 (residues 20-144 of SEQ ID NO: 22) with the germline human VH4-4 gene product (SEQ ID NO: 117).

FIG. 1F shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.73.2 (residues 20-145 of SEQ ID NO: 26) with the germline human VH 3-23 gene product (SEQ ID NO: 118).

FIG. 1G shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 6.77.1 (residues 20-146 of SEQ ID NO: 30) with the germline human VH 3-21 gene product (SEQ ID NO: 119).

FIG. 1H shows the predicted alignment of the amino acid sequences of the heavy chains of antibodies 7.16.6 and 7.26.4 (residues 20-144 of SEQ ID NOS: 34 and 42, respectively) with the germline human VH 1-18 gene product (SEQ ID NO: 120).

FIG. 1I shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 7.20.5 (residues 20-146 of SEQ ID NO: 38) with the germline human VH4-4 gene product (SEQ ID NO: 121).

FIG. 1J shows an alignment of the predicted amino acid sequence of the heavy chain of antibody 9.8.2 (residues 20-136 of SEQ ID NO: 46) with the germline human VH3-33 gene product (SEQ ID NO: 122).

FIG. 1K shows the predicted alignment of kappa light chain amino acid sequences of antibodies 1.7.2 and 1.8.2 (residues 21-132 of SEQ ID NOS: 4 and 8, respectively) with the germline human A3 gene product (SEQ ID NO: 123).

FIG. 1L shows an alignment of the predicted kappa light chain amino acid sequence of antibody 6.14.2 (residues 23-130 of SEQ ID NO: 12) with the germline human O12 gene product (SEQ ID NO: 124).

FIG. 1M shows an alignment of the predicted kappa light chain amino acid sequence of antibody 6.22.2 (residues 20-127 of SEQ ID NO: 16) with the germline human A26 gene product (SEQ ID NO: 125).

FIG. 1N shows an alignment of the predicted kappa light chain amino acid sequence of antibody 6.34.2 (residues 23-130 of SEQ ID NO: 20) with the germline human O12 gene product (SEQ ID NO: 126).

FIG. 10 shows an alignment of the predicted kappa light chain amino acid sequence of antibody 6.67.1 (residues 21-135 of SEQ ID NO: 24) with the germline human B3 gene product (SEQ ID NO: 127).

FIG. 1P shows the predicted alignment of the kappa light chain amino acid sequence of antibody 6.73.2 (residues 23-132 of SEQ ID NO: 28) to the germline human O12 gene product (SEQ ID NO: 128).

FIG. 1Q shows the predicted alignment of the kappa light chain amino acid sequence of antibody 6.77.1 (residues 21-133 of SEQ ID NO: 32) with the germline human A2 gene product (SEQ ID NO: 129).

FIG. 1R shows the predicted alignment of kappa light chain amino acid sequences of antibodies 7.16.6 and 7.26.4 (residues 21-133 of SEQ ID NOS: 36 and 44, respectively) with the germline human A2 gene product (SEQ ID NO: 130).

FIG. 1S shows an alignment of the predicted kappa light chain amino acid sequence of antibody 7.20.5 (residues 21-132 of SEQ ID NO: 40) with the germline human A3 gene product (SEQ ID NO: 131).

FIG. 1T shows an alignment of the predicted kappa light chain amino acid sequence of antibody 9.8.2 (residues 25-132 of SEQ ID NO: 48) with the germline human O18 gene product (SEQ ID NO: 132).

Figure 2 is a CLUSTAL alignment of predicted human anti-MAdCAM antibody heavy and kappa light chain amino acid sequences.

FIG. 2A is a CLUSTAL alignment (residues 1-132 of SEQ ID NO: 4, 8 and 40, residues 1-133 of SEQ ID NO: 36, 44 and 32, residues 1-135 of SEQ ID NO: 24, residues 1-130 of SEQ ID NO: 20, residues 1-132 of SEQ ID NO: 28, residues 1-130 of SEQ ID NO: 12, residues 1-132 of SEQ ID NO: 48, and residues 1-127 of SEQ ID NO: 16, respectively, in that order of appearance) and radial trees of predicted kappa light chain amino acid sequences, showing the degree of similarity between anti-MAdCAM antibody kappa light chains.

FIG. 2B is a CLUSTAL alignment (residues 20-144 of SEQ ID NO: 34, residues 20-138 of SEQ ID NO: 4 and 6, residues 20-122 of SEQ ID NO: 10, residues 20-145 of SEQ ID NO: 26, residues 20-146 of SEQ ID NO: 30, residues 20-139 of SEQ ID NO: 14, residues 20-143 of SEQ ID NO: 18, residues 20-136 of SEQ ID NO: 46, residues 20-146 of SEQ ID NO: 38, and residues 20-144 of SEQ ID NO: 22, respectively, in that order of appearance) and radial trees of predicted heavy chain amino acid sequences, showing the degree of similarity between anti-MAdCAM antibody heavy chains.

FIG. 3 is a CLUSTAL alignment of the amino acid sequences of the 2N-terminal domains of macaca brachycan (SEQ ID NO: 50) and human (residues 1-225 of SEQ ID NO: 107) MAdCAM that form the α 4 β 7 binding domain. According to Tan et al, Structure (1998) 6: 793 comparison of beta strands was performed using 801.

Figure 4 is a graph showing the dose effect of purified biotinylation 1.7.2 and 7.16.6 on human peripheral blood lymphocyte adhesion to frozen human liver endothelial sections expressing MAdCAM.

Fig. 5 shows a two-dimensional graphical representation of diversity data based on the MAdCAM epitopes bound by 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 collected in table 7. anti-MAdCAM antibodies within the same round showed the same reactivity pattern, belonging to the same epitope bin (bin) and likely recognized the same epitope on MAdCAM. anti-MAdCAM antibody clones within overlapping circles are unable to bind simultaneously and therefore are likely to recognize overlapping epitopes on MAdCAM. The non-integrated circles represent anti-MAdCAM antibody clones with distinct spatial epitope separations.

Figure 6 shows sandwich ELISA data for anti-MAdCAM antibody 1.7.2 and Alexa 488-labeled 7.16.6, showing that two antibodies capable of detecting different epitopes on MAdCAM can be used to detect soluble MAdCAM for diagnostic purposes.

FIG. 7 Using anti-MAdCAM mAb 7.16.6 in a cynomolgus monkey model, showing that inhibitory anti-MAdCAM antibody (1mg/kg) is directed against circulating peripheral alpha₄β₇ ⁺The effect of the number of basocytes, expressed as a fold increase relative to control IgG2a mAb or vector.

Detailed description of the invention

Definitions and general techniques

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by those of ordinary skill in the art. Also, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used and the techniques thereof in connection with, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and are described with reference to various general or more specific references that are cited and discussed throughout the specification, 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 is incorporated herein by reference. Enzymatic reactions and purification techniques are performed as commonly done in the art or as described herein, according to the manufacturer's instructions. Standard techniques are used for chemical synthesis, chemical analysis, preparation, formulation and delivery of drugs, and treatment of patients.

Unless otherwise indicated, the following terms will be understood to have the following meanings:

the term "polypeptide" encompasses natural or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or multimeric.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, due to its source or derivative source, (1) is not associated with the naturally associated component with which it is associated in its native state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) is not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. Proteins can 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 60-75% of the sample exhibits a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically constitute about 50%, 60%, 70%, 80%, or 90% W/W of the protein sample, more typically about 95%, and preferably more than 99% pure. Protein purity or homogeneity can be indicated by a number of methods well known in the art, such as subjecting a protein sample to polyacrylamide gel electrophoresis followed by staining the gel with dyes well known in the art to visualize individual polypeptide bands. For some purpose, higher resolution may be provided using HPLC or other methods well known in the art of purification.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the naturally occurring sequence. In some embodiments, a fragment is at least 5,6, 8, or 10 amino acids long. In further embodiments, fragments are at least 14 amino acids long, more preferably at least 20 amino acids long, typically 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 comprising a fragment of at least 25 amino acids which has substantial identity to a portion of the amino acid sequence and has at least one of the following properties: (1) specific binding to MAdCAM under appropriate binding conditions, (2) inhibition of α₄β₇Integrin and/or L-selectin bind to MAdCAM, or (3) can reduce MAdCAM cell surface expression in vitro or in vivo. Typically, polypeptide analogs contain conservative amino acid substitutions (or insertions or deletions) relative to the naturally occurring sequence. Analogs are typically at least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long or longer, and they can often be as long as the full-length naturally occurring polypeptide.

Preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity of the formed protein complex, (4) alter the binding affinity, or (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include different muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the form of intermolecular contact structure domain outside the polypeptide part). Conservative amino acid substitutions should not significantly alter the structural characteristics of the parent sequence (e.g., the substituted amino acid does not tend to disrupt the helix present in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized secondary and tertiary Structures of polypeptides are in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H.Freeman and Company, New York (1984)); introduction to Protein Structure (c. branden and j. hooze, eds., Garland Publishing, New York, n.y. (1991)) and Thornton et al Nature, 354: 105(1991), each of which is incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptidomimetics" 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 is incorporated herein by reference. Such compounds are often developed by means of computerized molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent therapeutic or prophylactic effects. Generally, peptidomimetics are structurally similar to exemplary polypeptides (i.e., polypeptides having desirable biochemical properties or pharmacological activity), such as human antibodies, but which have one or more optional linkages such as: -CH₂NH-、-CH₂S-、-CH₂-CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-and-CH₂SO-peptide bonds are replaced by methods well known in the art. Systematic replacement of one or more amino acids of the consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can also be used to produce more stable peptides. Furthermore, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variant may be produced by methods known in the art (Rizo and Gierasch Ann. Rev. biochem. 61: 387(1992), which are incorporated herein by reference); for example by adding internal cysteine residues capable of forming intramolecular disulphide bonds to cyclise the peptide.

An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the antibody isotypes IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, while the heavy chain also comprises a "D" region of about 10 or more amino acids. See, generally, Fundamental Immunology, ch.7(Paul, w., ed., 2nd ed. raven Press, n.y. (1989)) (which is incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form antibody binding sites, so that an intact immunoglobulin has two binding sites.

Immunoglobulin chains exhibit the same general structure of relatively conserved Framework Regions (FRs) joined by three hypervariable regions (also known as complementarity determining regions or CDRs). The CDRs of both chains of each pair are arranged through the framework regions to form an epitope-specific binding site. From N-terminus to C-terminus, both the light and heavy chains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. According to 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 its entirety, assigns amino acids to each domain.

"antibody" refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes for specific binding with an intact antibody. In one embodiment, the antibody is an antigen-binding portion thereof. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab ', F (ab')₂Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient for the polypeptide to specifically bind an antigen. Fab fragments are monovalent fragments consisting of the VL, VH, CL and CH1 domains; f (ab)₂A fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; the Fd fragment consists of the VH and CH1 domains; the Fv fragment consists of the VL and VH domains of a single arm of an antibody; while dAb fragments (Ward et al, Nature, 341: 544-546(1989)) consist of VH domains.

As used herein, an antibody referred to as, for example, 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, or 9.8.2 is a monoclonal antibody produced by a hybridoma of the same name. For example, antibody 1.7.2 is produced by hybridoma 1.7.2. The antibodies designated 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod are monoclonal antibodies whose sequences have been modified from their respective parents by site-directed mutagenesis.

Single chain antibodies (scFv) are antibodies in which the VL and VH regions are paired by a synthetic linker sequence to form a monovalent molecule that enables the VL and VH regions to be prepared as a single protein chain (Bird et al, Science, 242: 423-. Bispecific antibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker sequence is used that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger, P et al, Proc. Natl. Acad. Sci. USA, 90: 6444-. One or more CDRs from an antibody of the invention can be integrated into the molecule covalently or non-covalently so that it is an immunoadhesin that specifically binds to MAdCAM. Immunoadhesins can integrate a CDR as part of a larger polypeptide chain, can covalently link a CDR to another polypeptide chain, or can non-covalently integrate a CDR. The CDRs allow the immunoadhesin to specifically bind to a particular antigen of interest.

The antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be the same or different from each other. For example, a naturally occurring 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 components that accompany it in its native state (including other naturally associated antibodies), (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Examples of isolated antibodies include anti-MAdCAM antibodies that have been affinity purified using MAdCAM, anti-MAdCAM antibodies produced in vitro by hybridomas or other cell lines, and human anti-MAdCAM antibodies derived from transgenic mammals or plants.

As used herein, the term "human antibody" refers to antibodies in which the variable and constant region sequences are human sequences. The term encompasses antibodies having sequences derived from human genes that have been altered (e.g., to reduce potential immunogenicity, enhance affinity, eliminate cysteine or glycosylation sites that may cause undesirable folding, etc.). The term encompasses such antibodies recombinantly produced in non-human cells that can result in glycosylation of the antibody that is atypical of human cells. The term also encompasses antibodies produced in transgenic mice that contain some or all of the human immunoglobulin heavy and light chain loci.

In one aspect, the invention provides humanized antibodies. In some embodiments, the humanized antibody is derived from non-human species of antibody, which in heavy and light chain framework domain and constant region in certain amino acid has been mutated in order to avoid or cancel in human immune response. In some embodiments, humanized antibodies can be produced by fusing constant domains from human antibodies to variable domains of a non-human species. Examples of how to generate humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293. In some embodiments, the humanized anti-MAdCAM antibodies of the invention comprise the amino acid sequence of one or more framework regions of one or more human anti-MAdCAM antibodies of the invention.

In another aspect, the invention includes "chimeric antibodies". In some embodiments a chimeric antibody refers to an antibody comprising one or more regions from one antibody and one or more regions from one or more other antibodies. In a preferred embodiment, one or more CDRs are derived from a human anti-MAdCAM antibody of the invention. In a more preferred embodiment, all of the CDRs are derived from a human anti-MAdCAM antibody of the invention. In another preferred embodiment, the CDRs from more than one human anti-MAdCAM antibody of the invention are mixed and matched in the chimeric antibody. For example, a chimeric antibody may comprise CDR1 from the light chain of a first human anti-MAdCAM antibody, which may be combined with CDR2 and CDR3 from the light chain of a second human anti-MAdCAM antibody, and the CDRs of the heavy chain may be derived from a third anti-MAdCAM antibody. Furthermore, the framework regions may be derived from one of the same anti-MAdCAM antibody, from one or more different antibodies, such as a human antibody, or from a humanized antibody

A "neutralizing antibody," "inhibitory antibody," or antagonist antibody is an inhibitory alpha₄β₇Or express alpha₄β₇Or any other cognate ligand or cognate ligand-expressing cell binds to MAdCAM for at least about 20% of the antibody. In a preferred embodiment, the antibody inhibits alpha₄β₇Integrins or expression of alpha₄β₇Binds to MAdCAM by at least 40%, more preferably 60%, even more preferably 80%, 85%, 90%, 95% or 100%. This reduction in binding can be measured by any method known to one of ordinary skill in the art, for example in an in vitro competitive binding assay. Measurement of expression alpha₄β₇Examples of the reduction of cell binding to MAdCAM in (a) are shown in example I.

Fragments or analogs of the antibodies can be readily prepared by those of ordinary skill in the art in light of the teachings herein. Preferred fragments or analogs have the amino-and carboxy-termini near the boundaries of the functional domains. Structural and functional domains can be determined by comparing nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computer-aided comparison methods are used to identify motifs or predicted protein conformation domains that are identified to be present in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al, Science, 253: 164 (1991)).

The term "surface plasmon resonance" as used herein refers to an optical phenomenon that enables analysis of real-time biospecific interactions by detecting changes in protein concentration within a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, n.j.). For further description, see Jonsson, u, et al, 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 dissociation rate constant for dissociation of an antibody from an antibody/antigen complex.

The term "K_d"refers to the dissociation constant of a particular antibody-antigen interaction. An antibody can be said to bind an antigen when the dissociation constant is ≦ 1 μ M, preferably ≦ 100nM, and most preferably ≦ 10 nM.

The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface clusters of molecules such as amino acids or carbohydrate side chains and generally have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, the interaction points span amino acid residues that are separated from each other on the protein.

As used herein, 20 conventional amino acids and abbreviations thereof 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 reference. Stereoisomers of these 20 conventional amino acids (e.g., D amino acids), unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). According to standard usage and convention, in polypeptide notation as used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction.

The term "polynucleotide" as referred to herein refers to a polymeric form of nucleotides (i.e., ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide) that are at least 10 bases in length. The term includes both single-stranded and double-stranded forms of DNA.

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

The term "oligonucleotide" as referred to herein includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides that typically comprise 200 bases or less in length. Preferably the oligonucleotide is 10-60 bases long, most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20-40 bases long. While oligonucleotides may be double stranded, as used in the construction of gene mutants, oligonucleotides are typically single stranded, as used in probes; the oligonucleotide of the invention may be a sense or antisense oligonucleotide.

The term "naturally occurring nucleotide" as referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" referred to herein includes nucleotides having a modified or substituted sugar group or the like. The term "oligonucleotide linkage" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphororaniladate, 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, nucleic acids res, 16: 3209 (1988); zon et al, Anti-Cancer Drug Design 6: 539 (1991); zon et al, Oligonucleotides and antigens: a Practical Approach, pp.87-108(F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); stec et al, U.S. Pat. No.5,151,510; uhlmann and peyman, Chemical Reviews, 90: 543(1990), the disclosures of which are incorporated herein by reference. The oligonucleotide may include a label for detection, if desired.

"operably linked" sequences include expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or control the gene of interest at a distance. The term "expression control sequence" as used herein refers to a polynucleotide sequence necessary to effect expression and processing of a coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; 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 secretion. Depending on the host organism, the nature of such control sequences varies; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequences" is intended to include (at a minimum) all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

The term "vector" as used herein means a nucleic acid molecule capable of transporting additional nucleic acid to a 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 additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication 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 thus can be 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 present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, e.g., viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It is understood that the term is 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 generations 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 term "host cell" as used herein.

The term "selectively hybridize" as referred to herein refers to detectably and specifically binding. Polynucleotides, oligonucleotides, and fragments thereof according to the invention can selectively hybridize to a nucleic acid strand under hybridization and wash conditions that minimize the apparent amount of detectable binding to non-specific nucleic acids. "high stringency" or "highly stringent" conditions can be used to obtain selective hybridization conditions 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 at a hybridization temperature of 42 ℃ in a hybridization buffer of 6 XSSPE or SSC, 50% formamide, 5 XDenhardt's reagent, 0.5% SDS, 100. mu.g/ml denatured fragmented salmon sperm DNA for 12-16 hours, wherein one polynucleotide can be immobilized to a solid surface such as a membrane, followed by two washes with a wash buffer of 1 XSSC, 0.5% SDS at 55 ℃. See also Sambrook et al, supra, pp.9.50-9.55.

The term "percent sequence identity" in the context of nucleotide sequences refers to the residues in two sequences that are the same when aligned in the maximum correspondence. The length of the sequence identity comparison may exceed a stretch of at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are many different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using the programs FASTA, Gap or Bestfit in the Wisconsin package version 10.3 of Accelrys, Inc. of san Diego, Calif. FASTA, which includes programs such as FASTA2 and FASTA3, provides alignments and percent sequence identities of regions of optimal overlap between 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); which is incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleotide sequences can be determined using FASTA with its default parameters (field size 6 and NOPAM factor for scoring matrix) or using Gap with its default parameters, as provided in version 10.3 of the Wisconsin package, which is incorporated herein by reference.

Unless otherwise indicated, reference to a nucleotide sequence includes the complementary strand thereof. Thus, reference to a nucleic acid molecule having a particular sequence should be understood to encompass the complementary strand thereof, having the sequence complementary thereto.

In the field of molecular biology, researchers use the terms "percent sequence identity", "percent sequence similarity", and "percent sequence homology" interchangeably. In the present application, these terms shall have the same meaning only with respect to nucleotide sequences.

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

The term "significant identity" when applied to a polypeptide means that two peptide sequences, when optimally aligned, for example by the programs GAP or BESTFIT using default GAP weights (GAP weights), have 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, the residue positions that are not identical differ by conservative amino acid substitutions. "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case 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 up to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol., 24: 307-31(1994), which is incorporated herein by reference. Examples of groups of amino acids with side chains having similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; and 6) the side chains containing sulfur are cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science, 256: 1443-45(1992), which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences by similarity measures assigned to different substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit" which can determine sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from different organism species or between a wild-type protein and its mutant protein using default parameters. See, e.g., version 10.3 of the Wisconsin package. Polypeptide sequences may also be compared using the FASTA program in Wisconsin package version 10.3, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identities of regions of best overlap between query and search sequences (Pearson (1990); Pearson (2000)). When comparing the sequences of the invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, in particular blastp or tblastn, with default parameters. See, e.g., Altschul et al, j.mol.biol.215: 403-; altschul et al, Nucleic Acids Res.25: 3389 and 402(1997), which are incorporated herein by reference.

The length of polypeptide sequences to which homology is compared is generally 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 databases containing sequences from a large number of different organisms, it is preferred to compare the amino acid sequences.

As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable label, such as an antibiotic that incorporates a radiolabeled amino acid or is attached to a label that is detectable against organismsA biotin moiety for detection of a biotin protein (e.g., streptavidin containing a fluorescent label or enzymatic activity detectable by optical or colorimetric methods) is attached to the polypeptide. In another embodiment, the label or tag may be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide tags include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent labels (e.g., FITC, rhodamine, rare earth phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal 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, podophyllotoxin, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroanthracycline dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, and the like, Lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, the labels are linked by spacer arms of different lengths to reduce potential steric hindrance.

The term "agent" is used herein to denote a compound, a mixture of compounds, a biological macromolecule, or an extract made from a biological material. The term "agent or drug" as used herein refers to a compound or composition that, when properly administered to a patient, results in the desired therapeutic effect. Other Chemical Terms are used herein according to conventional usage in The art, as exemplified by The McGraw-HillDirectionof Chemical Terms (Parker, S., Ed., McGraw-Hill, san Francisco (1985)), which is incorporated herein by reference.

The term "anti-inflammatory" agent or "immunomodulatory" agent is used herein to refer to an agent having functional properties that inhibit inflammation, including inflammatory diseases, in a subject, including a human. In various embodiments of the invention, the inflammatory disease may be, but is not limited to, inflammatory diseases of the gastrointestinal tract including crohn's disease, ulcerative colitis, diverticular disease, gastritis, liver disease, primary biliary fibrosis, sclerosing cholangitis. Inflammatory diseases also include, but are not limited to, abdominal diseases (including peritonitis, appendicitis, biliary tract diseases), acute transverse myelitis, allergic dermatitis (including allergic skin, allergic eczema, cutaneous atopy, atopic eczema, atopic dermatitis, skin inflammation, inflammatory eczema, inflammatory dermatitis, flea skin, miliaria dermatitis, miliaria eczema, dermatophagoides pteronyssinus skin), ankylosing spondylitis (reiter's syndrome), asthma, airway inflammation, atherosclerosis, arteriosclerosis, biliary atresia, bladder inflammation, breast cancer, cardiovascular inflammation (including vasculitis, rheumatoid nail-fold infarction, leg ulcer, polymyositis, chronic vasculitis, pericarditis, chronic obstructive pulmonary disease), chronic pancreatitis, peri-neuroinflammation, colitis (including amoeblitis, infectious colitis, bacterial colitis, chronic inflammatory bowel disease, peri-inflammatory bowel disease, colitis (including amoebic colitis), infectious colitis, bacterial colitis, chronic, Crohn's colitis, ischemic colitis, ulcerative colitis, idiopathic proctocolitis, inflammatory bowel disease, pseudomembranous colitis), collagen vascular disorders (rheumatoid arthritis, SLE, progressive systemic sclerosis, mixed connective tissue disease, diabetes), Crohn's disease (Crohn's disease, granulomatous ileitis, ileal colitis, inflammation of the digestive system), demyelinating diseases (including myelitis, multiple sclerosis, disseminated brain sclerosis, acute disseminated encephalomyelitis, perivenous demyelination, vitamin B12 deficiency, Guillain-Barre syndrome, MS-related retroviruses), dermatomyositis, diverticulitis, exudative diarrhea, gastritis, granulomatous hepatitis, granulomatous inflammation, cholecystitis, insulin-dependent diabetes, liver inflammatory diseases (primary biliary cirrhosis, liver fibrosis, chronic inflammation of the liver, chronic inflammation of the, Hepatitis, sclerosing cholangitis), lung inflammation (idiopathic pulmonary fibrosis, eosinophilic granuloma of the lung, lymphoproliferative pulmonary disease X, bronchiolitis, acute bronchitis), venereal lymphogranuloma, malignant melanoma, mouth/tooth diseases (including gingivitis, periodontal disease), mucositis, musculoskeletal system inflammation (myositis), non-alcoholic steatohepatitis (non-alcoholic fatty liver disease), eye & orbital inflammation (including uveitis, optic neuritis, peripheral rheumatoid ulcer, peripheral corneal inflammation), osteoarthritis, osteomyelitis, pharyngeal inflammation, polyarthritis, proctitis, psoriasis, radiation injury, sarcoidosis, sickle cell neocatathy, thrombophlebitis, systemic inflammatory response syndrome, thyroiditis, systemic lupus erythematosus, graft versus host disease, acute burn, behcet's syndrome, Sjogren's syndrome.

The terms patient and subject include human and veterinary subjects.

Human anti-MAdCAM antibodies and characterization thereof

In one embodiment, the invention provides anti-MAdCAM antibodies comprising human CDR sequences. In a preferred embodiment, the invention provides human anti-MAdCAM antibodies. In some embodiments, human anti-MAdCAM antibodies are produced by immunizing a non-human transgenic animal, such as a rodent, wherein the genome of the animal comprises human immunoglobulin genes such that the transgenic animal produces human antibodies. In some embodiments, the invention provides anti-MAdCAM antibodies that do not bind complement.

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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod. In another preferred embodiment, the anti-MAdCAM antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68 (with or without a signal sequence), or a variable region of any one of the 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 the group consisting of SEQ ID NOs: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64 (with or without a signal sequence), or an amino acid sequence of a variable region, or one or more CDRs from said amino acid sequence. Also encompassed by the invention are human anti-MAdCAM antibodies comprising the amino acid sequence from CDR1 to CDR3 of any one of the above-mentioned sequences. The invention further provides anti-MAdCAM antibodies comprising one or more FR regions of any of the above-mentioned sequences.

The invention further provides an anti-MAdCAM antibody comprising one of the aforementioned amino acid sequences, in which one or more modifications have been made. In some embodiments, cysteines in the antibody that may be chemically reactive are replaced with additional residues (such as, but not limited to, alanine or serine). In one embodiment, the substitution is at a non-canonical cysteine. Substitutions may be made in the CDRs or framework regions of the variable region of the antibody or in the constant region. In some embodiments, the cysteine is classical (canonical).

In some embodiments, the amino acid substitution is made to remove a potential proteolytic site in the antibody. This site may be present in the variable region CDRs or framework or constant regions of the antibody. Substitution of cysteine residues and removal of proteolytic sites can reduce heterogeneity in antibody products. In some embodiments, asparagine-glycine pairs forming potential deamidation sites can be removed by altering one or both of the residues. In some embodiments, amino acid substitutions are made to add or remove potential glycosylation sites in the variable region of the antibodies of the invention.

In some embodiments, the cleavage of the C-terminal lysine of the heavy chain of the anti-MAdCAM antibody of the invention. In various embodiments of the invention, the heavy and light chains of the anti-MAdCAM antibody may optionally include signal sequences.

In one aspect, the invention provides 12 inhibitory human anti-MAdCAM monoclonal antibodies and hybridoma cell lines producing them. Table 1 lists the nucleic acids encoding the full-length heavy and light chains (including the signal sequences) and the corresponding sequence identifiers for the full-length deduced amino acid sequences (SEQ ID NO:).

TABLE 1

In another aspect, the invention provides modified forms of certain of the above human anti-MAdCAM monoclonal antibodies. Table 2 lists the sequence identifiers of the DNA and protein sequences of the modified antibodies.

TABLE 2

Classes and subclasses of anti-MAdCAM antibodies

The antibody may be an IgG, IgM, IgE, IgA or IgD molecule. In a preferred embodiment, the antibody is of the IgG class and is IgG₁、IgG₂、IgG₃Or IgG₄Sub-classes. In a more preferred embodiment, the anti-MAdCAM antibody is an IgG₂Or IgG₄Sub-classes. In another preferred embodiment, the anti-MAdCAM antibody is of the same class and subclass as antibody 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod, being an IgG₂(ii) a Or the same class and subclass as 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1, or 9.8.2, is IgG₄。

The class and subclass of anti-MAdCAM antibodies can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for the particular class and subclass of antibody. These antibodies are commercially available. ELISA, western blotting and other techniques allow the class and subclass to be determined. Alternatively, class and subclass can be determined by sequencing all or part of the constant regions of the heavy and/or light chains of the antibody, comparing their amino acid sequences to known amino acid sequences of immunoglobulins of various classes and subclasses, and identifying the class and subclass of the antibody as the class exhibiting the highest sequence identity.

Species and molecular selectivity

In another aspect of the invention, the anti-MAdCAM antibody exhibits species and molecular selectivity. In one embodiment, the anti-MAdCAM antibody binds to human, cynomolgus or dog MAdCAM. In some embodiments, the anti-MAdCAM antibody does not bind to a new century monkey species such as marmoset. Following the teachings of the present specification, the species selectivity of anti-MAdCAM antibodies can be determined using methods well known in the art. For example, species selectivity can be determined using western blotting, FACS, ELISA, or immunohistochemistry. In a preferred embodiment, one can use immunohistochemistry to determine species selectivity.

In some embodiments, an anti-MAdCAM antibody that specifically binds MAdCAM is at least 10-fold, preferably at least 20, 30, 40, 50, 60, 70, 80, or 90-fold, and most preferably at least 100-fold more selective for MAdCAM than for VCAM, fibronectin, or any other antigen. In a preferred embodiment, the anti-MAdCAM antibody does not exhibit any significant binding to VCAM, fibronectin or any other antigen other than MAdCAM. The selectivity of anti-MAdCAM antibodies for MAdCAM can be determined using methods well known in the art in accordance with the teachings of the present specification. For example, one can determine this selectivity using western blotting, FACS, ELISA, or immunohistochemistry.

Binding affinity of anti-MAdCAM antibodies to MAdCAM

In another aspect of the invention, the anti-MAdCAM antibody specifically binds to MAdCAM with high affinity. In one embodiment, when surface plasmon resonance is used, such as BIAcore measured at 3X 10 anti-MAdCAM antibody^-8K of M or less_dSpecifically binds to MAdCAM. In a more preferred embodiment, the antibody is at 1 × 10^-8Or less or 1X 10^-9K of M or less_dSpecifically binds to MAdCAM. In an even more preferred embodiment, the antibody is at 1 × 10^-10K of M_dOr less K_dSpecifically binds to MAdCAM. In other preferred embodiments, the antibodies of the invention are present at 2.66X 10^-10M or less, 2.35X 10^-11M or less or 9X 10^-12K of M or less_dSpecifically binds to MAdCAM. In another preferred embodiment, the antibody is at 1 × 10^-11K of M or less_dSpecifically binds to MAdCAM. In another preferred embodiment, the antibody has substantially the same K 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod_dSpecifically binds to MAdCAM. K with reference antibody in the same assay_dIn comparison, having a K "substantially identical to that of the reference antibody_d"K of antibody_dIs. + -.100 pM, preferably. + -.50 pM, more preferably. + -.20 pM, still more preferably. + -.10 pM,. + -.5 pM or. + -.2 pM. In another preferred embodiment, the antibody has substantially the same K as an antibody comprising one or more variable regions 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod_dBinds to MAdCAM. In another preferred embodiment, the antibody binds to a polypeptide comprising an amino acid sequence selected from the group consisting of 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 or 68, or a variable region thereof, and a pharmaceutically acceptable carrier or excipient_dBinds to MAdCAM. In another preferred embodiment, the antibody is conjugated to a peptide comprising one or more C from an antibodySubstantially identical K of antibody to DR_dBinds to MAdCAM, said certain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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, or 68.

The binding affinity of an anti-MAdCAM antibody to MAdCAM can be determined by any method known in the art. In one embodiment, binding affinity can be determined by competitive ELISA, RIA or surface plasmon resonance such as BIAcore. In a more preferred embodiment, the binding affinity is determined by surface plasmon resonance. In an even more preferred embodiment, the binding affinity and dissociation rate can be determined by BIAcore. An example of determining binding affinity is described in example II below.

Half-life of anti-MAdCAM antibodies

According to another object of the invention, the anti-MAdCAM antibody has a half-life of at least 1 day in vivo or in vitro. In a preferred embodiment, the antibody or portion thereof has a half-life of at least 3 days. In a more preferred embodiment, the antibody or portion thereof has a half-life of 4 days or more. In another embodiment, the antibody or portion thereof has a half-life of 8 days or more. As discussed below, in another embodiment, the antibody or antigen binding portion thereof is derivatized or modified to provide it with a longer half-life. In another preferred embodiment, the antibody may comprise a point mutation to increase serum half-life as described in WO00/09560, published 24.2.2000.

Antibody half-life can be measured by any method known to those of ordinary skill in the art. For example, antibody half-life may be measured by western blotting, ELISA or RIA at appropriate time periods. Antibody half-life can be measured in any suitable animal, e.g., a primate such as a cynomolgus monkey or a human.

Identification of MAdCAM epitopes recognized by anti-MAdCM antibodies

The invention also provides human anti-MAdCAM antibodies that bind to the same antigen or epitope as the human anti-MAdCAM antibodies provided herein. In addition, the present invention provides human anti-MAdCAM antibodies that compete or cross-compete with human anti-MAdCAM antibodies. 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, 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 human anti-MAdCAM antibody comprises one or more variable regions or one or more CDRs from 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, 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 human anti-MAdCAM antibody comprises an amino acid sequence selected from the group consisting of 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 or 68 (with or without a signal sequence) or a variable region thereof. In another preferred embodiment, the human anti-MAdCAM antibody comprises one or more CDRs from a polypeptide comprising an amino acid sequence selected from the group consisting of 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 or 68. In a highly preferred embodiment, the anti-MAdCAM antibody is another human antibody.

Various methods known in the art can be used to determine whether an anti-MAdCAM antibody binds to the same antigen as another anti-MAdCAM antibody. For example, one known anti-MAdCAM antibody can be used to capture the antigen, elute the antigen from the anti-MAdCAM antibody, and then determine whether the test antibody will bind to the eluted antigen. Whether an antibody competes with an anti-MAdCAM antibody can be determined 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 capable of binding to MAdCAM at the same time as the anti-MAdCAM antibody, the test antibody binds to a different epitope than the anti-MAdCAM antibody. However, if the test antibody does not bind to MAdCAM at the same time, the test antibody competes with the human anti-MAdCAM antibody. The experiment can be performed using ELISA or surface plasmon resonance or preferably with BIAcore. To test whether an anti-MAdCAM antibody cross competes with another anti-MAdCAM antibody, the competition method described above can be used in both directions, i.e., to determine whether a known antibody blocks the test antibody, or vice versa.

Light and heavy chain gene utilization

The invention also provides anti-MAdCAM antibodies comprising a light chain variable region encoded by the human kappa gene. In a preferred embodiment, the light chain variable region is encoded by the human vka 2, A3, a26, B3, O12 or O18 gene family. In various embodiments, the light chain comprises no more than 11, no more than 6, or no more than 3 amino acid substitutions from a germline human vka 2, A3, a26, B3, O12, or O18 sequence. In a preferred embodiment, the amino acid substitution is a conservative substitution.

SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 provide the amino acid sequence of the full length kappa light chain of 12 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. FIG. 1K-1T is an alignment of the light chain variable region amino acid sequences of 12 anti-MAdCAM antibodies and their germline sequences derived therefrom. Fig. 2A shows an alignment of the light chain variable region amino acid sequences of the kappa light chains of the 12 anti-MAdCAM antibodies with respect to each other. One of ordinary skill in the art, in light of the teachings of this specification, can determine the differences between germline sequences and antibody sequences of additional anti-MAdCAM antibodies. SEQ ID NO: 54. 58, 62, 66 or 68 provide the amino acid sequences of the full length kappa light chains of 5 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 substitutions, respectively, from their parent anti-MAdCAM antibody, i.e., 6.22.2, 6.34.2, 6.67.1, 6.77.1 or 7.26.4.

In a preferred embodiment, the VL of the anti-MAdCAM antibody comprises the same mutation, relative to the germline amino acid sequence, as the VL of any one or more 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod body or 7.26.4-mod. The invention includes anti-MAdCAM antibodies that utilize the same human vk and human Jk genes as the exemplified antibodies. In some embodiments, the antibody comprises one or more mutations from the same germline as the one or more exemplary antibodies. In some embodiments, the antibody comprises different substitutions at the same one or more positions as the one or more exemplary antibodies. For example, the VL of an anti-MAdCAM antibody may contain one or more amino acid substitutions identical to those present in antibody 7.16.6, and another amino acid substitution identical to antibody 7.26.4. In this way, different characteristics of antibody binding can be mixed and matched to alter, for example, the affinity of the antibody for MAdCAM or its off-rate from antigen. In another embodiment, the mutation occurs at the same position as the position of the mutation found in any one or more of the VLs 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod, but with conservative amino acid substitutions instead of with the same amino acid. For example, an amino acid substitution in one 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod, or 7.26.4-mod may conservatively replace an aspartic acid if the amino acid substitution is glutamic acid, as compared to the germline. Similarly, if the amino acid substitution is serine, a threonine may be conservatively substituted.

In another preferred embodiment, the light chain comprises an amino acid sequence identical to 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another very preferred embodiment, the light chain comprises the same amino acid sequence as the light chain CDR region 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, 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 having at least one CDR region of a 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 or 7.26.4-mod light chain. In another preferred embodiment, the light chain comprises amino acid sequences having CDRs from different light chains using the same Vk and Jk genes. In a more preferred embodiment, the CDRs from the 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, 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 a sequence selected from SEQ ID NOs: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 64, 66 or 68, with or without a signal sequence. In another embodiment, the light chain comprises an amino acid sequence consisting of a sequence selected from the group consisting of SEQ ID NOs: 3.7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67 (with or without a signal sequence), or by a nucleotide sequence encoding an amino acid sequence having 1-11 amino acid insertions, deletions or substitutions compared thereto. Preferably, the amino acid substitution is a conservative amino acid substitution. In another embodiment, the antibody or portion thereof comprises a lambda light chain.

The invention also provides anti-MAdCAM antibodies or portions thereof comprising human VH gene sequences or sequences derived from human VH genes. In one embodiment, the heavy chain amino acid sequence is derived from the human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33, or 4-4 gene family. In various embodiments, the heavy chain comprises no more than 15, no more than 6, or no more than 3 amino acid changes from a germline human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33, or 4-4 gene sequence.

SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 provide the amino acid sequences of the full-length heavy chains of the 12 anti-MAdCAM antibodies. FIGS. 1A-1J are alignments of the amino acid sequences of the heavy chain variable regions of 12 anti-MAdCAM antibodies and their germline sequences derived therefrom. Fig. 2B shows an alignment of the amino acid sequences of the heavy chain variable regions of the 12 anti-MAdCAM antibodies with each other. Based on the teachings of the present specification and the nucleotide sequences of the present invention, one of ordinary skill in the art can determine the encoded amino acid sequences of the 12 anti-MAdCAM antibody heavy chains and germline heavy chains, and can determine differences between germline and antibody sequences. SEQ ID NO: 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, which were modified by amino acid substitutions from their parent anti-MAdCAM antibodies 6.22.2, 6.34.2 and 6.67.1, respectively. An additional modified anti-MAdCAM antibody 7.26.4-mod having the amino acid sequence of SEQ ID NO: 42, full length heavy chain amino acid sequence.

In a preferred embodiment, the VH of the anti-MAdCAM antibody comprises the same mutation, relative to the germline amino acid sequence, as the VH of any one or more 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. Similar to the discussion above, the antibody comprises one or more mutations from the same germline as the one or more exemplary antibodies. In some embodiments, the antibody comprises different substitutions at one or more of the same positions as the one or more exemplary antibodies. For example, the VH of an anti-MAdCAM antibody may contain one or more amino acid substitutions identical to those present in antibody 7.16.6, and another amino acid substitution identical to antibody 7.26.4. In this way, one can mix different characteristics of antibody binding to alter, for example, the affinity of the antibody for MAdCAM or its dissociation rate from antigen. In another embodiment, the amino acid substitution occurs at the same position as a substitution found in any one or more VH of the 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod from the germline, but with a different residue, which substitution is a conservative substitution compared to the reference antibody.

In another preferred embodiment, the heavy chain comprises an amino acid sequence identical to 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another very preferred embodiment, the heavy chain comprises the same amino acid sequence as the heavy chain CDR region 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, 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 the amino acid sequence of at least one CDR region from 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, 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 amino acid sequences having CDRs from different heavy chains. In a more preferred embodiment, the CDRs from the 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, 6.22.2-mod, 6.34.2-mod, 6.67-mod, 6.77.1-mod or 7.26.4-mod. In another preferred embodiment, the heavy chain comprises a sequence selected from SEQ ID NOs: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64, with or without a signal sequence. In another embodiment, the heavy chain comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63, or a nucleotide sequence encoding an amino acid sequence having 1-15 amino acid insertions, deletions, or substitutions as compared to the above. In another embodiment, the substitution is a conservative amino acid substitution.

Antibodies and antibody-producing cellsMethod for generating a system

Immunization method

In one embodiment of the invention, human antibodies are produced by immunizing a non-human animal containing some or all of the human immunoglobulin heavy and light chain loci with a MAdCAM antigen. In a preferred embodiment, the non-human animal is a XENOMOUSE animal that is an engineered mouse strain containing a large fragment of a human immunoglobulin locus and defective in mouse antibody production. See, e.g., Green et al, Nature Genetics 7: 13-21(1994) and U.S. Pat. Nos.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 0009560 and WO 00/037504. The XENOMOUSE^TMAnimals produce fully human antibody repertoires as adults and produce antigen-specific human mabs. Second generation XENOMOUSE^TMAnimals contained about 80% of the repertoire of human antibody V genes by introducing germline-configured YAC fragments of the megabase-sized human heavy chain locus and kappa light chain locus. In other embodiments, XENOMOUSE^TMMice contain approximately all of the human heavy and lambda light chain loci. See Mendez et al, Nature Genetics 15: 146-: 483-495(1998), the disclosure of which is incorporated herein by reference.

The invention also provides methods for producing anti-MAdCAM antibodies from non-human, non-mouse animals by immunizing non-human transgenic animals comprising human immunoglobulin loci. One can produce these animals using the methods described immediately above. The methods disclosed in these documents may be modified as described in U.S. patent 5,994,619 ("the' 619 patent"), which is incorporated herein by reference. The' 619 patent describes a method for generating new Cultured Inner Cell Mass (CICM) cells and cell lines, which are 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 for producing transgenic animals capable of delivering heterologous DNA to their progeny. In a preferred embodiment, the non-human animal may be a rat, sheep, pig, goat, cow or horse.

In another embodiment, the non-human animal containing a human immunoglobulin locus is an animal having a "minilocus" (minilocus) of a human immunoglobulin. In the minilocus approach, the exogenous Ig locus is mimicked by the inclusion of a single gene from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region and an additional constant region (preferably a gamma constant region) constitute one construct for insertion into an animal. The process is described in particular in U.S. Pat. Nos.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, which are incorporated herein by reference.

The advantage of the small locus approach is that it is rapid, and constructs comprising portions of the Ig locus can be rapidly produced and introduced into animals. However, one potential drawback of the small locus approach is that there may not be sufficient immunoglobulin diversity to support complete B cell development, and thus antibody production may be low.

To produce human anti-MAdCAM antibodies, a non-human animal containing some or all of the human immunoglobulin loci is immunized with a MAdCAM antigen and the antibodies or antibody-producing cells are isolated from the animal. The MAdCAM antigen may be isolated and/or purified MAdCAM and is preferably human MAdCAM. In another embodiment, the MAdCAM antigen is a MAdCAM fragment, preferably the extracellular domain of MAdCAM. In another embodiment, the MAdCAM antigen is a fragment comprising 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 animals can be accomplished 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 animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, for example, Harlow and Lane and U.S. patent 5,994,619. In a preferred embodiment, the MAdCAM antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering the polypeptide in localized deposits, or they may contain substances that stimulate the host to secrete chemotactic factors for macrophages and other components of the immune system. Preferably, if the polypeptide is administered, the vaccination schedule will comprise two or more administrations of the polypeptide, spread over several weeks.

Example I provides immunization of XENOMOUSE with full-length human MAdCAM in phosphate buffered saline^TMAnimal protocol.

Production of antibodies and antibody-producing cell lines

After immunizing an animal with MAdCAM antigen, antibodies and/or antibody-producing cells can be obtained from the animal. Sera containing anti-MAdCAM antibodies can be obtained from the animal by exsanguinating or by sacrificing the animal. The serum can be used as it is obtained from an animal, the immunoglobulin fraction can be obtained from the serum, or anti-MAdCAM antibodies can be purified from the serum.

In another embodiment, an antibody-producing immortalized cell line can be prepared from an immunized animal. Following immunization, the animals are 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 culturing them under conditions that select for immortalized cells, subjecting them to the effects of an oncogenic or mutant compound, fusing them with immortalized cells such as myeloma cells, and inactivating tumor suppressor genes. See, e.g., Harlow and Lane, supra. In embodiments involving myeloma cells, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After immortalization and selection with antibiotics, the immortalized cells or culture supernatant thereof are screened using MAdCAM, a portion thereof, or cells expressing MAdCAM. In a preferred embodiment, the primary screening can be performed using enzyme-linked immunoassays (ELISA) or Radioimmunoassays (RIA), preferably using ELISA. Examples of ELISA screens are provided in PCT publication No. WO 00/37504, which is incorporated herein by reference.

In another embodiment, antibody-producing cells can be prepared from a human having an autoimmune disorder and expressing an anti-MAdCAM antibody. Cells expressing anti-MAdCAM antibodies can be isolated by isolating leukocytes and subjecting them to Fluorescence Activated Cell Sorting (FACS) or by panning on plates coated with MAdCAM or portions thereof. These cells can be fused with human non-secreted myeloma to produce human hybridomas expressing human anti-MAdCAM antibodies. In general, this is a less preferred embodiment, since it is possible that anti-MAdCAM antibodies have a low affinity for MAdCAM.

Cells, such as hybridomas, that produce anti-MAdCAM antibodies are selected, cloned, and further screened for desirable characteristics, including healthy cell growth, high antibody production, and desirable antibody characteristics, as discussed further below. Hybridomas can be cultured and expanded in vivo in syngeneic animals, in animals lacking the immune system, such as nude mice, or in cell culture in vitro. Methods for 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 B cells of the spleen are fused to a myeloma derived from the same species as the non-human animal. More preferably, the immunized animal is XeNOMOUS^TMAnimals and myeloma cell lines are non-secretory mouse myelomas, e.g., the myeloma cell line is P3-X63-AG8-653 (ATCC). See, e.g., example I.

Accordingly, in one embodiment, the invention provides a method for generating a cell line capable of producing a human monoclonal antibody or fragment thereof against MAdCAM, the method 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 the transgenic animal; (d) immortalizing the antibody-producing cell; (e) establishing an independent monoclonal population of immortal antibody-producing cells; and (f) screening the immortalized antibody-producing cells or culture supernatant thereof to identify antibodies to MAdCAM.

In one aspect, the invention provides hybridomas that produce human anti-MAdCAM antibodies. In a preferred embodiment, the hybridoma is a mouse hybridoma, as described above. In another embodiment, the hybridoma is produced in a non-human, non-mouse species such as rat, sheep, pig, goat, cow, or horse. In another embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused to a human cell expressing an anti-MAdCAM antibody.

Nucleic acids, vectors, host cells and recombinant methods for producing antibodies

Nucleic acids

Nucleic acid molecules encoding anti-MAdCAM antibodies of the invention are provided. In one embodiment, the nucleic acid molecule encodes the heavy and/or light chain of an anti-MAdCAM immunoglobulin. In a preferred embodiment, a single nucleic acid molecule encodes the heavy chain of an anti-MAdCAM immunoglobulin and another nucleic acid molecule encodes the light chain of an anti-MAdCAM immunoglobulin. In a more preferred embodiment, the encoded immunoglobulin is a human immunoglobulin, preferably human IgG. The encoded light chain may be a lambda chain or a kappa chain, preferably a kappa chain.

In a preferred embodiment the nucleic acid molecule encoding the variable region of the light chain comprises the germline sequence of the human vka 2, A3, a26, B3, O12 or O18 gene or a variant of said sequence. In a preferred embodiment, the nucleic acid molecule encoding the light chain comprises a sequence derived from a human jk 1, jk2, jk 3, jk 4 or jk 5 gene. In a preferred embodiment, the nucleic acid molecule encoding the light chain encodes no more than 11 amino acid changes from the germline a2, A3, a26, B3, O12 or O18 vk gene, preferably no more than 6 amino acid changes, and more preferably no more than 3 amino acid changes. In a more preferred embodiment, the nucleic acid encoding the light chain is a germline sequence.

The invention provides nucleic acid molecules encoding a light chain variable region (VL) comprising up to 11 amino acid changes compared to a germline sequence, wherein the amino acid changes are identical to amino acid changes in the VL from the germline sequence of one 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The invention also provides nucleic acid molecules comprising a nucleotide sequence encoding an amino acid sequence of the light chain variable region 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. The invention also provides nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of one or more 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, 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 molecule comprises a nucleotide sequence encoding the amino acid sequences of all 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, 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 molecule comprises a nucleotide sequence encoding SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68 or a nucleotide sequence comprising the amino acid 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 molecule comprises a nucleotide sequence encoding SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, or a nucleotide sequence comprising the amino acid sequence of one or more 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 a more preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding seq id NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66, 68, or a nucleotide sequence comprising the amino acid sequences of all 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 invention also provides nucleic acid molecules encoding amino acid sequences of VLs having amino acid sequences identical to those described above, in particular to those comprising SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical in VL of the amino acid sequence. The invention also provides a polypeptide having an amino acid sequence substantially similar to SEQ ID NO: 3.7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65, or 67, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

In another embodiment, the invention provides nucleic acid molecules that hybridize under highly stringent conditions to a nucleic acid molecule encoding a VL as described above, particularly to a nucleic acid molecule comprising a nucleotide sequence encoding a VL as set forth in SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68, or a pharmaceutically acceptable salt thereof. The invention also provides methods of treating a mammal under highly stringent conditions with a composition comprising SBQ ID NO: 3.7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 53, 57, 61, 65 or 67.

The invention also provides nucleic acid molecules encoding the variable regions of the heavy chains (VH) utilizing the human VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33 or 4-4VH genes. In some embodiments, the nucleic acid molecule encoding a VH gene further utilizes a gene of the human JH4 or JH6 family. In some embodiments, the nucleic acid molecule encoding a VH gene utilizes a JH4b or JH6b gene. In another embodiment, the nucleic acid molecule comprises a sequence derived from a human D3-10, 4-23, 5-5, 6-6, or 6-19 gene. In a more preferred embodiment, the nucleic acid molecule encoding a VH comprises no more than 15 amino acid changes, preferably no more than 6 amino acid changes, and more preferably no more than 3 amino acid changes, from a germline VH 1-18, 3-15, 3-21, 3-23, 3-30, 3-33, or 4-4 gene. In a highly preferred embodiment, the nucleic acid molecule encoding a VH comprises at least one amino acid change compared to the germline sequence, wherein the amino acid change is identical to an amino acid change in the heavy chain self germline sequence 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In a more preferred embodiment, the VH comprises no more than 15 amino acid changes from germline sequence, wherein the changes are consistent with a change in the VH from germline sequence for one 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, 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 molecule comprises a nucleotide sequence encoding an amino acid sequence of a 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, 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 molecule comprises a nucleotide sequence encoding the amino acid sequence of one or more 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, 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 molecule comprises a nucleotide sequence encoding the amino acid sequences of all 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, 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 molecule comprises a nucleic acid sequence encoding SEQ id no: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64, or a nucleotide sequence comprising the amino acid sequence of one of SEQ ID NOs: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63. In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64, or a nucleotide sequence comprising the amino acid sequence of one or more CDRs of any one of SEQ ID NOs: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63. In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64, or a nucleotide sequence comprising the amino acid sequences of all the CDRs of any one of SEQ ID NOs: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63, or a pharmaceutically acceptable salt thereof. In some embodiments the nucleic acid molecule comprises a nucleotide sequence encoding a contiguous region of the heavy or light chain of any of the above-mentioned anti-MAdCAM antibodies starting from CDR1 and ending at CDR 3.

In another embodiment, the nucleic acid molecule encodes an amino acid sequence of a VH that is identical to one of the amino acid sequences encoding a VH as described immediately above, in particular to a VH comprising the amino acid sequence of SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or 64 is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. The invention also provides a polypeptide having an amino acid sequence substantially similar to SEQ ID NO: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63, is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

In another embodiment, the nucleic acid molecule encoding a VH hybridizes under highly stringent conditions to the nucleotide sequence encoding a VH described above, in particular to a nucleic acid molecule comprising SEQ ID NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60 or 64. The invention also provides methods of producing a polypeptide that hybridizes under highly stringent conditions to a nucleic acid comprising SEQ ID NO: 1.5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 51, 55, 59, or 63, and a nucleic acid molecule encoding a VH.

The nucleotide sequence encoding one or both of the entire heavy and light chains of an anti-MAdCAM antibody, or the variable region thereof, can be obtained from any source that produces anti-MAdCAM antibodies. Methods for 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 can be used to generate cDNA for use in Polymerase Chain Reaction (PCR) or cDNA cloning of antibody genes.

In one embodiment of the invention, the nucleic acid molecule may be obtained from a hybridoma expressing an anti-MAdCAM antibody as described above, preferably a hybridoma having as one of its fusion partners a transgenic animal cell expressing a human immunoglobulin gene, e.g., xenomine^TMAn animal, a non-human mouse transgenic animal, or a cell of a non-human, non-mouse transgenic animal. In another embodiment, the hybridoma is derived from a non-human, non-transgenic animal, which can be used, for example, to humanize the antibody.

A nucleic acid molecule encoding the entire heavy chain of an anti-MAdCAM antibody can be constructed by fusing a nucleic acid molecule encoding the entire variable region of the heavy chain or an antigen-binding domain thereof to a nucleic acid molecule encoding the constant region of the heavy chain. Similarly, a nucleic acid molecule encoding an anti-MAdCAM antibody light chain can be constructed by fusing a nucleic acid molecule encoding a light chain variable region or an antigen-binding domain thereof with a nucleic acid molecule encoding a constant region of a light chain. Nucleic acid molecules encoding the VH and VL regions can be converted to full-length antibody genes by inserting them into expression vectors that already encode the heavy and light chain constant regions, respectively, such that the VH fragment is operatively linked to a heavy chain constant region (CH) fragment within the vector and the VL fragment is operatively linked to a light chain constant region (CL) fragment within the vector. Alternatively, nucleic acid molecules encoding a VH or VL chain are converted to full-length antibody genes by linking, e.g., linking, nucleic acid molecules encoding a VH chain to nucleic acid molecules encoding a CH chain using standard molecular biology techniques. The same results can be achieved using nucleic acid molecules encoding VL and CL chains. The sequences of human heavy and light chain constant region genes are known in the art. See, for example, Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed., NIHPubl. No.91-3242 (1991). The nucleic acid molecules encoding the full-length heavy and/or light chains can then be expressed by cells into which the nucleic acid molecules have been introduced and the anti-MAdCAM antibodies isolated.

In a preferred embodiment, the nucleic acid encoding the heavy chain variable region encodes seq id NO: 2.6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 52, 56, 60, or 64, and the nucleic acid molecule encoding the light chain variable region encodes the amino acid sequence of SEQ ID NO: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 54, 58, 62, 66 or 68.

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

As described below, the nucleic acid molecules can be used to recombinantly express large quantities of anti-MAdCAM antibodies. As described further below, the nucleic acid molecules can also be used to produce chimeric antibodies, single chain antibodies, immunoadhesins, bispecific antibodies, mutated antibodies and antibody derivatives. Also described below, the nucleic acid molecule can be used to humanize an antibody if the nucleic acid molecule is derived from a non-human, non-transgenic animal.

In another embodiment, the nucleic acid molecules of the invention can be used as probes or PCR primers for specific antibody sequences. For example, nucleic acid molecule probes can be used in diagnostic methods or nucleic acid molecule PCR primers can be used to amplify DNA regions that are particularly useful for isolating nucleotide sequences for generating anti-MAdCAM antibody variable regions. In a preferred embodiment, the nucleic acid molecule is an oligonucleotide. In a more preferred embodiment, the oligonucleotides are derived from hypervariable regions of the heavy and light chains of the antibody of interest. In a more preferred embodiment, the oligonucleotide encodes all or part of one or more CDRs.

Carrier

The invention provides a vector comprising a nucleic acid molecule of the invention encoding a heavy chain or an antigen-binding portion thereof. The invention also provides a vector comprising a nucleic acid molecule of the invention encoding a light chain or an antigen-binding portion thereof. The invention also provides vectors comprising nucleic acid molecules encoding the fusion proteins, modified antibodies, antibody fragments, and probes thereof.

To express the antibody or antibody portion of the present invention, DNA encoding partial or full-length light and heavy chains obtained as described above is inserted into an expression vector, and thus the gene is 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, YAC, EBV-derived episomes, and the like. The antibody genes are ligated into a vector such that the transcriptional and translational control sequences in the vector exert their intended function of regulating the transcription and translation of the antibody genes. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The genes for the antibody light chain and the genes for the antibody heavy chain may be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody gene can be inserted into the expression vector by standard methods, such as linking the antibody gene fragment to complementary restriction sites on the vector, or blunt end ligation if there are no restriction sites.

A convenient vector is one which encodes a fully functional human CH or CL immunoglobulin sequence, by designing appropriate restriction sites to allow any VH or VL sequence to be readily inserted and expressed as described above. In such vectors, splicing typically occurs between the splice donor site of the inserted J region and the splice acceptor site preceding the human C region, as well as in the splice region present in the human CH exon. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may 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 gene, the recombinant expression vector of the present invention carries regulatory sequences that control the expression of the antibody chain gene in a host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of host cell to be transformed, the level of protein expression desired, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements which direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers from retrovirus LTR, Cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian virus 40(SV40) (e.g., SV40 promoter/enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Pat. Nos.5,168,062, 4,510,245, and 4,968,615, each of which is incorporated herein by reference. Methods for expressing antibodies in plants, including descriptions of promoters and vectors and transformation of plants, are known in the art. See, for example, U.S. patent 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Selectable marker genes facilitate the selection 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 a selectable marker gene confers resistance to a host cell into which the vector has been introduced, e.g., against G418, hygromycin or methotrexate. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR with methotrexate selection/amplification)^-Host cells) and neo genes (for G418 selection), as well as glutamate synthase genes.

Non-hybridoma host cells and methods for recombinantly producing proteins

Nucleic acid molecules encoding the heavy and/or light chains of anti-MAdCAM antibodies or antigen-binding portions thereof, and vectors comprising these nucleic acid molecules, can be used to transform suitable mammalian, plant, bacterial or yeast host cells. Transformation can be accomplished by any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, biolistic injection, and direct microinjection of DNA into the nucleus. Alternatively, the nucleic acid molecule may be introduced into a mammalian cell by a viral vector. Methods for transforming cells are well known in the art. See, for example, U.S. patent nos.4,399,216, 4,912,040, 4,740,461 and 4,959,455 (which patents are incorporated herein by reference). Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines useful 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 alia, 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 many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have 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 a recombinant expression vector encoding a heavy chain or antigen-binding portion thereof, a light chain and/or an antigen-binding portion thereof is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a period of time sufficient for the antibody to be expressed in the host cell, or more preferably, for the antibody to be secreted into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, tobacco, Arabidopsis, duckweed, maize, wheat, potato, and the like. Bacterial host cells include E.coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Saccharomyces methanolica.

Moreover, many known techniques can be used to enhance expression of the antibodies of the invention from the producing cell line (or other portions therefrom). For example, the glutamine synthetase gene expression system (GS system) is a common method for improving expression under certain conditions. The GS system is discussed in whole or in part in european patents nos. 0216846, 0256055, 0338841 and 0323997.

It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by or comprising the amino acid sequences provided herein are part of the invention, and glycosylation of the antibody need not be considered.

Transgenic animals and plants

The invention also provides transgenic non-human animals and transgenic plants comprising one or more nucleic acid molecules of the invention useful for producing antibodies of the invention. Antibodies can be produced and recovered in goat, cow, horse, pig, rat, mouse, rabbit, hamster, or other mammalian tissue or body fluids, such as milk, blood, or urine. See, for example, U.S. patent nos.5,827,690, 5,756,687, 5,750,172 and 5,741,957. As described above, a non-human transgenic animal comprising a human immunoglobulin locus can be immunized with MAdCAM or a portion thereof. Methods for producing antibodies in plants are described, for example, in U.S. Pat. Nos. 6,046,037 and 5,959,177, which are incorporated herein by reference.

In another embodiment, non-human transgenic animals and transgenic plants can be produced by introducing one or more nucleic acid molecules of the invention into an animal or plant by standard transgenic techniques. See Hogan, supra. The transgenic cell used to produce the transgenic animal may be an embryonic stem cell, a somatic cell, or a fertilized egg cell. The transgenic non-human organism may be chimeric, non-chimeric heterozygote, and non-chimeric homozygote. See, e.g., Hogan et al, Manipastinghe Mouse Embryo: a Laboratory Manual 2ed., Cold Spring harborPress (1999); jackson et al, Mouse Genetics and Transgenics: APracial Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: a Laboratory Handbook, academic Press (1999). In another embodiment, the transgenic non-human organism may have targeted breaks and substitutions encoding the heavy and/or light chains of interest. In a preferred embodiment, the transgenic animal or plant comprises and expresses nucleic acid molecules encoding a heavy chain and a light chain that combine to specifically bind to MAdCAM, preferably human MAdCAM. In another embodiment, the transgenic animal or plant comprises a nucleic acid molecule encoding a modified antibody, such as a single chain antibody, a chimeric antibody, or a humanized antibody. anti-MAdCAM antibodies can be produced in any transgenic animal. In a preferred embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cow, or horse. The non-human transgenic animal expresses the encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

Phage display libraries

The present invention provides a method for producing an anti-MAdCAM antibody or an antigen-binding portion thereof, comprising the steps of synthesizing a human antibody library on a bacteriophage, screening the library with MAdCAM or a portion thereof, isolating the phage that binds MAdCAM, and obtaining the antibody from the phage. A method of preparing an antibody library comprising the steps of: immunizing a non-human host animal containing a human immunoglobulin locus with MAdCAM or an antigenic portion thereof to generate an immune response, and extracting cells responsible for the production of the antibody from the host animal; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector for expression of the antibody on the phage. The recombinant anti-MAdCAM antibodies of the invention can be obtained by this method.

In addition to the anti-MAdCAM antibodies disclosed herein, recombinant anti-MAdCAM human antibodies of the invention can be isolated by screening recombinant combinatorial antibody libraries, preferably scFv phage display libraries, prepared using human VL and VH cdnas prepared from mRNA isolated from human lymphocytes. Methods for preparing and screening such libraries are known in the art. Kits for generating Phage display libraries are commercially available (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 to generate and screen 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. WO92/20791; 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-, nuc 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 desirable characteristics, the epitope imprinting method described in Hoogenboom et al, PCT publication No. wo 93/06213 is used, first to select human heavy and light chain sequences having similar binding activity to MAdCAM using human anti-MAdCAM antibodies as described herein. Antibody libraries used in such methods are preferably antibodies such as mccaferty et al, PCT publication No. wo 92/01047, mccaferty et al, Nature, 348: 552 (1990); and Griffiths et al, EMBO J, 12: 725-734(1993) describe the preparation and screening of scFv libraries. The scFv antibody library is preferably screened using human MAdCAM as antigen.

Once the initial human VL and VH fragments have been selected, a "mix and match" assay is performed in which different pairs of initially selected VL and VH fragments are screened for MAdCAM binding to select preferred VL/VH pair combinations. In addition, to further improve antibody quality, the VL and VH segments of preferred VL/VH pairs are preferably randomly mutated in the CDR3 regions of VH and/or VL using methods analogous to the in vivo somatic mutation process responsible for antibody affinity maturation during innate immune responses. This in vivo affinity maturation can be achieved by amplifying the VH and VL regions using PCR primers complementary to VH CDR3 or VL CDR3 respectively, which have been "spiked" with a random mixture of four nucleotide bases at a position such that the resulting PCR product encodes VH and VL fragments, and random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL fragments can be screened again for binding to MAdCAM.

After screening and isolating the anti-MAdCAM antibodies of the invention from the recombinant immunoglobulin display library, the nucleic acids encoding the selected antibodies can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. The nucleic acid can be further manipulated, if desired, to produce other antibody formats of the invention, as described below. To express the recombinant human antibodies isolated by screening combinatorial libraries, DNA encoding the antibodies is cloned into a recombinant expression vector and introduced into mammalian host cells as described above.

Class conversion

Another aspect of the invention is to provide a mechanism for switching the class of anti-MAdCAM antibodies to another class. In one aspect of the invention, a nucleic acid molecule encoding a VL or VH is isolated such that it does not include any nucleotide sequences encoding CL or CH, using methods well known in the art. The nucleic acid molecule encoding the VL or VH is then operably linked to nucleotide sequences encoding CL or CH from different classes of immunoglobulin molecules. As described above, this can be accomplished using a vector or nucleic acid molecule comprising a sequence encoding CL or CH. For example, anti-MAdCAM antibodies, which were originally IgM, can be class-switched to IgG. Furthermore, class switching can be used to switch one IgG subclass to another, e.g., from IgG₄Conversion to IgG₂. A preferred method for producing an antibody of the invention comprising a desired isotype or antibody subclass comprises the steps of: isolating nucleic acid encoding an anti-MAdCAM antibody heavy chain and nucleic acid encoding an anti-MAdCAM antibody light chain, obtaining the variable region of the heavy chain, linking the heavy chain variable region to a heavy chain constant region of a desired isotype, expressing the light chain and the linked heavy chain in a cell, and collecting the anti-MAdCAM antibody having the desired isotype.

Antibody derivatives

The above-described nucleic acid molecules can be used to generate antibody derivatives using techniques and methods known to those of ordinary skill in the art.

Humanized antibodies

The immunogenicity of non-human antibodies can be reduced to some extent using humanization techniques, possibly using display techniques using appropriate libraries. It is understood that murine antibodies or antibodies from other species may be humanized or primatized using techniques well known in the art. See, e.g., Winter and Harris, immunological Today, 14: 43-46(1993) and Wright et al, Crit. reviews in immunol., 12125-168 (1992). The target antibody can be engineered by recombinant DNA techniques to replace C with the corresponding human sequence_H1、C_H2、C_H3. Hinge and/or framework regions (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, C is replaced by the corresponding human sequence of an anti-MAdCAM antibody of the invention_H1. Hinge region, C_H2、C_H3 and/or the framework regions humanize a non-human anti-MAdCAM antibody.

Mutated antibodies

In another embodiment, the nucleic acid molecules, vectors, and host cells can be used to produce mutant anti-MAdCAM antibodies. Antibodies may be mutated in the variable regions of the heavy and/or light chains to alter the binding characteristics of the antibody. For example, mutations in one or more CDR regions can be made to increase or decrease the K of the antibody to MAdCAM_d. 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, the mutation is made at an amino acid residue known to be altered in the variable region of the anti-MAdCAM antibody compared to the germline. In a more preferred embodiment, the one or more mutations are in the anti-MAdCAM 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, 6, compared to germline22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod or 7.26.4-mod. In another embodiment, the one or more mutations are present in the amino acid sequence of SEQ ID NO: 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 or 68, or a nucleotide sequence thereof, is represented in SEQ ID NO: 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, or 67 at known altered amino acid residues in the variable or CDR regions. In another embodiment, the nucleic acid molecule is mutated in one or more framework regions. Mutations can be generated in the framework or constant regions to increase the half-life of the anti-MAdCAM antibody. See, for example, WO00/09560, published at 24/2/2000, which is hereby incorporated by reference. In one embodiment, there may be 1,3 or 5 or 10 point mutations and no more than 15 point mutations. Mutations may also be made in the framework or constant regions to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent binding to another molecule, or to alter certain properties such as complement fixation. In a single mutated antibody, mutations may be generated in each of the framework, constant and variable regions. Alternatively, in a single mutated antibody, the mutation may be generated in only one of the framework, variable or constant regions.

In one embodiment, there are no more than 15 amino acid changes in the VH or VL region of the mutated anti-MAdCAM antibody compared to the anti-MAdCAM antibody prior to the mutation. In a more preferred embodiment, there are no more than 10 amino acid changes, more preferably no more than 5 amino acid changes, or even more preferably no more than 3 amino acid changes in the VH or VL region of the mutated anti-MAdCAM antibody. In further embodiments, there are no more than 15 amino acid changes, more preferably no more than 10 amino acid changes, and even more preferably no more than 5 amino acid changes in the constant region.

Modified antibodies

In another embodiment, a fusion antibody or immunoadhesin may be produced comprising an anti-MAdCAM antibody linked to all or part of another polypeptide. In a preferred embodiment, only the variable region of the anti-MAdCAM antibody is linked to the polypeptide. In another preferred embodiment, the VH domain of the anti-MAdCAM antibody is linked to a first polypeptide and the VL domain of the anti-MAdCAM antibody is linked to a second polypeptide, said second polypeptide being associated with the first polypeptide in such a way that the VH and VL regions can interact with each other to form an antibody binding site. In another preferred embodiment, the VH region is separated from the VL region by a linker sequence such that the VH and VL regions can interact with each other (see single chain antibody section below). The VH-linker-VL antibody is then linked to the polypeptide of interest. The fusion antibody can be used to direct the polypeptide to cells or tissues expressing MAdCAM. 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 easily visualized enzyme such as horseradish peroxidase. Furthermore, a fusion antibody in which two (or more) single-chain antibodies are linked to each other can be produced. This is useful if it is desired to produce bivalent or multivalent antibodies on a single polypeptide chain, or if it is desired to produce bispecific antibodies.

To generate single chain antibodies (scFv), the DNA fragments encoding VH-and VL are operably linked to additional encoding flexible linking sequences, such as encoding amino acid sequences (Gly)₄-Ser)₃(SEQ ID NO: 147) such that the VH and VL sequences are expressed as a continuous single-chain protein, the VH and VL regions are linked by a flexible linker sequence (see, e.g., Bidrd 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)). A single chain antibody is monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

In another embodiment, other modified antibodies can be made using nucleic acid molecules encoding anti-MAdCAM antibodies. For example, "kappa antibodies" (I11 et al, Protein Eng, 10: 949-57(1997)), "miniantibodies" (Martin et al, EMBOJ, 13: 5303-9(1994)), "bispecific antibodies" (Holliger et al, PNAS USA, 90: 6444-.

In another aspect, chimeric antibodies and bispecific antibodies can be produced. Chimeric antibodies can be produced that comprise CDRs and framework regions from different antibodies. In a preferred embodiment, the CDRs of the chimeric antibody comprise all the CDRs of the light or heavy chain variable regions of the human anti-MAdCAM antibody, while the framework regions are derived from one or more different antibodies. In a more preferred embodiment, the CDRs of the chimeric antibody comprise all the CDRs of the light and heavy chain variable regions of the human anti-MAdCAM antibody. The framework regions may be from another species and in a preferred embodiment are humanized. Alternatively, the framework region may be from another human antibody.

Bispecific antibodies can be generated that specifically bind to MAdCAM through one binding region and bind to a second molecule through another binding region. Bispecific antibodies can be produced by recombinant molecular biology techniques, or can be physically associated together. Furthermore, single chain antibodies containing more than one VH and VL can be generated that specifically bind to MAdCAM and another molecule. Such bispecific antibodies can be produced using well known techniques, e.g., for (i) and (ii), see, e.g., Fanger et al, Immunol Methods 4: 72-81(1994) and Wright and Harris, supra, for (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 molecule expressed at high levels on endothelial cells. In a more preferred embodiment, the other molecule is VCAM, ICAM or L-selectin.

In various embodiments, the modified antibodies described above can be prepared using one or more variable regions or one or more CDR regions 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, 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 antibody is prepared using one or more variable regions or one or more CDR regions whose amino acid sequences are set forth in SEQ ID NO: 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 or 68, or the nucleotide sequence thereof is set forth in SEQ ID NO: 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, or 67.

Derivatized and labeled antibodies

An antibody or antibody portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or portion thereof is derivatized and MAdCAM binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the human anti-MAdCAM antibodies described herein. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular entities that can mediate the association of the antibody or antibody portion with other molecules (e.g., streptavidin core region or poly-histidine tag), such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide.

One type of derivatized antibody may be produced by cross-linking two or more antibodies, of the same type or of different types, e.g., to produce a bispecific antibody. Suitable crosslinking agents include those that are heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate), having two groups of different reactivity separated by a suitable spacer. These cross-linking agents are available from Pierce chemical Company, Rockford, I11.

Another type of derivatized antibody is a labeled antibody. Detection agents that can be used in the derivatization of the antibodies or antibody moieties of the invention include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, rare earth phosphors, and the like. The antibody may also be labeled with an enzyme for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody is labeled with a detectable enzyme, it is detected by adding additional reagents for the enzyme to produce a reaction product that can be distinguished. For example, when horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobiphenyl can produce a colored reaction product that can be detected. Antibodies can also be labeled with biotin and detected by indirect measurement of avidin or streptavidin binding. The antibody may be labeled with a magnetic reagent such as gadolinium. The antibody may also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding region, epitope tag) recognized by a second reporter molecule. In some embodiments, the labels are linked by spacer arms of different lengths to reduce potential steric hindrance.

anti-MAdCAM antibodies can also be labeled with radiolabeled amino acids. Radiolabels are useful for diagnostic and therapeutic purposes. For example, the radiolabel may be used to detect tissue expressing MAdCAM by x-ray or other diagnostic techniques. Furthermore, the radiolabel may be used therapeutically as a toxin for diseased tissues or MAdCAM expressing tumors. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides-³H、¹⁴C、¹⁵N、 ³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I。

anti-MAdCAM antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological characteristics of the antibody, such as increasing serum half-life or enhancing tissue binding. The method is also applicable to any antigen binding fragment or variant of an anti-MAdCAM antibody.

Pharmaceutical composition and kit

In further aspects, the invention provides compositions comprising inhibitory human anti-MAdCAM antibodies and methods of treating a subject with the compositions. In some embodiments, the subject treated is a 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 comprise administration of one or more of the inhibitory anti-MAdCAM monoclonal antibodies or antigen-binding fragments thereof of the invention alone or in combination with a pharmaceutically acceptable carrier. The inhibitory anti-MAdCAM antibodies of the invention and compositions containing them can be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. Additional therapeutic agents include anti-inflammatory agents or immunomodulatory agents. These agents include, but are not limited to, topical and oral corticosteroids such as hydrocortisone, methyldydrohydrocortisone, NCX-1015 or budesonide; aminosalicylates such as 5-aminosalicylic acid, azosalicylic acid, balsalazide or NCX-456; immunomodulators such as azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, FK506, IL-10 (ilointerleukin), IL-11 (oproxil interleukin), IL-12, MIF/CD74 antagonists, CD40 antagonists, e.g., TNX-100/5-D12, OX40L antagonists, GM-CSF, pimecrolimus, or rapamycin; anti-TNF α agents such as infliximab, adalimumab, CDP-870, onacept, etanercept; anti-inflammatory agents, such as PDE-4 inhibitors (roflumilast, etc.), TACE inhibitors (DPC-333, RDP-58, etc.) and ICE inhibitors (VX-740, etc.) and IL-2 receptor antagonists such as daclizumab; selective adhesion molecule antagonists, such as natalizumab, MLN-02 or alicafensen; analgesics, such as, but not limited to, COX-2 inhibitors, e.g., rofecoxib, valdecoxib, celecoxib, P/Q-type voltage sensitive channel (α 2 δ) modulators, e.g., gabapentin and pregabalin, NK-1 receptor antagonists, cannabinoid receptor modulators, and δ opioid receptor agonists, as well as antineoplastic, antiangiogenic or chemotherapeutic agents. These additional agents may be included in the same composition or administered separately. In some embodiments, one or more inhibitory anti-MAdCAM antibodies of the invention can be used as a vaccine or adjuvant for a vaccine. In particular, since MAdCAM is expressed in lymphoid tissues, vaccine antigens can be advantageously targeted to lymphoid tissues by binding the antigens to the anti-MAdCAM antibodies of the present invention.

As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or retarding 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, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are surfactants, wetting agents or small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which improve the shelf life or the effect of the antibodies.

The compositions of the present invention may be in a variety of forms such as liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, lyophilized cakes, dry powders, liposomes and suppositories. The preferred form depends on the desired mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, e.g., compositions like those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, 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 subcutaneous injection.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, lyophilized cakes, dry powders, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile 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 ingredients enumerated above, as required, followed by sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile solution thereof. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. The desired properties of the solution can be maintained, for example, by using surfactants, and in the case of dispersants, the desired particle size is maintained by using surfactants, phospholipids and polymers. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as monostearate salts, polymeric materials, oils and gelatin.

Although the preferred route/mode of administration for many therapeutic applications is subcutaneous, intramuscular, intradermal, or intravenous infusion, the antibodies of the invention can be administered by a variety of methods known in the art. The skilled artisan will appreciate that the route and/or manner of administration will vary depending on the desired result.

In particular embodiments, the antibody compositions can be formulated with carriers that prevent rapid release of the antibody, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing 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 particular embodiments, the anti-MAdCAM antibodies of the invention can be administered orally, e.g., using an inert diluent or an absorbable food carrier. The compound (and other ingredients, as desired) may also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet of a subject. For oral therapeutic administration, the anti-MAdCAM antibody may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For administration of the compounds of the present invention by means other than parenteral administration, it may be necessary to coat the compound with a material that prevents inactivation of the compound or to administer it simultaneously with the compound.

The compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding portion of the invention. A "therapeutically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody portion can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount also refers to an amount by which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" is an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since the prophylactic dose is administered in a subject prior to or at an early stage of the disease, the prophylactically effective amount may be less than the therapeutically effective amount.

The dosage regimen may be adjusted to provide an optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally as the treatment situation requires. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects undergoing treatment; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the anti-MAdCAM antibody or portion thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of formulating such antibodies for use in treating sensitivity in an individual.

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

Another aspect of the invention provides a kit comprising an anti-MAdCAM antibody or antibody portion of the invention or a composition comprising such an antibody. In addition to the antibody or composition, the kit may also include a diagnostic or therapeutic agent. The kit may also include instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit comprises an antibody or a composition comprising the antibody and a diagnostic agent useful in the methods described below. In another preferred embodiment, the kit comprises an antibody or a composition comprising the antibody and one or more therapeutic agents useful in the methods described below.

Gene therapy

The nucleic acid molecule of the invention may be administered to a patient in need thereof by gene therapy. The therapy may be in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both the heavy and light chains may be administered to a patient. In a more preferred embodiment, the nucleic acid molecules are administered such that they stably integrate into the chromosome of the B cells, since these cells are specialized for the production of 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 recombinant viruses known to infect the cell type of interest. Typical vectors for gene therapy include liposomes, plasmids and viral vectors. Exemplary viral vectors are retroviruses, adenoviruses and adeno-associated viruses. Following infection in vivo or ex vivo, antibody expression levels can be monitored by sampling 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 and expressing an isolated nucleic acid molecule encoding an anti-MAdCAM antibody heavy chain or antigen-binding portion thereof. In another embodiment, the gene therapy method comprises the steps of: administering an isolated nucleic acid molecule encoding an anti-MAdCAM antibody light chain or an antigen-binding portion thereof and expressing the nucleic acid molecule. In a more preferred method, the gene therapy method comprises the steps of: administering and expressing an isolated nucleic acid molecule encoding an anti-MAdCAM antibody heavy chain or antigen-binding portion thereof of the invention and an isolated nucleic acid molecule encoding an anti-MAdCAM antibody light chain or antigen-binding portion thereof of the invention. The gene therapy method may further comprise the step of administering another anti-inflammatory agent or an immunomodulatory agent.

Diagnostic methods of use

anti-MAdCAM antibodies can be used to detect MAdCAM in a biological sample in vitro or in vivo. The anti-MAdCAM antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, western blotting, or immunoprecipitation. The anti-MAdCAM antibodies of the invention are useful for detecting MAdCAM in humans. In another embodiment, anti-MAdCAM antibodies can be used to detect MAdCAM in old continental primates such as macaques and macaques, orangutans, and apes. The invention provides a method for detecting MAdCAM in a biological sample comprising contacting the biological sample with an anti-MAdCAM antibody of the invention and detecting the antibody that binds to MAdCAM. In one embodiment, the anti-MAdCAM antibody is directly derivatized with a detectable label. In another embodiment, the anti-MAdCAM antibody (primary antibody) is unlabeled and a secondary or other molecule capable of binding the anti-MAdCAM antibody is labeled. Secondary antibodies are selected that specifically bind to the primary antibody of a particular species and class, as is well known to those skilled in the art. For example, if the anti-MAdCAM antibody is human IgG, the secondary antibody may be anti-human-IgG. Other molecules capable of binding to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available, such as from Pierce Chemical company.

Suitable labels for the antibody or secondary antibody have been disclosed above and include a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include 7-hydroxycoumarin, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of the magnetic agent include gadolinium; and examples of suitable radioactive materials include¹²⁵I、¹³¹I、³⁵S or³H。

In an alternative embodiment, MAdCAM can be detected in a biological sample by a competitive immunoassay using MAdCAM standards labeled with a detectable substance and unlabeled anti-MAdCAM antibodies. In this assay, a biological sample, labeled MAdCAM standards and anti-MAdCAM antibodies are mixed and the amount of labeled MAdCAM standards bound to unlabeled antibodies 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.

The immunoassays disclosed above can be used for many purposes. In one embodiment, anti-MAdCAM antibodies can be used to detect MAdCAM in cells in cell culture. In a preferred embodiment, anti-MAdCAM antibodies can be used to determine the level of cell surface MAdCAM expression following treatment of cells with various compounds. This method can be used to test compounds that can be used to activate or inhibit MAdCAM. In this method, a cell sample is treated with a test compound for a period of time while leaving another untreated, and cell surface expression can then be determined by flow cytometry, immunohistochemistry, western blotting, ELISA or RIA. Furthermore, the immunoassay can be scaled up for high throughput screening to detect the activation or inhibition of MAdCAM by a large number of compounds.

The anti-MAdCAM antibodies of the invention may also be used to determine MAdCAM levels on tissues or in cells derived from tissues. In a preferred embodiment, the tissue is diseased tissue. In a more preferred embodiment, the tissue is an inflamed gastrointestinal tract or a biopsy thereof. In a preferred method embodiment, the tissue or biopsy sample thereof is excised from the patient. The tissue or biopsy sample is then used in an immunoassay to detect, for example, MAdCAM levels, cell surface MAdCAM levels, or localization of MAdCAM by the methods described above. This method can be used to determine whether inflamed tissue expresses MAdCAM at high levels.

The diagnostic methods described above can be used to determine whether a tissue expresses high levels of MAdCAM, which can be predictive of a tissue that will respond well to treatment with anti-MAdCAM antibodies. Furthermore, the diagnostic method can also be used to determine whether treatment with an anti-MAdCAM antibody (see below) results in a tissue expressing lower levels of MAdCAM and can therefore be used to determine whether the treatment is successful.

The antibodies of the invention may also be used to localize tissues and organs expressing MAdCAM in vivo. In a preferred embodiment, anti-MAdCAM antibodies can be used to localize inflamed tissue. anti-MA of the inventionAn advantage of dCAM antibodies is that they do not produce an immune response upon administration. The method comprises the following steps: an anti-MAdCAM antibody or pharmaceutical composition thereof is administered to a patient in need of such a diagnostic test and the patient is subjected to an imaging assay to determine the localization of tissue expressing MAdCAM. Imaging analysis is well known in the medical arts and includes, but is not limited to, x-ray analysis, gamma scintigraphy, Magnetic Resonance Imaging (MRI), positron emission tomography, or Computed Tomography (CT). In another embodiment of the method, a biopsy sample is obtained from the patient to determine whether the target tissue expresses MAdCAM without subjecting the patient to imaging analysis. In a preferred embodiment, the anti-MAdCAM antibody may be labeled with a detectable agent capable of imaging in the patient. For example, the antibody may be labeled with a contrast agent useful for x-ray analysis, such as barium, or a magnetic contrast agent useful for MRI or CT, such as gadolinium chelates. Other labeling agents include, but are not limited to, radioisotopes such as⁹⁹Tc. In another embodiment, the anti-MAdCAM antibody is unlabeled and can be imaged by administering a secondary antibody or other molecule that is detectable and capable of binding to the anti-MAdCAM antibody.

The anti-MAdCAM antibodies of the invention may also be used to determine the level of soluble MAdCAM present in the blood, serum, plasma or other biological fluids of a donor, including but not limited to stool, urine, sputum or biopsy samples. In a preferred embodiment, the biological fluid is plasma. The biological fluid was then used in an immunoassay to determine the level of soluble MAdCAM. Soluble MAdCAM can be a surrogate marker for ongoing gastrointestinal inflammation and the assay method can be used as a diagnostic marker to measure disease severity.

The diagnostic methods described above can be used to determine whether an individual expresses high levels of soluble MAdCAM, which can indicate that the individual responds well to treatment with anti-MAdCAM antibodies. Furthermore, diagnostic methods can also be used to determine whether treatment with an anti-MAdCAM antibody (see below) or other agent of the disease causes an individual to express lower levels of MAdCAM and can therefore be used to determine whether the treatment is successful.

anti-MAdCAM antibody vs₄β₇Inhibition of MAdCAM-dependent adhesion:

in another embodiment, the invention provides binding to MAdCAM and inhibiting the binding of alpha₄β₇anti-MAdCAM antibodies that bind and adhere to MAdCAM in cells that are integrin or to other cognate ligands such as L-selectin. In a preferred embodiment, the MAdCAM is human MAdCAM and is in soluble form or expressed on the cell surface. In another preferred embodiment, the anti-MAdCAM antibody is a human antibody. In another embodiment, the antibody or portion thereof has an IC of no greater than 50nM₅₀Value suppression alpha₄β₇And MAdCAM binding. In a preferred embodiment, the IC₅₀Values were not greater than 5 nM. In a more preferred embodiment, the IC₅₀Values were less than 5 nM. In a more preferred embodiment, the IC₅₀Values of less than 0.05. mu.g/mL, 0.04. mu.g/mL, or 0.03. mu.g/mL. In another preferred embodiment, IC₅₀Values less than 0.5. mu.g/mL, 0.4. mu.g/mL, or 0.3. mu.g/mL. IC (integrated circuit)₅₀The value may be determined by any method known in the art. In general, ICs₅₀Values can be measured by ELISA or adhesion assays. In a preferred embodiment, IC is measured by an adhesion assay using cells or tissues that naturally express MAdCAM or that have been engineered to express MAdCAM₅₀The value is obtained.

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

In another embodiment, the invention provides anti-MAdCAM antibodies that bind to naturally expressed MAdCAM and inhibit lymphocyte binding to specialized gastrointestinal lymphoid tissue. In a preferred embodiment, the naturally expressed MAdCAM is human or primate MAdCAM and is in soluble form or expressed on the cell surface. In another preferred embodiment, the anti-MAdCAM antibody is a human antibody. In another embodiment, the antibody or portion thereof has an IC of no greater than 5mg/kg₅₀Value-inhibited intestine nourishing type alpha₄β₇ ⁺Lymphocytes are recruited to tissues expressing MAdCAM. In a preferred embodiment, the IC₅₀The value is not more than 1 mg/kg. In a more preferred embodiment, the IC₅₀The value is less than 0.1 mg/kg. In one embodiment, IC is determined using gamma scintigraphy or single photon emission electron computed tomography by measuring the dose-effect relationship of technetium-labeled peripheral blood lymphocytes recruitment to the gastrointestinal tract₅₀The value is obtained. In another embodiment, IC₅₀Values can be obtained using flow cytometry by measuring the enterotrophy alpha in the peripheral circulation₄β₇ ⁺Lymphocytes such as, but not limited to, CD4⁺α₄β₇ ⁺The increase in memory T-cells was measured as a function of the dose of anti-MAdCAM antibody.

In order that the invention may be more readily understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Example 1

Generation of anti-MAdCAM antibody-producing hybridomas

Antibodies of the invention were prepared, assayed, and selected according to this example.

Preparation of major immunogens

Preparation of two immunogens for XenoMouse^TMImmunization of mice:

(i)MAdCAM-IgG₁an Fc fusion protein and (ii) a cell membrane prepared from cells stably transfected with MAdCAM.

(i)MAdCAM-IgG₁Fc fusion protein

Expression vector construction

Extracellular immunization of mature MAdCAMEcoRI/BglII cDNA fragments of the immunoglobulin-like domain were excised from pINCY Incyte clone (3279276) and cloned into the EcoRI/BamHI sites of pIG1 vector (Simmons, D.L (1993) Cellular Interactions in Development: A Practical Approach, ed.Hartley, D.A (Oxford Univ.Press, Oxford), pp.93-127.) to generate in-frame IgG₁Fc fusion. The resulting insert was excised with EcoRI/NotI and cloned into pCDNA3.1+ (Invitrogen). The MAdCAM-IgG in the vector₁FccDNA sequencing confirmation. MAdCAM-IgG₁The amino acid sequence of the Fc fusion protein is shown below:

MAdCAM-IgG₁fc fusion protein:

MDFGLALLLAGLLGLLLG

QPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ

ID NO：107)

underlining: signal peptide

Bold type: MAdCAM extracellular domain

Recombinant protein expression/purification:

CHO-DHFR cells were treated with MAdCAM-IgG₁pCDNA3.1+ vector transfection of Fc fusion protein cDNA and Selective expression of MAdCAM-IgG in Iscove's Medium containing 600. mu.g/mL G418 and 100ng/mL methotrexate₁Stable cloning of Fc fusion proteins. For protein expression, stably expressing MAdCAM-IgG in Iscove's Medium containing 10% Low IgG fetal bovine serum (Gibco), non-essential amino acids (Gibco), 2mM glutamine (Gibco), sodium pyruvate (Gibco), 100. mu.g/mL G418, and 100ng/mL methotrexate was inoculated in a hollow fiber bioreactor₁Fc, and was used to produce concentrated culture medium supernatants. Purification of MAdCAM-IgG from the collected supernatant by affinity chromatography₁An Fc fusion protein. Briefly, the supernatant was applied to a HiTrap G protein Sepharose (5mL, Pharmacia) column (2mL/min), washed with 25mM Tris pH8, 150mM NaCl (5 column volumes) and eluted with 100mM glycine pH2.5(1 mL/min), immediately neutralizing the fractions to pH 7.5 with 1M Tris pH 8. Will contain MAdCAM-IgG₁Fractions of Fc fusion protein were identified by SDS-PAGE, pooled together and applied to a polyacrylamide dextran S100 column (Pharmacia) pre-equilibrated with 35mM Bis Tris pH 6.5, 150mM NaCl. Gel filtration was performed at 0.35 mL/min, and MAdCAM-IgG was collected in about 3X 5mL fractions₁Fc fusion protein peak. These samples were mixed and applied to a Resource Q (6mL, Pharmacia) column, pre-equilibrated in 35mM BisTris pH 6.5. The column was washed with 5 column volumes of 35mM Bis Tris pH 6.5, 150mM NaCl (6 mL/min) and the MAdCAM-IgG eluted with 35mM Bis Tris pH 6.5, 400mM NaCl₁Fc fusion protein was added to the 4-6mL fraction. At this stage, the protein was 90% pure and migrated as a single band of about 68kD by SDS-PAGE. For use as an immunogen and for all subsequent assays, the material buffer was changed to 25mM HEPES pH 7.5, 1mM EDTA, 1mM DTT, 100mM NaCl, 50% glycerol and stored in aliquots at-80 ℃.

(ii)Cell membrane stably expressing MAdCAM

Will contain the disclosed MAdCAM sequence (Shyjan AM et al, J immunol.,156the SacI/NotI fragment at nucleotides 645 and 1222 of 2851-7(1996) was PCR amplified from the colonic cDNA library and cloned into the SacI/NotI site of the pIND-Hygro vector (Invitrogen). The SacI fragment containing the additional 5' coding sequence was isolated from pCDNA3.1 MAdCAM-IgG₁Fc was subcloned into this construct to generate full-length madcam cdna. The KpnI/NotI fragment containing the MAdCAM cDNA was then cloned into the corresponding site in pEF5FRTV5GWCAT vector (Invitrogen) and replaced the CAT coding sequence. The cDNA insert was verified by sequencing and used for transfection by Flp recombinase technology according to the manufacturer's instructions to generate a single stably expressed clone in FlpIn NIH 3T3 cells (Invitrogen). By which support of alpha₄β₇ ⁺Ability of JY human lymphoblastoid B lines to bind (Chan BM et al, J.biol.chem.,267：8366-70(1992)) selecting stably expressing clones, which is summarized below. Stable clones of MAdCAM-expressing CHO cells were prepared in the same manner using FlpIn CHO cells (Invitrogen).

FlpIN NIH-3T3 cells expressing MAdCAM were grown in Dulbecco's modified Eagles medium (Gibco) containing 2mM L-glutamine, 10% donor calf serum (Gibco) and 200. mu.g/mL hygromycin B (Invitrogen) and expanded in roller bottles. FlpIN CHO cells expressing MAdCAM were grown in Ham's F12/Dulbecco's modified Eagles medium (Gibco) containing 2mM L-glutamine, 10% donor calf serum (Gibco) and 350. mu.g/mL hygromycin B (Invitrogen) and expanded in roller bottles. Cell harvest non-enzymatic cell dissociation solution (Sigma) was used and scraped, washed in phosphate buffered saline and centrifuged. Two passes at 25mM BisTris pH8, 10mM MgCl₂0.015% (w/v) aprotinin, 100U/mL bacitracin homogenized and centrifuged to prepare cell membranes from the cell pellet. Resuspend the final pellet in the same buffer and 50 × 10⁶Cell equivalents were aliquoted into the thick-walled eppendorf and spun at > 100,000g to generate cells for XenoMouse mouse immunizationThe membrane precipitates the particles. The supernatant was decanted and the cell membranes were stored in eppendorf at-80 ℃ until needed. By SDS-PAGE and use of the N-terminal residue ([ C ] against MAdCAM]-KPLQVEPPEP) (SEQ ID NO: 134) western blotting of the generated rabbit anti-peptide antibody confirmed the expression of the protein in the cell membrane. Immunization and hybridoma generation:

about eight to ten weeks old XENOMOUSE^TMPurified recombinant MAdCAM-IgG for mice₁Fc fusion protein (10. mu.g/dose/mouse) or from CHO or NIH 3T3 cells stably expressing MAdCAM (10X 10)⁶Cells/dose/mouse) were immunized intraperitoneally or in their hind footpads. This dose is repeated five to seven times in three to eight weeks. Mice received a final injection of human MAdCAM extracellular domain in PBS 4 days prior to fusion. Spleen and lymph node lymphocytes from immunized mice were fused with a non-secretory myeloma P3-X63-Ag8.653 cell line and received HAT selection as previously described (Galfre and Milstein, methods enzymol.73: 3-46 (1981)). Recovery of a series of fully secreted MAdCAM-specific human IgG₂Kappa and IgG₄Kappa antibody hybridomas and subcloning them. Twelve hybridoma subclones producing MAdCAM-specific monoclonal antibodies, i.e., 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 recovered and tested using the assays described below. Parental lines 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 of derivative subclone hybridoma lines 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 all had anti-MAdCAM activity. ELISA assay

Using MAdCAM-IgG₁Fc fusion protein capture antibodies antigen-specific antibodies were detected in mouse sera and hybridoma supernatants by ELISA assays as described (Coligan et al, Unit2.1, "Enzyme-linked immunological assays," in Current protocols Iin immunology (1994)). For the use of MAdCAM-IgG₁Fc fusion protein immunization of animals against human IgG by flow cytometry₁Is not specific toReactivity and ability to bind to FlpIn CHO MAdCAM cells antibodies were screened.

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

MAdCAM-IgG at 100. mu.L/well in plates containing buffer (100mM sodium carbonate/sodium bicarbonate buffer, pH 9.6)₁Fc fusion protein (4.5. mu.g/mL) was coated onto ELISA plates overnight at 4 ℃. After incubation, the coating buffer was removed and the plate was blocked with 200 μ L/well blocking buffer (5% BSA, 0.1% Tween20, phosphate buffered saline) and incubated for 1 hour at room temperature. The blocking buffer was removed and 50 μ Ι/well of hybridoma supernatant or other serum or supernatant (e.g., positive control) was added at room temperature for 2 hours. After incubation, the plate was washed with PBS (3X 100. mu.L/well) and diluted with HRP-conjugated secondary antibody (i.e., 1: 1000 anti-IgG) in PBS₂Mouse anti-human IgG of antibody₂HRP (SB Cat. No.9060-05) or 1: 1000 anti-IgG₄Mouse anti-human IgG of antibody₄HRP (Zymed Cat. No.3840)) detects binding of hybridoma mAbs. The plates were incubated for 1 hour at room temperature, washed in PBS (3X 100. mu.L/well) and washed with 100. mu.L of OPD (o-phenylenediamine (DAKO S2405) + 5. mu.L of 30% H₂O₂12mL) color development. The plate was developed for 10-20 min with 100. mu.L of 2MH₂SO₄The reaction was terminated. Plates were read at 490 nm.

Adhesion determination:

antibodies binding to MAdCAM-IgG1 Fc fusion protein were demonstrated by ELISA using alpha₄β₇ ⁺JY cells and (i) MAdCAM-IgG₁The Fc fusion protein or (ii) MAdCAM-CHO cells were evaluated for antagonistic activity in adhesion assays.

(i)MAdCAM-IgG₁Fc fusion protein assay

100 μ L of 4.5 μ g/mL MAdCAM-IgG purified in Dulbecco's PBS₁The Fc fusion protein solution was adsorbed to a 96-well black Microfluor "B" type U bottom (Dynex #7805) plate overnight at 4 ℃. The plate coated with MAdCAM was then inverted and excess liquid was aspiratedAfter this time, blocking was performed in 10% BSA/PBS at 37 ℃ for at least 1 hour. During this period, cultured JY cells were counted by trypan blue exclusion (which should be about 8X 10)⁵cells/mL) and at 20X 10⁶Cells/assay plate were pipetted into a 50mL centrifuge tube. JY cells were cultured in RPMI1640 medium (Gibco) containing 2mM L-glutamine and 10% heat-inactivated fetal bovine serum (Life Technologies #10108-165) at 1-2X 10 every 2-3 days⁵inoculation/mL to prevent culture differentiation. The cells were washed twice at 2X 10 by centrifugation (240g) with RPMI1640 medium (Gibco) containing 2mM L-glutamine (Gibco)⁶cells/mL the final cell pellet was resuspended in RPMI1640 for calcein AM loading. Calcein AM (Molecular Probes # C-3099) diluted 1: 200 in DMSO (approximately 15. mu.g final concentration) was added to the cells and protected from light during incubation (30 min at 37 ℃). During this cell incubation step, the antibodies tested were diluted as follows: for single dose experiments, antibody was made up to 3 μ g/mL (1 μ g/mL final concentration) in BSA (Sigma # A3059) in PBS 0.1 mg/mL; for the whole IC₅₀The antibody was diluted in 0.1mg/mL BSA/PBS at the highest concentration of 3. mu.g/mL (1. mu.g/mL final concentration) and then double diluted across the plate (1: 2 ratio). The last well of the row was used to detect total binding, so 0.1mg/ml BSA in PBS was used.

After blocking, the contents of the plate were flicked off and 50 μ L of antibody/control was added to each well and the plate was incubated at 37 ℃ for 20 minutes. During this period, the calcein-loaded JY cells were washed once by centrifugation with RPMI1640 medium containing 10% fetal bovine serum and once with 1mg/mL BSA/PBS, resuspending the final cell pellet to 1X 10 in 1mg/mL BSA/PBS⁶and/mL. Add 100. mu.L of cells to each well of the U-bottomed plate, seal the plate, centrifuge briefly (centrifuge at 1000rpm for 2 minutes) and then incubate the plate at 37 ℃ for 45 minutes. At the end of this period, the plates were washed with Skatron plate washer and Wallac Victor²1420 multiple tag reader measures fluorescence (excitation lambda 485nm, emission lambda 535nm, counting from the top of the plate, 8mm from the bottom of the plate, at normal emission calibre for 0.1 sec). For each antibody concentration, hundredThe specific binding is expressed as the percentage of the maximal fluorescence response absent any antibody minus the fluorescence associated with non-specific binding. IC (integrated circuit)₅₀The value is defined as the concentration of anti-MAdCAM antibody at which the adhesion response decreases to 50% of the response in the absence of anti-MAdCAM antibody. Can be integrated with₅₀Values < 0.1. mu.g/mL inhibited JY cell binding to MAdCAM-IgG₁Antibodies to the Fc fusion protein can be considered to have potent antagonistic activity and further subjected to MAdCAM-CHO adhesion assay. All twelve antibodies tested showed potent antagonistic activity (table 3). Monoclonal antibodies 1.7.2, 1.8.2, 7.16.6, 7.20.5 and 7.26.4 were derived from IgG₂Kappa lineage, while monoclonal antibodies 6.14.2, 6.22.2, 6.34.2, 6.67.1, 6.73.2, 6.77.1 and 9.8.2 are derived from IgG₄The kappa lineage.

(ii) MAdCAM-CHO cell adhesion assay

JY cells were cultured as above. CHO cells expressing MAdCAM were generated with the pEF5FRT MAdCAM cDNA construct and using the Flp recombinase technique (Invitrogen) as described above. Single stable clones of MAdCAM-expressing CHO cells were selected based on their ability to support JY cell adhesion and to support the binding of rabbit anti-peptide antibodies generated against the N-terminus of MAdCAM and described above, the binding of which was determined by flow cytometry. CHO cells expressing MAdCAM were cultured in DMEM/F12 medium (Gibco #21331-020) containing 2mM L-glutamine, 10% fetal bovine serum (Gibco) and 350. mu.g/mL hygromycin B (Invitrogen) and split 1: 5 every 2/3 days. For adhesion assays, MAdCAM-expressing CHO cells were cultured at 4X 10 in 200. mu.L medium⁴Cells/well were seeded in 96-well black plates-clear bottom (Costar #3904) and incubated at 37 deg.C/5% CO₂Incubate under conditions overnight.

The following day, hybridoma supernatants or purified monoclonal antibodies were diluted in 1mg/mLBSA/PBS starting from a concentration of 30 μ g/mL (equivalent to a final concentration of 10 μ g/mL) as described above. For MAdCAM CHO plates, the contents of the plate were popped off and 50. mu.L of antibody/control was added to each well, incubated at 37 ℃Plate 20 minutes. The last well of the row was used to determine the total binding amount, so 0.1mg/mL BSA in PBS was used. JY cells loaded with calcein AM were prepared as above to reach 1X 10 in 1mg/mL BSA/PBS⁶Final concentration of/mL, then 100 μ L was added to the plate after a 20 minute incubation period with the antibody. The plates were then incubated at 37 ℃ for 45 minutes, then washed on a Tecan plate washer (PW 384) and fluorescence measured using a Wallac plate reader as described above. For each antibody concentration, percent adhesion was expressed as the percentage of the maximum fluorescence response in the absence of any antibody minus the fluorescence associated with non-specific binding. Can be integrated with₅₀An antibody with a value < 1 μ g/mL that inhibits binding of JY cells to MAdCAM CHO cells can be considered to have potent antagonist activity. As before, the IC is put into₅₀The value was defined as the concentration of anti-MAdCAM antibody at which the adhesion response had dropped to 50% of the response in the absence of anti-MAdCAM antibody. IC 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 and 9.8.2 in this assay₅₀The efficacy is described in table 3 below.

TABLE 3 IC of exemplary anti-MAdCAM antibodies₅₀Value of

To measure antagonist potency of anti-MAdCAM mAb in a flow-based assay (flow-based assay) under shear stress conditions designed to mimic the microvascular environment on the high endothelial venules supplying gut-associated lymphoid tissue, MAdCAM-expressing CHO cells were plated on glass microscope slides (50 x 4mm) and allowed to attach to form a confluent monolayer (approximately 2.5 x10 mm)⁵A cell). The cells are then contacted with a series of cellsThe concentration (0.1-10 u g/mL) of affinity purified mAb before connecting to the flow determination system at 37 degrees C temperature in 20 minutes. Isotype matching of IgG₂Or IgG₄mAb (10. mu.g/mL) was used as a negative control. Normal donor Peripheral Blood Lymphocytes (PBLs) were infused onto the cell monolayer under a constant shear stress of 0.05 Pa. Videography experiments and calculation of total lymphocyte adhesion (rolling + firm adhesion). All tested monoclonal antibodies showed potent antagonists under the conditions described.

(iii) Stamper-Woodruff assay

To visualize the MAdCAM⁺Vascular, biotinylated anti-MAdCAM mAb was generated on 1-2mg of affinity-purified protein using a 20 molar excess of biotin-nhs (pierce) in phosphate buffered saline according to the manufacturer's instructions. The reaction was allowed to stand at room temperature (30 minutes), desalted using a PD-10(Pharmacia) column and the concentration of protein was determined.

Normal liver lymph nodes were removed from donor organs, flash frozen in liquid nitrogen and stored at-70 ℃ until use. 10 μm frozen sections were cut, air dried on poly-L-lysine coated slides, and fixed in acetone prior to assay. Sections were blocked with avidin-biotin blocking system (DAKO) and then incubated with a range of concentrations (1-50. mu.g/mL) of biotinylated anti-MAdCAM mAb (2 hours) at room temperature. Isotype matched IgG₂Or IgG₄mAb (50. mu.g/mL) was used as a negative control and blocked anti-beta₇Antibody (50. mu.g/mL) served as a positive control.

Peripheral blood lymphocytes from normal donors were labeled with mouse anti-human CD2mAb (DAKO) to enable subsequent visualization of adherent cells. Will be 5X 10⁵PBL was added to each lymph node section and incubated for 30 minutes, followed by gentle rinsing to avoid adherent cell shedding. The sections were then restoded in acetone and re-incubated with biotinylated anti-MAdCAM mAb (10. mu.g/mL), followed by re-incubation with biotinylated goat-anti-mouse mAb (to recognize CD 2-labeled PBL and unstained MAdCAM⁺Vessel) and then streptabicomplex/HRP (DAKO) re-incubation. MAdCAM was finally visualized by adding DAB substrate (DAKO) to the sections⁺Vascular and CD 2-labeled PBL, brown reaction products showed areas of positive staining. By counting 50 MAdCAM-1 attached to hepatic porta fascicles, veins or sinusoids⁺The number of lymphocytes in the vessel quantifies lymphocyte adhesion. The PBL adhesion was then taken as 100% in the absence of any antibody and the data expressed as mean values were normalized to percent adhesion. Data were compiled based on n-3 different PBL donors and for different liver lymph node donors. With blocking anti-beta₇Representative data for biotinylated purified monoclonal antibodies 1.7.2 and 7.16.6 compared to antibody controls are depicted in fig. 4.

And (3) selective determination:

VCAM and fibronectin are structurally similar to MAdCAM and are sequence homologous. By determining their blockade of alpha₄β₁ ⁺/α₅β₁ ⁺The ability of Jurkat T-cells (ATCC) to bind to their cognate cell adhesion molecules assessed the MAdCAM-specificity of affinity purified anti-MAdCAM mabs. A solution of 100. mu.L of 4.5. mu.g/mL fibronectin cell-binding fragment (110Kd, Europa Bioproducts, Inc., Cat. No. UBF4215-18) or VCAM (Panvera) in Dulbecco's PBS was adsorbed to a 96-well black Microfluor "B" U-shaped-bottom (Dynex #7805) plate overnight at 4 ℃. The coated plate was then inverted and excess liquid was aspirated, after which it was blocked in 10% BSA/PBS at 37 ℃ for at least 1 hour. During this time, cultured Jurkat T cells were counted using trypan blue exclusion and loaded with calcein AM dye as previously described for the JY cells above. The antibodies used in the assay were diluted from the highest concentration of 10. mu.g/mL in BSA in 0.1mg/mL PBS. The last well of the row was used to determine the total binding amount, so 0.1mg/ml BSA in PBS was used. Leptospirin (Bachem, Cat. No. H-9010) prepared in PBS was used to block alpha at the highest concentration of 100nM₅β₁Fibronectin interaction. anti-CD 106mAb (clone 51-10C9, BDPharmingen Cat. No.555645) was used to block alpha at the highest concentration of 1. mu.g/mL₄β₁the/VCAM interaction.

After blocking, the contents of the plate were flicked off and 50 μ L of antibody/control was added to each well and the plate was incubated at 37 ℃ for 20 minutes. The calcein-loaded Jurkat T cells were washed once as before, and the final cell pellet was resuspended to 1X 10 in 1mg/mLBSA/PBS⁶and/mL. Add 100. mu.L of cells to each well of the U-bottomed plate, seal the plate, centrifuge briefly (2 min at 1000 rpm) and incubate the plate at 37 ℃ for 45 min. At the end of this period, the plate was washed with a Skatron plate washer and with a Wallac Victor²1420 multiple tag reader measures fluorescence (excitation lambda 485nm, emission lambda 535nm, counting from the top of the plate, 8mm from the bottom of the plate, with normal emission calibre for 0.1 sec). For each antibody, the extent of inhibition is shown below in table 4 (-negligible inhibition of adhesion,. full inhibition of adhesion). All exemplified mabs were potent and selective anti-MAdCAM antagonists, exhibiting a selectivity for MAdCAM that greatly exceeded selectivity for VCAM and fibronectin by more than 100-fold.

TABLE 4 comparative Selectivity of anti-MAdCAM antibodies to MAdCAM over other cell adhesion molecules fibronectin and VCAM

Hybridomas were deposited at 9 months and 9 days 2003 in the european collection of cell culture collections (ECACCs), h.p. aat CAMR, Porton Down, Salisbury, wiltshire sp 40 JG, with the following accession numbers:

hybridoma cell And (4) preservation No.

1.7.2 03090901

1.8.2 03090902

6.14.2 03090903

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:

determination of affinity constant of fully human anti-MAdCAM monoclonal antibody by BIAcore

We performed affinity measurements of purified antibodies by surface plasmon resonance using a BIAcore 3000 instrument according to the protocol provided by the manufacturer.

Scheme 1

For kinetic analysis, high density mouse anti-human (IgG) was prepared on a CM5BIAcore sensor chip using conventional amine coupling₂And IgG₄) Antibody surface. Hybridoma supernatants were 10, 5, 2-fold diluted in HBS-P (10mM HEPES pH 7.4, 150mM NaCl, 0.005% surfactant P20) running buffer containing 100. mu.g/mL BSA and 10mg/mL carboxymethyl dextran or used neat. Each mAb was captured to a separate surface with a 1 minute contact time and washed for 5 minutesWash to stabilize mAB baseline. Then the MAdCAM-IgG₁Fc (141nM) fusion protein was injected over the entire surface for one minute followed by 3 minutes of dissociation. Data were normalized to the number of antibodies captured on each surface and evaluated with a langmuir 1: 1 global fit using a baseline drift model present on BIAevaluation software provided by BIAcore.

Scheme 2

Affinity purified mAb was immobilized on dextran layer on CM5 biosensor chip using amine coupling. The chip was prepared using acetate buffer at pH 4.5 as the fixation buffer and a protein density of 2.5-5.5kRU was obtained. MAdCAM-IgG in flow buffer₁Samples of Fc-fusion proteins were prepared at concentrations ranging from 0.2-55nM (including 0nM solution containing only running buffer as zero reference). Samples were randomized and injected in duplicate for 3 min each across 4 flow cells using HBS-EP (10mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA, 0.005% surfactant P20) as running buffer. A flow rate of 100. mu.L/min was used to minimize mass transport limitation (mass transport). Monitoring MAdCAM-IgG₁Dissociation of Fc fusion protein for 180 min by 6 sec injection of 25mM H₃PO₄Surfaces were regenerated (50. mu.L/min) or 10mM (6.22.2), 20mM (6.67.1, 6.73.2, 6.77.1) to 25mM (6.34.2) and 45mM NaOH (6.14.2) and the data were analyzed using the BIAevaluation (v3.1) software package.

Table 5 lists affinity measurements for representative anti-MAdCAM antibodies of the invention:

TABLE 5 determination of the affinity constant K by surface plasmon resonance (BIAcore)_d

Kinetic analysis indicated that the antibodies prepared according to the invention had high affinity 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, either as regions of linear (primary) sequence or structural (secondary) sequence. The Luminex epitope box-and-box (binding), BIAcore box-and-box, and species immunohistochemistry analyses were used together to elucidate the functional epitope characteristics of anti-MAdCAM antibodies.

Epitope frame merging method based on Luminex:

a microbead (Calbiochem M11427) that binds MxhIgG2, 3,4 was bound to the first unknown anti-MAdCAM antibody. We added 150 μ L of the first unknown antibody dilution (0.1 μ g/mL diluted in hybridoma medium) to the wells of a 96-well tissue culture plate. Stock solutions of microbeads were gently vortexed and diluted to 0.5X 10 in supernatant⁵Concentration of beads/mL. The beads were placed in the supernatant on a shaker overnight at 4 ℃ in the dark.

Each well of a 96-well microtiter filter plate (Millipore # MABVN1250) was pre-wetted by adding 200 μ L of wash buffer (PBS containing 0.05% Tween 20) and removed by aspiration. Then, 0.5X 10 was added at 50. mu.L/well⁵Microbeads/mL are stored in the filter plate and the plate wells are washed with wash buffer (2X 100. mu.L/well). MAdCAM-IgG diluted in hybridoma medium (0.1. mu.g/mL) was added at 60. mu.L/well₁An Fc antigen. The plates were covered and incubated for one hour at room temperature with gentle shaking. Wells were washed twice by adding 100. mu.L/well wash buffer followed by aspiration. Next, we added a second unknown anti-MAdCAM antibody (0.1. mu.g/mL) diluted in hybridoma culture medium at 60. mu.L/well. Shaking at room temperature in the darkPlates were run for two hours. The wells were then washed twice by adding 100 μ L/well wash buffer followed by aspiration. Next, biotinylated MxhIgG2, 3,4 (0.5. mu.g/mL) was added at 60. mu.L/well. The plates were shaken at room temperature for one hour in the dark. Wells were washed twice by adding 100. mu.L/well wash buffer followed by aspiration. mu.L of 1. mu.g/mL MxhIgG2, 3,4 streptavidin-PE (Pharmacia #554061) diluted in hybridoma medium was added to each well. The plates were shaken in the dark at room temperature for twenty minutes. Wells were washed twice by adding 100. mu.L/well wash buffer followed by aspiration. Subsequently, each well was resuspended in 80 μ L of blocking buffer (PBS with 0.5% bovine serum albumin, 0.1% TWEEN and 0.01% thimerosal) and the resuspended beads were carefully pipetted up and down.

Luminex 100 and accompanying software (b) were usedCorporation) read plate luminescence readings. Based on the luminescence data obtained for the various test anti-MAdCAM antibodies, the anti-MAdCAM antibodies were grouped according to their binding specificity. The anti-MAdCAM antibodies tested were divided into a series of epitope bins (epitope bins) as shown in table 8.

BIAcore box and method:

in a similar manner to that described above, BIAcore can also be used to determine epitope specificity of the anti-MAdCAM antibodies of the invention used as exemplars. The 9 anti-MAdCAM 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 on the dextran layer of the CM5 biosensor chip independent flow cell using amine coupling method. The fixation buffer was 10mM 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 10mM acetate buffer pH 5.5 (clones 6.67.1 and 6.77.1). In all cases a protein density of about 3750RU was obtained. The deactivation of the unreacted N-hydroxysuccinimide ester was carried out with 1M ethanolamine hydrochloride, pH 8.5.

MAdCAM-IgG₁The Fc fusion protein was diluted to a concentration of 1.5. mu.g/mL (about 25nM) in HBS-EP running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% polysorbate 20). It was then injected across the first flow cell at a volume of 50 μ L at an injection rate of 5 μ L/min. After injection was complete, the first antibody probe was added to the same flow cell. All test antibodies were diluted in HBS-EP to a concentration of about 20. mu.g/mL and also injected at a flow rate of 5. mu.L/min in a volume of 50. mu.L. When no binding of the test antibody was observed, the next test clone was injected immediately thereafter. When binding occurs, the sensor surface is regenerated to remove MAdCAM-IgG₁Fc fusion proteins and test antibodies. Depending on the immobilized antibody and the test antibody present, a variety of regeneration solutions are used. A summary of the regeneration conditions used is described in Table 6

TABLE 6 summary of regeneration conditions for BIAcore epitope mapping

(flow rate 50. mu.L/min in all regeneration steps)

After regeneration, MAdCAM-IgG is bound again₁Fc fusion protein and injection of additional test antibody. These steps were performed until all clones were injected with MAdCAM-IgG binding₁Surface of immobilized antibody of Fc fusion protein. A new flow cell with different immobilized antibodies and bound MAdCAM was then used for probing with these nine test clones. Based on their close primary amino acid sequence homology of the heavy and kappa light chains, with SEQ ID numbers 2, 4,6, 8, respectively, anti-MAdCAM antibodies 1.7.2 and 1.8.2 are expected to recognize the same MAdCAM epitope. Therefore, only 1.7.2 was evaluated by BIAcore reaction matrix. Antibodies 6.14.2 and 6.73.2 were omitted from the assay, but all other anti-MAdCAM antibody pairs were tested in this manner in combination. An arbitrary 100RU level was chosen as the threshold between binding/non-binding andand a reaction matrix was generated based on whether binding was observed (table 7).

TABLE 7 BIAcore epitope box-and-frame reaction matrix

The reaction matrix for all combinations of antibody pairs. Indicates that the antibody probe does not bind, x indicates that binding is observed (above the selection threshold level of 100 RU)

The matrix diagonal (shaded gray) of table 7 represents the binding data for the same probe pair. In all cases, the antibody was self-blocking except at two clones 7.16.6 and 9.8.2. Antibodies 7.16.6 and 9.8.2 did not cross-compete. The loss of self-blocking was probably due to mAb-induced conformational changes in the fusion protein that allowed the mAb to additionally bind to the second site of MAdCAM-IgFc.

Grouping clones showing the same reactivity pattern produced at least six different epitope bins, as shown graphically (fig. 5).

Further accurate identification of the epitope sequence of MAdCAM 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 a spotted peptide library array (Reineek et al, curr. Topicsin Microbiol. and Immunol 243: 23-36(1999), M.Famulok, E-LWinnacker, C-H Wong eds., Springer-Verlag, Berlin), phage or bacterial flagellin/fliC expression library display or simple MALDI-TOF analysis of limited proteolysis followed by binding protein fragments.

Immunohistochemical assay:

frozen tissue specimens of OCT or sucrose-embedded ileum (peyer's patch), mesenteric lymph node, spleen, stomach, duodenum, jejunum, colon were used as positive staining controls for anti-madcam mab. To use human IgG₂ mAbStaining of human sections yielded biotinylated derivatives of anti-MAdCAM mAb. 10 μm frozen tissue sections were excised onto L-lysine coated slides and placed directly into 100% acetone at 4 deg.C (10 min) and then 3% hydrogen peroxide in methanol (10 min) and washed with PBS between steps. Slides were blocked with biotin blocking system (DAKO Cat. No. X0590), then incubated with primary antibody in PBS (1: 100-1: 1000) (1 hour), washed with PBS-Tween 20 (0.05%) and then visualized for binding with HRP-streptavidin (BD Bioscience Cat. No.550946, 30 minutes) and DAB substrate (Sigma Cat. No. D5905). For IgG₄mAb using HRP-conjugated mouse anti-human IgG₄(Zymed Cat.No.3840) secondary antibody. The slides were counterstained with mayer's hematoxylin alum stain (1 min), washed and then mounted in DPX.

Binding affinities were compared for multiple species (mouse, rat, rabbit, dog, pig, cynomolgus and human tissues). By immunohistochemistry, anti-MAdCAM antibodies were not reactive to rat, rabbit and pig tissues, and when analyzed by ELISA, were not cross-reactive to recombinant mouse MAdCAM. Data for human, cynomolgus and dog tissues are presented in tabular form as follows, table 8:

TABLE 8 mode of cross-reactivity of anti-MAdCAM antibodies to MAdCAM species orthologs

n.d: not determined

According to the annotation, the binding of anti-MAdCAM antibodies to specialized endothelial structures and lymphoid tissues is indicated by shading. Epitope bins based on Luminex epitope analysis and a pattern of MAdCAM cross-reactivity for each antibody are shown. The Luminex epitope box-and-law 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) are derived from separate independent assays that differ from 1.7.2, 1.8.2, 7.16.6, 7.20.5, 7.26.4, and 9.8.2 (bold), as indicated by differences in font character.

All of the anti-MAdCAM antibodies tested had the ability to recognize an epitope of human MAdCAM expressed on the vascular endothelial compartment of the gastrointestinal tract. Except for 1.7.2 and 1.8.2, all other anti-MAdCAM antibodies tested were able to specifically bind to the vascular endothelial compartment of the gastrointestinal tract of cynomolgus monkeys. Certain other anti-MAdCAM antibodies, namely 6.14.2 and 6.67.1, also have the ability to specifically recognize dog MAdCAM orthologs as well as MAdCAM of cynomolgus monkeys.

Generation of CHO cell line expressing functionally active chimeric cynomolgus/human MAdCAM:

the difference in binding affinity of certain anti-MAdCAM antibodies to human and cynomolgus MAdCAM led us to determine whether a structural basis could be derived for this observation.

Based on the published amino acid sequence of macaca brachypus (Shyjan AM et al, J Immunol., 156, 2851-7(1996)), primers were designed for PCR amplification of macaca brachypus MADCAlpha₄β₇A binding domain sequence. Total RNA was prepared from frozen excised cynomolgus mesenteric lymph nodes (approximately 200mg) using the Trizol method (Invitrogen) according to the manufacturer's instructions. Mu.g was reverse transcribed with oligo-dT as primer and AMV reverse transcriptase (Promega). A portion of the reverse transcription product was subjected to PCR treatment with primers forward 5'-AGC ATGGAT CGG GGC CTG GCC-3' (SEQ ID NO: 111) and reverse 5'-GTG CAG GACCGG GAT GGC CTG-3' (SEQ ID NO: 112) using GC-2 polymerase in 1M GC melt (Clontech) at an annealing temperature of 62 ℃. The appropriate size RT-PRC product was excised and purified from a 1% agarose gel after electrophoresis, followed by TOPO-TA cloning (Invitro)gen) between the EcoRI sites of the pCR2.1 vector. The insert sequence was confirmed by sequencing. The nucleotide and predicted translated amino acid sequences thereof are shown in SEQ ID NOs 49 and 50, respectively.

Predicted alpha of human and cynomolgus MAdCAM when aligned (this sequence alignment is provided in figure 3)₄β₇The amino acid sequence of the binding domain shows a high degree of sequence identity (90.8%). To generate cell lines expressing functionally active macaque MAdCAM that mimic the anti-MAdCAM binding pattern described in table 8, the pcr2.1 vector was mapped to macaque α₄β₇The Sac I fragment of the binding domain sequence was subcloned directly into the C-terminal human MAdCAMpI ND-Hygro construct containing the carboxy-terminal mucin stem and transmembrane domain as described above. The sequence and orientation were verified and the KpnI/NotI fragment was then cloned into the pEF5FRTV5GWCAT vector (Invitrogen), replacing the CAT coding sequence and used for transfection according to the manufacturer's instructions to generate a single stably expressed clone In Flp In CHO cells (Invitrogen).

Binding of anti-MAdCAM antibody clones to CHO cells expressing cynomolgus/human MAdCAM chimeras was assessed by flow cytometry, and functional activity of anti-MAdCAM antibodies was determined using a JY cell adhesion assay very similar to that described above. The binding and functional activity of anti-MAdCAM antibodies is expressed in table 9.

Table 9 correlation between functional activity of a panel of anti-MAdCAM antibodies in the cynomolgus/human MAdCAM-CHO/JY adhesion assay and binding to human and cynomolgus/human MAdCAM CHO cells as measured by FACS.

Overall, there was a good correlation between the ability of a given anti-MAdCAM antibody to bind to human or cynomolgus MAdCAM, as determined by immunohistochemistry (table 8) by recombinant cell-based binding, and its functional activity (table 9). For example, anti-MAdCAM antibodies 1.7.2, 1.8.2 and 6.73.2, showed a consistent lack of binding to cynomolgus tissues and cells expressing chimeric cynomolgus/human MAdCAM proteins. anti-MAdCAM antibodies 1.7.2, 1.8.2 and 6.73.2 also did not have the ability to detect functional blocking activity in the cynomolgus/human MAdCAM/JY adhesion assay.

Similar methods can be used to determine the epitopes of anti-MAdCAM antibodies 6.14.2 and 6.67.1 that recognize dog MAdCAM.

Example IV:

use of anti-MAdCAM mAb to detect circulating soluble MAdCAM as a diagnostic method for disease

anti-MAdCAM antibodies can be used to detect circulating soluble MAdCAM (smadcam). Detection of sMAdCAM in clinical plasma, serum samples, or other biological fluids such as, but not limited to, stool, urine, sputum, may be a useful surrogate disease biomarker for underlying diseases including, but not limited to, inflammatory bowel disease.

Based on the epitope box-and-box data (tables 7 and 8), anti-MAdCAM antibodies 1.7.2 and 7.16.6 were shown to recognize different epitopes on human MAdCAM. ELISA plates were coated with 50. mu.g/mL of a 1.7.2 solution in 100. mu.L/well Phosphate Buffered Saline (PBS) overnight at 4 ℃. After incubation, the plates were blocked with PBS blocking buffer (200 μ L/well) containing 10% milk for 1.5 hours. After incubation, the plate was washed with PBS (2X 100. mu.L/well) and serially diluted with MAdCAM-IgG1-Fc fusion protein from a maximum concentration of 50. mu.g/mL in PBS to about 5ng/mL to a final volume of 100. mu.L, which was added to the plate and incubated for 2 hours at room temperature. In a similar manner, MAdCAM-IgG1-Fc protein can be diluted in plasma or serum or some other such relevant biological fluid and used to determine the expression of soluble MAdCAM in clinical samples, as described below. As a negative control, only buffer was added to wells containing the primary anti-MAdCAM antibody. Thereafter, the plate was washed with PBS (3X 100. mu.L/well) and then incubated with Alexa 488-labeled 7.16.6 (100. mu.L, 5. mu.g/mL) in the dark. Alexa 488-labeled 7.16.6 was generated using a commercially available kit (Molecular Probes, A-20181) according to the protocol provided by the manufacturer.

The plate was washed with PBS containing 0.05% Tween-20, and the binding of labeled 7.16.6 to the captured soluble MAdCAM was measured by fluorescence (Wallac Victor)²1420 multiple mark reader, excitation lambda 485nm, emission lambda 535nm, counting from the top of the plate 3mm from the bottom, with normal emission calibre lasting 0.1 sec). When fluorescence is plotted as a function of concentration of MAdCAM-IgG1-Fc fusion protein, see fig. 6, it is shown that 1.7.2 and labeled 7.16.6 can be used for diagnostic purposes to determine circulating soluble MAdCAM levels expressed in biological fluids or clinical samples. This sandwich ELISA method is not limited to the use of 1.7.2 and 7.16.6, but any combination of anti-MAdCAM antibodies that recognize different epitopes on MAdCAM can be used, as outlined by the data and explanations of table 7 and fig. 5. Similar strategies can be applied to develop similar assays, such as immunohistochemistry and western blotting, using other described anti-MAdCAM antibodies, with different partners, variants, labels, etc.

Example V:

amino acid Structure of anti-MAdCAM mAbs prepared according to the invention

In the following discussion, structural information related to anti-MAdCAM mAb prepared according to the present invention is provided.

To analyze the structure of the mabs produced according to the invention, we cloned genes encoding heavy and light chain fragments from specific hybridoma clones. Cloning and sequencing of the genes was performed as follows:

approximately 2X 10 from immunized Xenomouse-derived mice using the Fast-Track kit (Invitrogen)⁵Poly (A) + mRNA was isolated from each hybridoma cell. PCR was performed after cDNA generation using random primers. Human VH or V.kappa.family specific primers (Marks et al, ' Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable gene and amplification of family-specific Oligonucleotide probes '; Eur. J. Immunol., 21, 985-ch.991 (1991)) or the common human VH primer MG-30(5 ' -CAG GTGCAG CTG GAG CAG TCI GG-3(SEQ ID NO: 108)) were used in combination with specific primers for human C.gamma.2, MG40-d (5 ' -GCT GAG GGA GTA GAG TCC TGA GGA-3(SEQ ID NO: 109)) or C.gamma.4 constant region, MG-40d (5 ' GCT GAG GGA GTA GAG TCC TGA GGA CTG T-3(SEQ ID NO: 110)) or C.kappa.P 2 (h.kappa.P 2; as described previously in Greene et al 1994). The sequences of human mAb-derived heavy or kappa chain transcripts from hybridomas were obtained by direct sequencing of PCR products generated from poly (a +) RNA using the primers described above. The PCR products were cloned into pCR2.1 vector using TOPO-TA cloning kit (Invitrogen) and both strands were sequenced using Prism dye termination sequencing kit and ABI 377 sequencer. The entire sequence was analyzed by alignment with 'V BASE sequence directory' (Tomlinson et al, J.mol.biol., 227, 776-.

Further, each 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, 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod was subjected to full-length DNA sequencing. For this purpose, RNeasy kit (Qiagen) was used, from about 3 to 6X 10⁶Hybridoma cells isolate total RNA. mRNA was reverse transcribed using oligo-dT and AMV based reverse transcriptase system (Promega). V BASE was used to design 5' specific amplification primers containing optimized Kozak sequence and ATG start codon (underlined), and for specific heavy chains and kappaStrand 3' reverse primer, as described in table 10.

Table 10: PCR primer pairs for amplification of cDNA from anti-MAdCAM mAb expressing hybridomas and primers for anti-MAdCAM antibody modified variant construction.

The primer pair was used to amplify the cDNA using Expand high fidelity Taq polymerase (Roche) and the PCR product was cloned into pCR2.1 TOPO-TA (Invitrogen) for subsequent sequencing. Clones verifying the heavy and kappa light chain sequences were then cloned into the pEE6.1 and pEE12.1 vectors (LONZA) using XbaI/EcoRI and HindIII/EcoRI sites, respectively.

Gene utilization analysis

Table 11 shows the heavy and kappa light chain gene utilization for each hybridoma summarized in the present invention.

Table 11: heavy and kappa light chain gene utilization

Sequence analysis

To further examine the antibody structure, the predicted amino acid sequence of the antibody was obtained from the cDNA obtained from the clone.

Sequence identification numbers (SEQ ID NOS: 1-48 and 51-68) provide anti-MAdCAM antibodies 1.7.2(SEQ ID NOS 1-4), 1.8.2(SEQ ID NOS 5-8), 6.14.2(SEQ ID NOS 9-12), 6.22.2(SEQ ID NOS 13-16), 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 NOS33-36), 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 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) and the nucleotide and amino acid sequence of the kappa light chain. For each cloned anti-MAdCAM antibody sequence, the sequence of the signal peptide sequence (or the bases encoding this sequence) is shown in lower case and underlined.

Fig. 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 sequences of the respective germline gene products. The positions of the CDR1, CDR2, and CDR3 sequences of the antibody are underlined, the differences between the expressed sequences and the corresponding germline sequences are indicated in bold, and are indicated in the germline sequences as (-) compared to where the germline sequences have an addition in the expressed sequences.

Fig. 1K-1T provides sequence alignments between the predicted kappa light 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 sequences of the respective germline gene products. The positions of the CDR1, CDR2, and CDR3 sequences of the antibody are underlined, the differences between the expressed sequences and the corresponding germline sequences are indicated in bold, and are indicated in the germline sequences as (-) compared to where the germline sequences have an addition in the expressed sequences.

Post-translational modification: presence of glycosylation and deamidation:

the effect of some changes in the expressed anti-MAdCAM antibody sequence, as compared to the derived germline sequence, was to introduce residues for potentially acceptable N-linked glycosylation (Asn-X-Ser/Thr) and deamidation (Asn-Gly) (see table 12). The nucleic acid sequences encoding the kappa light chain variable domain amino acid sequences 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(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) are predicted to have the presence of N-linked glycosylation. Using SDS-PAGE and Pto-QCombinations of Emerald 488 glycoprotein (Molecular Probes) staining were studied for the presence of this post-translational modification 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. mu.g of reduced anti-MAdCAM antibody was loaded onto a 4-12% SDS-polyacrylamide gel using MOPS buffer. After electrophoresis, the gel was fixed in 50% MeOH, 5% acetic acid and washed in 3% acetic acid. Then oxidizing any saccharide on the gel with periodic acid and using Pro-QEmerald 488 glycoprotein staining kit (Molecular Probes). After the final washing step, the glycoprotein staining was visualized by setting the wavelength to 473nm with a fluorescence scanner.

After glycoprotein staining, the gel was stained for total protein with SYPRO Ruby protein gel dye and analyzed with a fluorescence scanner setting wavelength of 473 nm. All 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 stained positively, indicating the presence of glycosylation. As an additional confirmation, anti-MAdCAM antibody 7.26.4 was subjected to trypsin/chymotrypsin digestion, LC-MS/MS analysis confirmed the presence of modified tryptic peptides and additionally confirmed kappa light chain glycosylation.

The specific Asn-Gly sequences in the CDR1 regions of anti-MAdCAM antibodies 1.7.2, 1.8.2, 6.22.2, and 7.20.5 render these regions susceptible to deamidation. Deamidation at neutral pH introduces a negative charge and can also lead to β -isomerization, which can affect the properties of the antibody. For anti-MAdCAM antibodies 1.7.2, 1.8.2 and 7.20.5, the presence of deamidated Asn-isoaspartic acid residues was assessed by mass spectrometry after capturing the isoaspartic acid side chain with MeOH.

Briefly, for the anti-MAdCAM antibody 1.7.2, the status of trypsin/Asp-N peptide SSQSLLQSNGYNYL (SEQ ID NO: 69) (1573.7Da) was selected for monitoring by LC-MS/MS. anti-MAdCAM antibody 1.7.2 was reduced in 10mM DTT, alkylated in 5mM sodium iodoacetate and then buffer exchanged to tryptic digestion buffer (50mM Tris-HCl, 1mM CaCl₂pH 7.6). The antibody was then mixed with sequencing grade modified trypsin (Promega) in a 1: 20 protease: protein ratio. The protein was digested in trypsin at 30 ℃ for 15 hours, and the resulting peptides were then separated by HPLC using C-18 RPC on an Ettan LC system. Will contain³³Asn peptide (4032Da) was collected from the column and diluted in Asp-N digestion buffer (50mM sodium phosphate buffer, pH 8.0). The endoprotease Asp-N (Roche) is then added in a Peptidase ratio of about 10: 1.

Acetyl chloride (100. mu.L) was added to a methanol sample (1mL, -20 ℃) and the mixture was warmed to room temperature. The trypsin + Asp-N digest was dried in a Speed-Vac and then 5. mu.L of methanol/acetyl chloride was added (45 min, room temperature) before drying again in a Speed-Vac. The resulting residue was reconstituted in 0.1% TFA and the peptide was initially analyzed on a Voyager-DESTR MALDI-TOF mass spectrometer using nitrocellulose thin layer sample preparation method, or reverse phase purification, using C18 ziptips (millipore) followed by mixing with a-nitrile based matrix droplets. Methylated peptide mixtures were also analyzed on a Deca XP Plus Ion Trap mass spectrometer as above using LC-MS/MS. The eluate was directly plunged into Ion Trap MS and the peptide was subsequently analyzed by MS and MS/MS. The MS was set to analyze all ions between 300-2000 Da. And then will be the strongest in any particular scanThe ions were subjected to MS/MS analysis. TABLE 12 post-translational modification of anti-MAdCAM antibodiesTable 12 discloses SEQ ID NOs: 135-146. Mutagenesis study:

the primary amino acid sequence of the anti-MAdCAM antibodies exemplified in the present invention can be modified by site-directed mutagenesis to remove potential sites for post-translational modifications (e.g., glycosylation, deamidation) or to alter isotype background, or to design other alterations that can improve therapeutic utility. For example, PCR was used to design changes to anti-MAdCAM antibodies 6.22.2, 6.34.2, 6.67.1, 6.77.1, and 7.26.4, to restore certain framework sequences to germline sequences, to remove potential glycosylation sites, and/or to change isotype background to human IgG₂. pCR2.1 TOPO-TA cloned cDNA (100ng), corresponding to heavy chain nucleotide SEQ ID NO: 13. 17, 21 and 29, and the kappa light chain nucleotide SEQ id no: 15. 19, 23, 31 and 43, which were used as templates in a series of PCRs using overlap-extension and a set of primer pairs described in table 10.

6.22.2 heavy chain: using Expand Taq polymerase and the nucleotide sequence SEQ ID NO: PCR2.1 TOPO-TA cDNA template (100ng), indicated at 13, PCR primer pair 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 PCR products (1) and (2), respectively. Products (1) and (2) were purified and combined (about 50ng each) with VH3-33 and VK6.22.2_ CS primers in a third PCR step to produce a modified 6.22.2 heavy chain V-domain. This modified variant contained the His/Phe mutation in FR1 and introduced an XbaI restriction site to allow in-frame cloning into a pEE6.1-derived vector, designated pEE6.1CH, which contained the corresponding human IgG₂A constant region. The final PCR fragment was cloned into the XbaI site of pEE6.1CH, its orientation checked and the full-length sequence of the insert verified. The nucleotide sequence of the modified 6.22.2 heavy chain is set forth in SEQ ID NO: 51 and the corresponding amino acid sequence is found in SEQ ID NO: 52, respectively. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.22.2 kappa light chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: the PCR primer pair 6.22.2_ VK _ F1 and revKappa (1) and A26 and 6.22.2_ VK _ R1(2) were used to generate PCR products (1) and (2), respectively, using pCR2.1 TOPO-TA cDNA template (100ng) denoted 15. The products (1) and (2) were purified and combined (about 50ng each) with a26 and revKappa primers in a third PCR step to generate a modified 6.22.2 kappa light chain V-domain. The modified variants contain Asn/Asp and Gly/Ser changes to the FR3 sequence. The resulting PCR product was cloned into pEE12.1 using the HindIII/EcoR1 site and the full length sequence was verified. The nucleotide sequence of the modified 6.22.2 κ light chain may be found in SEQ ID NO: 53 and the corresponding amino acid sequence is found in SEQ ID NO: 54, respectively. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.34.2 heavy chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: PCR2.1 TOPO-TA cDNA template (100ng), indicated at 17, PCR primer pair 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 PCR products (1) and (2), respectively. Products (1) and (2) were purified and combined (about 50ng each) with VH3-30 and VK6.22.2_ CS primers in a third PCR step to produce a modified 6.34.2 heavy chain V-domain. This modified variant contained the Ser/Arg mutation in FR3 and introduced an XbaI restriction site to allow in-frame cloning into a pEE6.1-derived vector, designated pEE6.1CH, which contained the corresponding human IgG₂A constant domain. The final PCR fragment was cloned into the XbaI site of pee6.1ch, checked for orientation and checked for the full-length sequence of the insert. The nucleotide sequence of the modified 6.34.2 heavy chain may be found in SEQ ID NO: 55 and the corresponding amino acid sequence is found in SEQ ID NO: 56 (c). Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.34.2 kappa light chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: the PCR primer pairs O12 and 6.34.2_ VK _ R1(1), 6.34.2_ VK _ F1 and 6.34.2_ VK _ R2(2) and 6.34.2_ VK _ F2 and revKappa (3) were used to generate PCR products (1), (2) and (3), respectively, using pCR2.1 TOPO-TA cDNA template (100ng) denoted 19. The products (1), (2) and (3) were purified and (1) and (2) (approximately 50ng each) were combined with O12 and 6.34.2_ VK _ R2 primers in a third PCR step to produce PCR product (4). The PCR products (2) and (3) (about 50ng each) were combined with 6.34.2_ VK _ F1 and revKappa in the fourth PCR step to produce PCR product (5). The PCR products (4) and (5) were purified and combined (about 50ng each) with primer O12 and revKappa to produce a modified 6.34.2 kappa light chain V-domain. The modified variant contained Asn/Ser changes in CDR1, Phe/Tyr changes in FR2, and Arg-Thr/Ser-Ser, Asp/Glu, and Ser/Tyr changes in the FR3 sequence. The resulting PCR product was cloned into pEE12.1 using the HindIII/EcoR1 site and the full length sequence was verified. The nucleotide sequence of the modified 6.34.2 κ light chain may be found in SEQ ID NO: 57 and the corresponding amino acid sequence thereof is found in SEQ ID NO: 58, respectively. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.67.1 heavy chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: PCR2.1 TOPO-TA cDNA template (100ng), indicated at 21, PCR primer pair 6.67.1_ VH _ F1 and 6.67.1VH _ CS (1) and VH4-4 and 6.67.1_ VH _ R1(2) were used to generate PCR products (1) and (2), respectively. Products (1) and (2) were purified and combined (about 50ng each) with VH4-4 and VK6.67.1_ CS primers in a third PCR step to produce a modified 6.67.1 heavy chain V-domain. This modified variant contained the Ile-Leu-Ala/Met-Ser-Val transition in FR3 and introduced an XbaI restriction site to allow in-frame cloning into a pEE6.1-derived vector, designated pEE6.1CH, containing the corresponding human IgG₂A constant domain. The final PCR fragment was cloned into the XbaI site of pee6.1ch, checked for orientation and checked for the full-length sequence of the insert. The nucleotide sequence of the modified 6.67.1 heavy chain may be found in SEQ ID NO: 59 and the corresponding amino acid sequence is found in SEQ ID NO: 60 in. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.67.1 kappa light chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: pCR2.1 TOPO-TA cDNA template (100ng) denoted 23, PCR primer pair 6.67.1_ VK _ F1 and revKappa (1) and B and 6.67.1_ VK _ R1(2) were used to generate PCR products (1) and (2), respectively. The products (1) and (2) were purified and combined (about 50ng each) with B3 and revKappa primers in a third PCR step to generate a modified 6.67.1 kappa light chain V-domain. The modified variant comprises a Thr/Asn alteration in CDR1 and an Arg/Gly alteration in FR 2. The resulting PCR product was cloned into pEE12.1 using the HindIII/EcoR1 site and the full length sequence was verified. The nucleotide sequence of the modified 6.67.1 κ light chain may be found in SEQ ID NO: 61 and the corresponding amino acid sequence thereof is found in SEQ ID NO: 62 (c). Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.77.1 heavy chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: PCR primer pairs VH 3-21 and 6.22.2VH _ CS were used to generate individual PCR products, designated pcr2.1 TOPO-TA cDNA template (100 ng). The PCR product was digested with XbaI, gel purified and cloned into the XbaI site of pee6.1ch, checking orientation. The insert was subjected to a full length sequence check. The nucleotide sequence of the modified 6.77.1 heavy chain may be found in SEQ ID NO: 63 and the corresponding amino acid sequence thereof is found in SEQ ID NO: 64. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.77.1 kappa light chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: PCR primer pairs A2 and 6.77.1_ VK _ R1(1), 6.77.1_ VK _ VK _ F1 and 6.77.1_ R2(2) and 6.77.1_ VK _ F2 and revKappa (3) were used to generate PCR products (1), (2) and (3), respectively, using pCR2.1 TOPO-TA cDNA template (100ng) denoted 31. The products (1), (2) and (3) were purified and (1) and (2) (approximately 50ng each) were combined with a2 and 6.77.1_ VK _ R2 primers in a third PCR step to produce PCR product (4). PCR products (2) and (3) (each about 50ng) were subjected to a fourth PCR step) Was combined with 6.77.1_ VK _ F1 and revKappa primers to give PCR product (5). PCR products (4) and (5) were purified and combined (about 50ng each) with primer a2 and JK2 to produce a modified 6.77.1 kappa light chain V-domain. The modified variant comprises an Asn/Lys change in the CDR1, a Ser/Tyr change in the FR3 and a Cys/Ser residue change in the CDR3 sequence. The resulting PCR product was cloned into pEE12.1 using the HindIII/EcoR1 site and the full length sequence was verified. The nucleotide sequence of the modified 6.77.1 κ light chain may be found in SEQ ID NO: 65 and the corresponding amino acid sequence thereof is found in seq id NO: 66 (c). Changes in nucleotide and amino acid sequences compared to the parent are shown.

7.26.4 kappa light chain:using Expand Taq polymerase and a primer consisting of the nucleotide sequence SEQ ID NO: pCR2.1 TOPO-TA cDNA template (100ng), indicated at 43, PCR primer pair 7.26.4_ VK _ F1 and revKappa (1) and A2 and 7.26.4_ VK _ R1(2) were used to generate PCR products (1) and (2), respectively. The products (1) and (2) were purified and combined (about 50ng each) with a2 and revKappa primers in a third PCR step to generate a modified 7.26.4 kappa light chain V-domain. The modified variant comprises an Asn/Ser change in CDR 1. The resulting PCR product was cloned into pEE12.1 using the HindIII/EcoR1 site and the full length sequence was verified. The nucleotide sequence of the modified 7.26.4 κ light chain may be found in SEQ ID NO: 67 and the corresponding amino acid sequence thereof is found in SEQ ID NO: 68. Changes in nucleotide and amino acid sequences compared to the parent are shown.

6.22.2, 6.34.2, 6.67.1, 6.77.1 and 7.26.4 are 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 their heavy chain nucleotide sequences are represented as SEQ ID NOs: 51. 55, 59, 63 and 41, the corresponding amino acid sequences being SEQ ID NO: 52. 56, 60, 64 and 42, and the kappa light chain nucleotide sequence is SEQ ID NO: 53. 57, 61, 65 and 67, the corresponding amino acid sequences being SEQ ID NOs: 54. 58, 62, 66 and 68, functional eukaryotic expression vectors for each of the variants are assembled as follows: 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 using NotI/SalIIn general, the parental version of the heavy chain of 7.26.4 was excised from the pEE6.1 vector using NotI/SalI, and the purified fragment was cloned into the same site in the corresponding pEE12.1 vector containing the kappa light chain sequence of the 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod modified variants. The sequence of the vector was confirmed and purified amounts of the vector were used to transiently transfect HEK 293T cells. Briefly, 9 × 10⁶HEK 293T cells (which were seeded in T165 flasks and washed into Optimem the day before transfection) were transiently transfected with vector cDNAs (40. mu.g) corresponding to 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod using Lipofectamine PLU (Invitrogen) according to the manufacturer's instructions. Cells were incubated for 3 hours, and then transfection medium was replaced with DMEM (Invitrogen 21969-035) medium containing 10% ultra-low IgG fetal bovine serum (Invitrogen 16250-078) and L-glutamine (50 mL). After 5 days the culture supernatant was harvested, filter sterilized and the anti-MAdCAM antibody was purified by protein G sepharose affinity chromatography in a similar manner as described above. The amount of antibody recovered (20-100. mu.g) was quantified by Bradford assay.

The anti-MAdCAM activity of affinity purified antibodies corresponding to 6.22.2-mod, 6.34.2-mod, 6.67.1-mod, 6.77.1-mod and 7.26.4-mod was evaluated in MAdCAM-IgG1-Fc fusion assays as previously described. IC of these anti-MADCAM antibodies compared to the parent anti-MADCAM antibodies from which they are derived₅₀The values are shown in table 13. The above described amino acid substitutions have little effect on the activity of the modified anti-MAdCAM antibodies compared to their parent. The antibodies also retained their binding to CHO cells expressing recombinant human MAdCAM or cynomolgus/human MAdCAM chimeras.

Table 13 activity of modified 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 to their parent.

Example VI

Beta in peripheral circulation caused by blocking anti-MAdCAM antibodies₇ ⁺Increase of lymphocytesAdding

Assays were developed to determine and correlate the mechanistic effects of anti-MAdCAM antibodies and their circulating levels in the blood. Inhibitory anti-MAdCAM antibodies should have inhibitory expression of alpha₄β₇Effect of integrin leukocyte recruitment to the gastrointestinal tract. Carrying alpha₄β₇The leukocyte classes of integrins should therefore be limited to the peripheral circulation.

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

Purified anti-human MAdCAM mAb 7.16.6(1mg/kg) or vehicle (20mM sodium acetate, 0.2mg/mL polysorbate 80, 45mg/mL mannitol and 0.02mg/mL EDTA, pH 5.5) were evaluated in a similar manner by intravenous administration to two groups of cynomolgus monkeys (n-4/group) via saphenous vein. Blood samples were collected in EDTA by femoral vein puncture 3 days after dosing. And macaque alpha₄β₇Integrin cross-reactive LPAM-specific antibodies, which are not commercially available, were used₇Antibody (recognition of alpha)₄β₇And alpha_Eβ₇Integrins) replacement. According to the following table, table 15, antibodies (30 μ L) were added to tubes containing 100 μ L of cynomolgus monkey blood, mixed by gentle vortex and incubated at 4 ℃ for 20-30 minutes.

TABLE 15 antibodies for immunotyping of cynomolgus monkey blood (BD Pharmingen)

Directory number	Antibodies or isotypes
		555748	mIgG1，k-FITC
555844	mIgG2a，k-PE
		559425	mIgG1-PerCP
555751	mIgG1，k-APC
		555728	CD 28-FITC
555945	7-PE
		558814	CD 95-APC
550631	CD 4-PerCP

To each tube was added 1mL of a 1: 10 FACLyse solution (BD #349202), mixed by gentle swirling and incubated in the dark at room temperature for about 12 minutes until the red blood cells were lysed. Then 2mL of BD staining buffer (#554656) was added to each tube, mixed and centrifuged at 250Xg for 6-7 minutes at room temperature. The supernatant was decanted and the pellet resuspended in 3mL of staining buffer, mixed again and resuspended at 250Xg at room temperatureCentrifuge for 6-7 minutes. Cytofix buffer (BD #554655) containing w/v paraformaldehyde (100. mu.L) was added to the cell pellet particles from peripheral blood of monkeys and mixed well by a low/medium speed vortexer. Samples were stored in the dark at 4 ℃ until collected for FACSCalibur. Immediately prior to harvest, PBS (100 μ L) was added to all tubes. CD4 obtained by appropriate gated sorting and quadrant analysis⁺β₇ ⁺CD95loCD28⁺(naive), CD4⁺β₇ ⁺CD95hiCD28⁺(central memory cell), CD4⁺β₇-CD95hiCD28⁺(central memory cell), CD4⁺β₇ ⁺CD95hiCD28^-Absolute cell number (effector memory cells). Other T cell subsets, e.g. CD8⁺T Central memory cell (. beta.)₇ ⁺CD8⁺CD28⁺CD95⁺) And any other leukocytes carrying MAdCAM ligands, can also be assayed by this method with suitable antibodies. anti-MAdCAM mAb 7.16.6 elicits circulating CD4 compared to vehicle controls⁺β₇ ⁺CD95hiCD28⁺Central memory T cell levels were increased approximately 3-fold as shown in figure 7. For circulating CD4⁺β₇-CD95hiCD28⁺The central memory T cell population had no effect, indicating that the effect of anti-MAdCAM mAb 7.16.6 is specific for T cells homing to the gut. anti-MAdCAM mAb 7.16.6 in cynomolgus monkeys against circulating (. alpha.)₄)β₇ ⁺The influence of lymphocyte populations suggests that this is a strong alternative evidence for mechanistic biomarkers, particularly in terms of practical application in a clinical setting.

Sequence of

SEQ ID NO: 1-48 and 51-68 provide the nucleotide and amino acid sequences of the heavy and kappa light chains of 12 human anti-MAdCAM antibodies, macaque MAdCAM alpha₄ β₇The nucleotide and amino acid sequences of the binding domains and the nucleotide and amino acid sequences of the five modified human anti-MAdCAM antibodies.

SEQ ID NO: 1-48 provides the nucleotide and amino acid sequences of the heavy and kappa light chains of 12 human monoclonal anti-MAdCAM antibodies: 1.7.2(SEQ ID NOS: 1-4), 1.8.2(SEQ ID NOS: 5-8), 6.14.2(SEQ ID NOS: 9-12), 6.22.2(SEQ ID NOS: 13-16), 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), and 9.8.2(SEQ ID NOS: 45-48).

SEQ ID NO: 49-50 provide macaque MADCAM alpha₄β₇Nucleotide and amino acid sequences of the binding domain.

SEQ ID NO: 51-68 provides the nucleotide and amino acid sequences of the heavy and kappa light chains of modified monoclonal anti-MAdCAM antibodies: 6.22.2(SEQ ID NOS: 51-54), modified 6.34.2(SEQ ID NOS: 55-58), modified 6.67.1(SEQ ID NOS: 59-62), modified 6.77.1(SEQ ID NOS: 63-66); and modified monoclonal anti-MAdCAM antibodies: the nucleotide and amino acid sequences of the modified kappa light chain of 7.26.4(SEQ ID NOS: 67-68). SEQ ID NOS: 70-106 and 108-110 provide various primer sequences.

Claims (31)

1. Hybridoma cell line 7.16.6, having ECACC accession No. 03090909.

2. A human monoclonal antibody that specifically binds to a mucosal addressin cell adhesion molecule (MAdCAM), wherein the monoclonal antibody has the same amino acid sequence as an antibody produced by hybridoma cell line 7.16.6 having ECACC accession No. 03090909.

3. Human monoclonal antibodies that specifically bind MAdCAM and inhibit the binding of MAdCAM to alpha₄β₇Wherein the antibody consists of a heavy chain and a light chain, wherein the heavy and light chains comprise the heavy and light chain CDR1, CDR2 and CDR3 amino acid sequences, respectively, of an antibody produced by hybridoma cell line 7.16.6 having ECACC accession No. 03090909.

4. The antigen-binding portion of the human monoclonal antibody of claim 3, which specifically binds to MAdCAM and inhibits binding of MAdCAM to α₄β₇。

5. Human monoclonal antibodies that specifically bind MAdCAM and inhibit the binding of MAdCAM to alpha₄β₇Wherein the antibody consists of a heavy chain and a light chain, wherein the heavy and light chains comprise the heavy and light chain variable domain amino acid sequences, respectively, of an antibody produced by hybridoma cell line 7.16.6 having ECACC accession No. 03090909.

6. The antigen-binding portion of the human monoclonal antibody of claim 5, which specifically binds to MAdCAM and inhibits binding of MAdCAM to α₄β₇。

7. Human monoclonal antibodies that specifically bind MAdCAM and inhibit the binding of MAdCAM to alpha₄β₇Wherein the antibody is comprised of a heavy chain and a light chain, wherein the heavy chain CDR1 amino acid sequence is SYGIN, the heavy chain CDR2 amino acid sequence is WISVYSGNTNYAQKVQG, the heavy chain CDR3 amino acid sequence is EGSSSSGDYYYGMDV, the light chain CDR1 amino acid sequence is KSSQSLLHTDGTTYLY, the light chain CDR2 amino acid sequence is EVSNRFS, and the light chain CDR3 amino acid sequence is MQNIQLPWT.

8. The antigen-binding portion of the human monoclonal antibody of claim 7, which specifically binds to MAdCAM and inhibits the binding of MAdCAM to α₄β₇。

9. Human monoclonal antibodies that specifically bind to MAdCAM and inhibitMAdCAM binding to alpha₄β₇Wherein the antibody consists of a heavy chain and a light chain, wherein the amino acid sequences of the heavy and light chain variable domains are SEQ ID NOs: 34 and the amino acid sequence of residues 20 to 144 of SEQ ID NO: 36 from residues 21 to 133.

10. The antigen-binding portion of the human monoclonal antibody of claim 9, which specifically binds MAdCAM and inhibits the binding of MAdCAM to α₄β₇。

11. A human monoclonal antibody consisting of a heavy chain and a light chain, wherein the amino acid sequences of the heavy and light chains are SEQ ID NOs: 34 and SEQ ID NO: 36.

12. a human monoclonal antibody consisting of a heavy chain and a light chain, wherein the amino acid sequences of the heavy and light chains are SEQ ID NOs: 34 and SEQ ID NO: 36, and wherein the heavy chain C-terminal lysine is cleaved.

13. Human monoclonal antibody according to any one of claims 3, 5,7 and 9, which is an IgG, IgM, IgE, IgA or IgD molecule or a bispecific antibody.

14. The antigen-binding portion of any one of claims 4,6, 8 and 10 which is a Fab fragment, a Fab 'fragment, a F (ab')₂Fragment, F_VFragments or single chain antibodies.

15. A pharmaceutical composition comprising an effective amount of the human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or the antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 and a pharmaceutically acceptable carrier.

16. Use of a human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or an antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 for the manufacture of a medicament for the treatment of an inflammatory disease.

17. The use according to claim 16, wherein the inflammatory disease is an inflammatory disease of the gastrointestinal tract.

18. The use according to claim 17, wherein the gastrointestinal inflammatory disease is inflammatory bowel disease.

19. The use according to claim 18, wherein the inflammatory bowel disease is crohn's disease or ulcerative colitis.

20. An isolated cell line producing the human monoclonal antibody of any one of claims 2, 3, 5,7, 9 and 11-13 or the antigen binding portion of any one of claims 4,6, 8, 10 and 14.

21. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or heavy chain antigen-binding portion or the light chain or light chain antigen-binding portion or both of the human monoclonal antibody of any of claims 2, 3, 5,7, 9, and 11-13.

22. A vector comprising the nucleic acid molecule of claim 21, wherein said vector optionally comprises an expression control sequence operably linked to said nucleic acid molecule.

23. A host cell, wherein said host cell comprises the vector of claim 22 or the nucleic acid molecule of claim 21.

24. A method for producing a monoclonal antibody or antigen-binding portion thereof that specifically binds MAdCAM, the method comprising culturing the host cell of claim 23 or the cell line of claim 1 or 20 under suitable conditions and recovering the antibody or antigen-binding portion.

25. Use of a human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or an antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 for the manufacture of a medicament for inhibiting leukocyte adhesion, migration and infiltration into tissue.

26. Use of a human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or an antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 in the preparation of a composition for the diagnosis of a disorder characterized by circulating soluble human MAdCAM.

27. A diagnostic kit comprising a human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or an antigen-binding portion according to any one of claims 4,6, 8, 10 and 14.

28. A vaccine comprising an effective amount of the human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or the antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 and a pharmaceutically acceptable carrier.

29. The vaccine according to claim 28, wherein the vaccine is a mucosal vaccine.

30. Use of a human monoclonal antibody according to any one of claims 2, 3, 5,7, 9 and 11-13 or an antigen-binding portion according to any one of claims 4,6, 8, 10 and 14 for the preparation of a method for detecting expression of alpha in circulation in a subject₄β₇To the use of the composition for increasing the level of leukocytes.

31. The use of claim 30, wherein the expression is alpha₄β₇The white blood cells of (a) are lymphocytes.