Anti-HLA-DQ2.5 antibody

Abstract

The present invention provides anti-HLA-DQ2.5 antibodies. These antibodies have binding activity against complexes formed between HLA-DQ2.5 and gluten peptides, but have little binding activity against complexes formed between HLA-DQ2.5 and unrelated peptides. Furthermore, the antibodies have been found to inhibit T cell activation.

Description

Technical Field

[0001] The present invention relates to anti-HLA-DQ2.5 antibodies Background Art

[0002] Celiac disease is an autoimmune disorder in which gluten ingestion damages the small intestine in genetically susceptible patients (NPLs 1 to 5). Approximately 1% of the Western population, or 8 million people in the United States and European Union, is thought to have celiac disease; however, no significant treatment advances have been made since the disease was first recognized in the 1940s.

[0003] Human leukemia antigens (HLA) belonging to the major histocompatibility complex (MHC) class II include HLA-DR, HLA-DP, and HLA-DQ molecules, such as the HLA-DQ2.5 isotype (hereinafter referred to as "HLA-DQ2.5"), which forms heterodimers composed of α and β chains on the cell surface. Most (>90%) patients with celiac disease have the HLA-DQ2.5 haplotype allele (NPL 6). This isotype is believed to have a stronger affinity for gluten peptides. Like other isotypes, HLA-DQ2.5 presents processed antigens derived from exogenous sources to the T cell receptor (TCR) on T cells. As a result of digestion of gluten-rich foods (e.g., bread) in celiac disease patients, immunogenic gluten peptides (e.g., gliadin peptides) (NPL 2) are formed. These peptides are transported through the intestinal epithelium into the lamina propria and deamidated by tissue transglutaminases such as transglutaminase 2 (TG2). Deamidated gliadin peptides are processed by antigen-presenting cells (APCs), which load them onto HLA-DQ2.5. The loaded peptides are presented to HLA-DQ2.5-restricted T cells and activate innate and adaptive immune responses. This causes inflammatory damage to the small intestinal mucosa and symptoms including various types of gastrointestinal disorders, nutritional deficiencies, and systemic symptoms. Anti-HLA DQ neutralizing antibodies have been reported to inhibit the activation of T cells from celiac disease patients. (NPL 7)

[0004] The current treatment for celiac disease is a lifelong gluten-free diet (GFD). However, even with a GFD, it is difficult to completely eliminate gluten exposure. These patients can tolerate only approximately 10 to 50 mg / day of gluten (NPL 11). Cross-contamination during GFD production is widespread, and even in patients who are well compliant with the GFD, trace amounts of gluten can cause celiac disease symptoms. In situations where this risk of inadvertent gluten exposure exists, adjunctive therapy with the GFD is necessary.

[0005] Citation List

[0006] Non-patent literature

[0007] [NPL 1]N Engl J Med 2007;357:1731-1743

[0008] [NPL 2]J Biomed Sci.2012;19(1):88

[0009] [NPL 3]N Engl J Med 2003;348:2517-2524

[0010] [NPL 4]Gut 2003;52:960-965

[0011] [NPL 5]Dig Dis Sci 2004;49:1479-1484

[0012] [NPL 6]Gastroenterology 2011;141:610-620

[0013] [NPL 7]Gut 2005;54:1217-1223

[0014] [NPL 8]Gastroenterology 2014;146:1649-58

[0015] [NPL 9]Nutrients 2013 Oct 5(10): 3975-3992

[0016] [NPL 10]J Clin Invest.2007;117(1):41-49

[0017] [NPL 11]Am J Clin Nutr 2007;85:160-6 Summary of the Invention

[0018] Technical issues

[0019] In the case where adjuvant therapy is required, the present invention provides anti-HLA-DQ2.5 antibodies.

[0020] Solutions to the Problem

[0021] Antigen-binding molecules of the invention, particularly monospecific and multispecific (eg, bispecific) antibodies, can bind to one or more complexes formed by HLA-DQ2.5 and gluten peptides.

[0022] More specifically, the present invention provides the following.

[0023] [1] An antigen-binding molecule having binding activity against at least one, two, three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide,

[0024] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0025] [1-2] The antigen-binding molecule of [1], wherein the antigen-binding molecule has binding activity against at least one, two, three, four, five, six, seven, eight or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide,

[0026] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0027] [2] The antigen-binding molecule of [1], wherein the antigen-binding molecule has binding activity against at least one, two, three, four, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide,

[0028] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0029] [2-2] The antigen-binding molecule of [2], wherein the antigen-binding molecule has binding activity against at least one, two, three, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide,

[0030] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0031] [3] The antigen-binding molecule of [1], which has binding activity against at least three, four, five, six, seven, eight, nine or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide,

[0032] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0033] [3-2] The antigen-binding molecule of [3], wherein the antigen-binding molecule has binding activity against at least three, four, five, six, seven, eight or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide,

[0034] The antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0035] [4] An antigen-binding molecule having binding activity to all of: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide,

[0036] The antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0037] [4-2] The antigen-binding molecule of [4], wherein the antigen-binding molecule has binding activity against all of: a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide,

[0038] The antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0039] [5] The antigen-binding molecule of [4], wherein the antigen-binding molecule has binding activity against all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; and a complex formed by HLA-DQ2.5 and a 26-mer gliadin.

[0040] wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0041] [6] The antigen-binding molecule of [5], wherein the antigen-binding molecule has binding activity to a complex formed by HLA-DQ2.5 and an immunodominant peptide associated with celiac disease.

[0042] [7] The antigen-binding molecule of [5], wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an immunodominant peptide associated with celiac disease; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide.

[0043] [8] The antigen-binding molecule described in [5], wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA- complex formed by HLA-DQ2.5 and α1b gliadin peptide; complex formed by HLA-DQ2.5 and γ4b gliadin peptide; complex formed by HLA-DQ2.5 and avenin 1 peptide; complex formed by HLA-DQ2.5 and avenin 2 peptide; complex formed by HLA-DQ2.5 and hordein 1 peptide; complex formed by HLA-DQ2.5 and hordein 2 peptide; complex formed by HLA-DQ2.5 and secalin 1 peptide; complex formed by HLA-DQ2.5 and secalin 2 peptide; complex formed by HLA-DQ2.5 and 14-mer 1 peptide; and complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0044] [5-2] The antigen-binding molecule of [5], wherein the antigen-binding molecule has binding activity against all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a 26-mer gliadin.

[0045] wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0046] [9] The antigen-binding molecule according to any one of [1] to [8], wherein the antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells.

[0047]

[10] The antigen-binding molecule of any one of [1] or [9], wherein the antigen-binding molecule has substantially no binding activity to HLA-DQ8, HLA-DQ2.2, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLADQ7.3, HLA-DR, or HLA-DP.

[0048]

[11] The antigen-binding molecule according to any one of [1] to

[10] , wherein the antigen-binding molecule has enhanced binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide.

[0049]

[12] The antigen-binding molecule of any one of [1] to

[11] , wherein the antigen-binding molecule is compared to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCs. B cells, wherein the antigen-binding molecule has stronger binding activity to at least one, two, three, four, five, six, seven, eight, nine or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide.

[0050] [12-2] The antigen-binding molecule described in

[12] , wherein the antigen-binding molecule is compared with at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a bovine Mycobacterium peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC. B cells, the antigen-binding molecules of the present invention have stronger binding activity to at least one, two, three, four, five, six, seven, eight or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide.

[0051]

[13] An antigen-binding molecule having binding activity against at least one, two, three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide. a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells,

[0052] The antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells.

[0053] [13-2]

[13] Antigen-binding molecules, wherein the antigen-binding molecules of the present invention have binding activity to at least one, two, three, four, five, six, seven, eight or all of the following: a complex formed by HLA-DQ2.5 and BC hordein peptide; a complex formed by HLA-DQ2.5 and γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and α1 gliadin peptide The present invention also provides a method for treating a gluten-restricted CD4+ T cell by blocking the interaction between the HLA-DQ2.5 / gluten peptide complex and the HLA-DQ2.5 / gluten peptide-restricted CD4+ T cell. In this case, the gluten peptide is a peptide in the complex bound by any of the above antigen-binding molecules.

[0054]

[14] The antigen-binding molecule of any one of [1] to [13-2], which is any one of the following (1) to (5):

[0055] (1) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0056] (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0057] (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0058] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule described in any one of (1) to (3);

[0059] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0060]

[15] The antigen-binding molecule according to any one of [1] to

[14] , wherein the antigen-binding molecule is a bispecific antigen-binding molecule.

[0061]

[16] The antigen-binding molecule described in

[15] , wherein the bispecific antigen-binding molecule is a bispecific antibody.

[0062]

[17] An antigen-binding molecule comprising at least two antigen-binding domains, wherein any one of the antigen-binding domains has binding activity against one or more complexes formed between HLA-DQ2.5 and an immunodominant peptide associated with celiac disease,

[0063] wherein any one of the antigen-binding domains has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell,

[0064] The antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0065]

[18] An antigen-binding molecule comprising at least two antigen-binding domains, wherein any one of the antigen-binding domains has binding activity against all of: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; and a complex formed by HLA-DQ2.5 and a BC hordein peptide,

[0066] wherein any one of the antigen-binding domains has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell,

[0067] The antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0068]

[19]

[18] The antigen-binding molecule described in any one of the antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and α1 gliadin peptide; a complex formed by HLA-DQ2.5 and α2 gliadin peptide; a complex formed by HLA-DQ2.5 and ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and BC hordein peptide; a complex formed by HLA-DQ2.5 and γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and γ2 gliadin peptide.

[0069]

[20]

[19] The antigen-binding molecule described in any one of the antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide.

[0070]

[21] An antigen-binding molecule comprising at least two antigen-binding domains, wherein any one of the antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and avenin 1 peptide; a complex formed by HLA-DQ2.5 and avenin 2 peptide; a complex formed by HLA-DQ2.5 and avenin 3 peptide; a complex formed by HLA-DQ2.5 and hordein 1 peptide; a complex formed by HLA-DQ2.5 and hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide,

[0071] wherein any one of the antigen-binding domains has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell,

[0072] The antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0073]

[22]

[21] An antigen-binding molecule, wherein any one of the antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and α1 gliadin peptide; a complex formed by HLA-DQ2.5 and α1b gliadin peptide; a complex formed by HLA-DQ2.5 and α2 gliadin peptide; a complex formed by HLA-DQ2.5 and ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and secalin 1 peptide; a complex formed by HLA-DQ2.5 and secalin 2 peptide; a complex formed by HLA-DQ2.5 and BC hordein peptide complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and avenin 1 peptide; a complex formed by HLA-DQ2.5 and avenin 2 peptide; a complex formed by HLA-DQ2.5 and avenin 3 peptide; a complex formed by HLA-DQ2.5 and hordein 1 peptide; a complex formed by HLA-DQ2.5 and hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide,

[0074]

[23] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain has binding activity to one or more complexes formed by HLA-DQ2.5 and gluten peptides, wherein the second antigen-binding domain has binding activity to one or more complexes formed by HLA-DQ2.5 and gluten peptides, wherein at least one gluten peptide in the complex bound by the first antigen-binding domain is different from at least one gluten peptide in the complex bound by the second antigen-binding domain.

[0075]

[24]

[23] , wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide.

[0076]

[25]

[23] , wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide.

[0077]

[26]

[23] , wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide,

[0078] wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells.

[0079]

[27]

[26] , wherein the antigen-binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide.

[0080]

[28] An antigen-binding molecule comprising a first antigen-binding domain having binding activity to a complex formed by HLA-DQ2.5 and a first gluten peptide and a second antigen-binding domain having binding activity to a complex formed by HLA-DQ2.5 and a second gluten peptide,

[0081] wherein the antigen-binding molecule has binding activity against at least two or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ complex formed by HLA-DQ2.5 and a hordein 1 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide.

[0082] wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells,

[0083] The antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0084]

[29]

[28] The antigen-binding molecule has binding activity to at least two or more of the following: a complex formed by HLA-DQ2.5 and α1 gliadin peptide; a complex formed by HLA-DQ2.5 and α1b gliadin peptide; a complex formed by HLA-DQ2.5 and α2 gliadin peptide; a complex formed by HLA-DQ2.5 and ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and secalin 1 peptide; a complex formed by HLA-DQ2.5 and secalin 2 peptide; a complex formed by HLA-DQ2.5 and BC hordein peptide; complexes formed by HLA-DQ2.5 and a γ1 gliadin peptide; complexes formed by HLA-DQ2.5 and a 26-mer gliadin peptide; complexes formed by HLA-DQ2.5 and a 14-mer 1 peptide; complexes formed by HLA-DQ2.5 and an α3 gliadin peptide; complexes formed by HLA-DQ2.5 and avenin 1 peptide; complexes formed by HLA-DQ2.5 and avenin 2 peptide; complexes formed by HLA-DQ2.5 and avenin 3 peptide; complexes formed by HLA-DQ2.5 and hordein 1 peptide; complexes formed by HLA-DQ2.5 and hordein 2 peptide; and complexes formed by HLA-DQ2.5 and a γ4b gliadin peptide.

[0085]

[30] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain,

[0086] wherein the first antigen-binding domain has binding activity against at least one or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; and a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0087] wherein the second antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and BC hordein peptide; a complex formed by HLA-DQ2.5 and γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an avenin 1 peptide; a complex formed by HLA-DQ2.5 and an avenin 2 peptide; a complex formed by HLA-DQ2.5 and an avenin 3 peptide; a complex formed by HLA-DQ2.5 and a hordein 1 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide,

[0088] wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells,

[0089] The antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0090]

[31]

[30] The antigen-binding molecule of

[31]

[30] , wherein the second antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and BC hordein peptide; a complex formed by HLA-DQ2.5 and γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and 33-mer gliadin peptide ; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an avenin 1 peptide; a complex formed by HLA-DQ2.5 and an avenin 2 peptide; a complex formed by HLA-DQ2.5 and an avenin 3 peptide; a complex formed by HLA-DQ2.5 and a hordein 1 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide.

[0091]

[32] The antigen-binding molecule of any one of

[17] to

[31] , wherein the antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells.

[0092]

[33] The antigen-binding molecule of any one of

[17] to

[32] , wherein the antigen-binding molecule has substantially no binding activity to HLA-DQ2.2, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLADQ7.3, HLA-DR or HLA-DP.

[0093]

[34] The antigen-binding molecule of any one of

[17] to

[33] , which has enhanced binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide.

[0094]

[35] The antigen-binding molecule of any one of

[17] to

[34] , wherein the antigen-binding molecule is compared to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC. B cells, wherein the antigen-binding molecule has stronger binding activity to at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18 or all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide. complexes formed by HLA-DQ2.5 and avenin 1 peptide; complexes formed by HLA-DQ2.5 and avenin 2 peptide; complexes formed by HLA-DQ2.5 and avenin 3 peptide; complexes formed by HLA-DQ2.5 and hordein 1 peptide; complexes formed by HLA-DQ2.5 and hordein 2 peptide; and complexes formed by HLA-DQ2.5 and γ4b gliadin peptide.

[0095]

[36] The antigen-binding molecule of any one of

[17] to

[35] , wherein the antigen-binding molecule is compared to at least one, two, three, four, five or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC. B cells, wherein the antigen-binding molecule has stronger binding activity to at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17 or all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and an α1 ...1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA A complex formed by A-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an avenin 1 peptide; a complex formed by HLA-DQ2.5 and an avenin 2 peptide; a complex formed by HLA-DQ2.5 and an avenin 3 peptide; a complex formed by HLA-DQ2.5 and a hordein 1 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide.

[0096]

[37] The antigen-binding molecule of any one of

[17] to

[36] , which is any one of the following (1) to (5):

[0097] (1) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0098] (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0099] (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0100] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of any one of (1) to (3);

[0101] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0102]

[38] The antigen-binding molecule of any one of

[17] to

[37] , wherein the antigen-binding molecule is a bispecific antigen-binding molecule.

[0103]

[39]

[38] The antigen-binding molecule of

[39] , wherein the bispecific antigen-binding molecule is a bispecific antibody.

[0104]

[40] The antigen-binding molecule of any one of

[37] to

[39] , which is any one of the following (a) to (d):

[0105] (a) an antigen-binding molecule comprising the following (i) and (iii),

[0106] (b) an antigen-binding molecule comprising the following (ii) and (iii),

[0107] (c) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of (a) or (b),

[0108] (d) an antigen-binding molecule that competes with the antigen-binding molecule of (a) or (b) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide,

[0109] (i) a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0110] (ii) a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0111] (iii) HCDR1 sequence of SEQ ID NO: 10, HCDR2 sequence of SEQ ID NO: 11, HCDR3 sequence of SEQ ID NO: 12, LCDR1 sequence of SEQ ID NO: 26, LCDR2 sequence of SEQ ID NO: 27, and LCDR3 sequence of SEQ ID NO: 28.

[0112] [40-1] The antigen-binding molecule of any one of [9],

[13] and

[32] , wherein the gluten peptides are one, two, three, four, five, six, seven, eight or all of the following: α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, BC hordein peptide, α1b gliadin peptide and γ4a gliadin peptide.

[0113] [40-2] The antigen-binding molecule of [40-1], wherein the gluten peptide is α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, and α1b gliadin peptide.

[0114] [40-3] The antigen-binding molecule of [40-1], wherein the gluten peptide is α2 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, BC hordein peptide, α1b gliadin peptide and γ4a gliadin peptide.

[0115] [40-4] The antigen-binding molecule of [40-1], wherein the gluten peptide is α2 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, and BC hordein peptide.

[0116] [40-5] The antigen-binding molecule of [40-1], wherein the gluten peptide is α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC hordein peptide, α1b gliadin peptide, γ4a gliadin peptide and γ2 gliadin peptide.

[0117] [40-5a] The antigen-binding molecule of [40-1], wherein the gluten peptide is α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC hordein peptide, α1b gliadin peptide and γ4a gliadin peptide.

[0118] [40-6] The antigen-binding molecule of [40-1], wherein the gluten peptide is α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC hordein peptide, and α1b gliadin peptide.

[0119]

[41] A nucleic acid encoding the antigen-binding molecule of any one of [1] to [40-6].

[0120]

[42] A vector into which the nucleic acid of

[41] is introduced.

[0121]

[43] A cell comprising the nucleic acid of

[41] or the vector of

[42] .

[0122]

[44] A method for producing an antigen-binding molecule by culturing the cells of

[43] .

[0123]

[45] The antigen-binding molecule of any one of the following (1) to (5):

[0124] (1) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0125] (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0126] (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0127] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of any one of (1) to (3);

[0128] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0129]

[46] The antigen-binding molecule of

[45] , which is any one of the following (a) to (d):

[0130] (a) an antigen-binding molecule comprising the following (i) and (iii),

[0131] (b) an antigen-binding molecule comprising the following (ii) and (iii),

[0132] (c) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of (a) or (b),

[0133] (d) an antigen-binding molecule that competes with the antigen-binding molecule of (a) or (b) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide,

[0134] (i) a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0135] (ii) a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0136] (iii) HCDR1 sequence of SEQ ID NO: 10, HCDR2 sequence of SEQ ID NO: 11, HCDR3 sequence of SEQ ID NO: 12, LCDR1 sequence of SEQ ID NO: 26, LCDR2 sequence of SEQ ID NO: 27, and LCDR3 sequence of SEQ ID NO: 28. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] [ Figure 1 ] Figure 1 Figure 2 shows an analysis of DQN0344xx / / IC17 binding to a complex formed by HLA-DQ2.5 and a gluten-derived peptide or an unrelated peptide. In the figure, "α," "γ," and "ω" are abbreviated as "a," "g," and "w." The same applies to other figures and other parts of this specification.

[0138] [ Figure 2 ] Figure 2 Shown are binding analyses of DQN0385ee / / IC17 to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0139] [ Figure 3 ] Figure 3 Shown are binding analyses of DQN0429cc / / IC17 to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0140] [ Figure 4 ] Figure 4 Shown are binding analyses of DQN0344xx / / DQN0385ee to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0141] [ Figure 5 ] Figure 5 Shown are binding analyses of DQN0344xx / / DQN0429cc to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0142] [ Figure 6 ] Figure 6 Shown are binding analyses of DQN0139bb / / IC17 to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0143] [ Figure 7 ] Figure 7 Shown are binding analyses of DQN0344xx to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0144] [ Figure 8 ] Figure 8Shown are binding analyses of DQN0385ee to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0145] [ Figure 9 ] Figure 9 Shown are binding analyses of DQN0429cc to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0146] [ Figure 10 ] Figure 10 Shown are binding analyses of DQN0139bb to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0147] [ Figure 11 ] Figure 11 Shown are analyses of IC17 binding to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides.

[0148] [ Figure 12 ] Figure 12 The binding analysis of the antibody to HLA-DQ5.1, HLA-DQ6.3, HLA-DR and HLA-DP is shown. The four strips from left to right show the results of HLA-DQ5.1, HLA-DQ6.3, HLA-DR and HLA-DP respectively.

[0149] [ Figure 13 ] Figure 13 Shown is the analysis of antibody binding to HLA-DQ2.5 positive PBMC-B cells.

[0150] [ Figure 14 ] Figure 14 Shown is the analysis of antibody binding to HLA-DQ2.5-positive PBMC-B cells.

[0151] [ Figure 15 ] Figure 15 is a summary of the above results. Figure 15 The numerical data are shown in Table 4.

[0152] [ Figure 16 ] Figure 16 is a summary of the above results. Figure 16 The numerical data are shown in Table 5.

[0153] [ Figure 17 ] Figure 17The neutralizing activity of the bivalent antibodies is shown. For DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, and IC17, the eight lanes from left to right show the results at antibody concentrations of 20, 5, 1.25, 0.3125, 0.078125, 0.019531, 0.004883, and 0.001221 μg / mL, respectively.

[0154] [ Figure 18 ] Figure 18 The neutralizing activity of the bispecific antibodies is shown. For DQN0344xx / / IC17, DQN0385ee / / IC17, DQN0429cc / / IC17, DQN0344xx / / DQN0385ee, DQN0344xx / / DQN0429cc, DQN0139bb / / IC17, and IC17, the eight lanes from left to right show the results at antibody concentrations of 20, 5, 1.25, 0.3125, 0.078125, 0.019531, 0.004883, and 0.001221 μg / mL, respectively.

[0155] [ Figure 19 ] Figure 19 The ELISA results of the initial screening are shown. The identified single hit (positive) B cell clone was able to specifically bind to IgG1 δ-GK and IgG4 δ-GK, but not IgG1 δ-K and IgG4 δ-K. Anti-keyhole limpet hemocyanin (KLH) rabbit monoclonal antibody was used as an isotype control.

[0156] [ Figure 20 ] Figure 20 The ELISA results of the secondary screening are shown. The single hit (positive) B cell clone identified was able to specifically bind to IgG1 δ-GK and IgG4 δ-GK, but not to IgG1 δ-GK-amide and IgG4 δ-GK-amide. Anti-KLH rabbit monoclonal antibody was used as an isotype control.

[0157] [ Figure 21 ] Figure 21 ELISA results for purified monoclonal antibodies are shown. YG55 specifically binds to IgG1 δ-GK and IgG4 δ-GK, but not IgG1 δ-GK-amide and IgG4 δ-GK-amide. Anti-KLH rabbit monoclonal antibody was used as an isotype control.

[0158] [ Figure 22 ] Figure 22Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / α1 gliadin-dependent Jurkat T cell activation.

[0159] [ Figure 23 ] Figure 23 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / α2 gliadin-dependent Jurkat T cell activation.

[0160] [ Figure 24 ] Figure 24 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / ω1 gliadin-dependent Jurkat T cell activation.

[0161] [ Figure 25 ] Figure 25 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN 0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / ω2 gliadin-dependent Jurkat T cell activation.

[0162] [ Figure 26 ] Figure 26 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / γ1 gliadin-dependent Jurkat T cell activation.

[0163] [ Figure 27 ] Figure 27 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / γ2 gliadin-dependent Jurkat T cell activation.

[0164] [ Figure 28 ] Figure 28 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / BC hordein-dependent Jurkat T cell activation.

[0165] [ Figure 29 ] Figure 29 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / α1b gliadin-dependent Jurkat T cell activation.

[0166] [ Figure 30 ] Figure 30 Shown are the inhibitory effects of DQN0344xx, DQN0385ee, DQN0429cc, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc on DQ2.5 / γ4a gliadin-dependent Jurkat T cell activation.

[0167] [ Figure 31 ] Figure 31 Shown is the inhibitory effect of DQN0344xx on DQ2.5 / gluten peptide-dependent Jurkat T cell activation.

[0168] [ Figure 32 ] Figure 32 Shown is the inhibitory effect of DQN0385ee on DQ2.5 / gluten peptide-dependent Jurkat T cell activation.

[0169] [ Figure 33 ] Figure 33 Shown is the inhibitory effect of DQN0429cc on DQ2.5 / gluten peptide-dependent Jurkat T cell activation.

[0170] [ Figure 34 ] Figure 34 Shown are the inhibitory effects of DQN0344xx / / DQN0385ee on DQ2.5 / gluten peptide-dependent Jurkat T cell activation.

[0171] [ Figure 35 ] Figure 35Shown are the inhibitory effects of DQN0344xx / / DQN0429cc on DQ2.5 / gluten peptide-dependent Jurkat T cell activation. DETAILED DESCRIPTION

[0172] The techniques and procedures described or referenced herein are commonly understood and routinely used by those skilled in the art using conventional methods, such as the widely utilized methodologies described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, editor, 1998) Academic Press; Animal Cell Culture (RI Freshney), editor, 1987); Introduction to Cell and Tissue Culture (JPMatherand PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths and DGNewell, editor, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (JMMiller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J.E. Colligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0173] I. Definition

[0174] For the purposes of this article, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0175] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0176] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess the alteration(s).

[0177] The term "anti-HLA-DQ2.5 antibody" refers to an antibody that binds to HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and gluten peptides with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting HLA-DQ2.5. In one embodiment, the extent of binding of the anti-HLA-DQ2.5 antibody to an unrelated antigen is less than about 10% of the binding of the antibody to HLA-DQ2.5 or an HLA-DQ2.5 / gluten peptide complex, as measured by, for example, radioimmunoassay (RIA). In certain embodiments, an antibody having "binding activity" for HLA-DQ2.5 or an HLA-DQ2.5 / gluten peptide complex has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0178] As used herein, the term "antigen binding molecules" refers to any molecule comprising an antigen binding site or any molecule having binding activity to an antigen, and can also refer to molecules such as peptides or proteins with a length of about five amino acids or more. Peptides and proteins are not limited to those from organisms, for example, they can be polypeptides produced by artificially designed sequences. They can also be any of naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc. A scaffold molecule comprising a known stable conformational structure such as an α / β barrel as a scaffold and wherein a part of the molecule is made into an antigen binding site is also an embodiment of the antigen binding molecules described herein. In some embodiments, "antigen binding molecules" are antibodies. The terms "antigen binding molecules" or "antibodies" herein are used in the broadest sense, and encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, as long as they exhibit desired antigen binding activity. In some embodiments, antibodies are multispecific antibodies. In some embodiments, multispecific antibodies are bispecific antibodies.

[0179] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0180] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0181] "Autoimmune disease" refers to a non-malignant disease or condition caused by and directed against an individual's own tissues. Autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, and especially exclude B-cell lymphomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to, celiac disease, inflammatory responses, such as inflammatory skin diseases (including psoriasis and dermatitis (e.g., atopic dermatitis)); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma, and other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalitis Myelitis; Sjögren's syndrome; juvenile diabetes mellitus; and immune reactions associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocytic diapedesis; inflammatory diseases of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemias (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune polyendocrine disease polyendocrinopathies); Reiter's disease; stiff-person syndrome; oculo-oral-genital triad syndrome; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0182] The term "celiac disease" refers to a hereditary autoimmune disease caused by ingestion of gluten in food, which damages the small intestine. Symptoms of celiac disease include, but are not limited to, gastrointestinal disturbances such as abdominal pain, diarrhea, and gastroesophageal reflux, vitamin deficiencies, mineral deficiencies, central nervous system (CNS) symptoms such as fatigue and anxiety and depression, bone symptoms such as osteomalacia and osteoporosis, skin symptoms such as skin inflammation, blood symptoms such as anemia and lymphopenia, and other symptoms such as infertility, hypogonadism, and developmental failure and short stature in children.

[0183] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0184] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0185] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0186] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) in the Fc region may be present or absent. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0187] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0188] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies that have a structure substantially similar to a native antibody structure or have heavy chains that contain an Fc region as defined herein.

[0189] As used herein, the term "gluten" refers collectively to a composite of storage proteins called prolamins found in wheat and other related cereals. In the intestinal lumen, gluten is degraded into so-called gluten peptides. Gluten peptides include, but are not limited to, gliadin from wheat, hordeins from barley, secalins from rye, and avenins from oats.

[0190] In celiac disease, gluten peptides are antigenic peptides recognized by disease-causing T cells. Meanwhile, immunodominance is a phenomenon in which an immune response is primarily triggered by a relatively small amount of antigenic peptides. Such antigenic peptides may be referred to as "immunodominant peptides." In celiac disease, such immunodominant peptides include, for example, α1 gliadin and α2 gliadin (both included in the sequence of 33-mer gliadin) and ω1 gliadin, ω2 gliadin, and BC hordein (a total of five peptides) (Science Translational Medicine, July 21, 2010: Vol. 2, No. 41, pp. 41-51). Alternatively, immunodominant peptides include, but are not limited to, α1 gliadin, α2 gliadin, ω1 gliadin, ω2 gliadin, BC hordein, γ1 gliadin, and γ2 gliadin (a total of seven peptides). In this article, such immunodominant peptides may be referred to as "immunodominant peptides associated with celiac disease." The type and total number of peptides are not particularly limited as long as they are significantly associated with celiac disease.

[0191] As used herein, the phrase "substantially no binding activity" refers to the activity of an antibody that binds to an uninterested antigen at a binding level that includes nonspecific or background binding but does not include specific binding. In other words, this antibody "does not have specific / significant binding activity" to the uninterested antigen. Specificity can be measured by any method mentioned in this specification or known in the art. The level of nonspecific or background binding can be zero, or can be close to zero instead of zero, or can be very low enough to be technically ignored by those skilled in the art. For example, when a technician cannot detect or observe any significant (or relatively strong) signal of binding between an antibody and an uninterested antigen in a suitable binding assay, it can be said that the antibody "substantially has no binding activity" or "does not have specific / significant binding activity" to the uninterested antigen. Alternatively, "substantially no binding activity" or "no specific / significant binding activity" can be rephrased as (to the uninterested antigen) "nonspecifically / significantly / substantially binding." Sometimes, the phrase "no binding activity" has substantially the same meaning as the phrase "substantially no binding activity" or "no specific / significant binding activity" in the art.

[0192] As used herein, "HLA-DR / DP" refers to "HLA-DR and HLA-DP" or "HLA-DR or HLA-DP." These HLAs are MHC class II molecules encoded by corresponding haplotype alleles at the human MHC class II locus. "HLA-DQ" is a general term for HLA-DQ isoforms, including HLA-DQ2.5, HLA-DQ2.2, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, and HLA-DQ8. In the present invention, in addition to HLA-DQ2.5, HLA-DQ2.2 and HLA-DQ7.5, HLA-DQ molecules include but are not limited to HLA-DQ molecules of known subtypes (isoforms), such as HLA-DQ2.3, HLA-DQ4.3, HLA-DQ4.4, HLA-DQ5.1, HLA-DQ5.2, HLA-DQ5.3, HLA-DQ5.4, HLA-DQ6.1, HLA-DQ6.2, HLA-DQ6.3, HLA-DQ6.4, HLA-DQ6.9, HLA-DQ7.2, HLA-DQ7.3, HLA-DQ7.4, HLA-DQ7.5, HLA-DQ7.6, HLA-DQ8, HLA-DQ9.2 and HLA-DQ9.3. Similarly, "HLA-DR(DP)" refers to the HLA-DR(DP) isoform.

[0193] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0194] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0195] A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subset of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subset is subgroup κI as described above in Kabat et al. In one embodiment, for VH, the subset is subgroup III as described above in Kabat et al.

[0196] "Humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically, two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

[0197] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain whose sequence is highly variable ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0198] (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0199] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0200] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0201] (d) A combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0202] In one embodiment, the HVR residues include those identified in the specification.

[0203] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0204] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules.

[0205] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0206] In the present invention, when evaluating the binding of anti-HLA-DQ2.5 antibodies to these HLA-DQ molecules, the CLIP peptide (e.g., SEQ ID NO: 45) can be used with appropriate HLA-DQ molecules such as HLA-DQ2.5, HLA-DQ2.2, and HLA-DQ7.5. Meanwhile, for HLA-DQ5.1, the DBY peptide (e.g., SEQ ID NO: 44) can be used for this purpose. This peptide is part of the DBY protein, which is an HLA-DQ5-restricted histocompatibility antigen.

[0207] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0208] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in cells that normally contain the nucleic acid molecule, but that is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0209] "Isolated nucleic acid encoding an anti-HLA-DQ2.5 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0210] The term "monoclonal antibody" as used herein refers to the antibody obtained from a substantially homologous antibody population, that is, the individual antibodies constituting the population are identical and / or in conjunction with the same epi-position, except possible variant antibodies, for example, variant antibodies containing naturally occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants typically exist in small quantities. In contrast to polyclonal antibody preparations (typically including different antibodies for different determinants (epi-topes)), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the attributive "monoclonal" indicates that the feature of an antibody is obtained from a substantially homologous antibody population, and should not be construed as requiring antibody to be prepared by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by various techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0211] A "naked antibody" is an antibody that is not conjugated to a heterologous moiety or radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0212] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, referred to as κ and λ, based on the amino acid sequence of its constant domain.

[0213] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance containing a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a base sequence, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule includes deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes nucleotide bases with derivatized sugar or phosphate backbone connections or modified chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as carriers for directly expressing antibodies of the present invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability and / or expression of the coding molecules of the RNA vector, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101823 B1).

[0214] " Percent (%) amino acid sequence identity " relative to a reference polypeptide sequence is defined as the percentage of the amino acid residue in the candidate sequence that is identical with the amino acid residue in the reference polypeptide sequence after the sequence is compared and a breach is introduced (if necessary) to achieve maximum percent sequence identity, without any conservative substitution being considered as a part for sequence identity. Comparisons for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm for achieving maximum alignment over the full length of the compared sequence.

[0215] The author of the ALIGN-2 sequence comparison computer program is Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington DC, 20559, along with user documentation, and is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a specific % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows:

[0216] Multiply 100 by the fraction X / Y

[0217] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in this program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0218] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation would be administered.

[0219] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0220] Unless otherwise indicated, the term "HLA-DQ2.5," as used herein, refers to any native HLA-DQ2.5 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed HLA-DQ2.5 as well as any form of HLA-DQ2.5 derived from processing in cells. The term also encompasses naturally occurring variants of HLA-DQ2.5, such as splice variants or allelic variants. The amino acid sequence of an exemplary HLA-DQ2.5 is publicly available in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) Accession No. 4OZG.

[0221] Herein, "TCR" refers to "T cell receptor", which is a membrane protein located on the surface of T cells (e.g., HLA-DQ2.5 restricted CD4+ T cells) and recognizes antigen fragments (e.g., gluten peptides) presented on MHC molecules including HLA-DQ2.5.

[0222] As used herein, "treatment" (and grammatical variants such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the disease.

[0223] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0224] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0225] II. Composition

[0226] In one aspect, the invention is based, in part, on the binding of anti-HLA-DQ2.5 antibodies to HLA-DQ2.5, which presents gluten peptides to T cells. In certain embodiments, antibodies that bind to HLA-DQ2.5 are provided.

[0227] A. Exemplary Anti-HLA-DQ2.5 Antigen Binding Molecules / Antibodies

[0228] In one aspect, the present invention provides isolated antigen-binding molecules or antibodies that have binding activity against HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and gluten peptides. In certain embodiments, the anti-HLA-DQ2.5 antibody ("antibody") has the following functions / characteristics.

[0229] The antibody has binding activity against HLA-DQ2.5 or the HLA-DQ2.5 / gluten peptide complex. In other words, the antibody binds to HLA-DQ2.5 or the HLA-DQ2.5 / gluten peptide complex. More preferably, the antibody has specific binding activity against HLA-DQ2.5 or the HLA-DQ2.5 / gluten peptide complex. In other words, the antibody specifically binds to HLA-DQ2.5 or the HLA-DQ2.5 / gluten peptide complex.

[0230] The antibody has substantially no binding activity against antigens of no interest, such as HLA-DQ2.2 / DQ5.1 / DQ6.3 / DQ7.3 / DQ7.5 / DQ8 / DR / DP. That is, the antibody does not substantially bind to the antigen of no interest. For example, the antibody has no specific binding activity or no significant binding activity against HLA-DR / DP. In other words, the antibody does not specifically bind to HLA-DR / DP or does not significantly bind to HLA-DR / DP. Similarly, the antibodies have substantially no binding activity to HLA-DQ molecules, such as HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. That is, the antibodies do not substantially bind to HLA-DQ molecules, such as HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. In other words, the antibodies have no specific / significant binding activity to HLA-DQ molecules, such as HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. That is, the antibodies do not specifically / significantly bind to HLA-DQ molecules, such as HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. These characteristics are preferred to prevent any substantial inhibitory effects on these non-target MHC class II molecules and to improve antibody PK in celiac disease patients (wherein the patients have HLA-DQ2.5).

[0231] *The characteristic of "substantially no binding activity" can be defined, for example, as described in the FACS results described herein. Under the measurement conditions described herein, an antibody that "has substantially no binding activity" to a specific antigen has an MFI (mean fluorescence intensity) value that is 250% or less, preferably 200% or less, and more preferably 150% or less of the MFI value of a negative control (for example, herein, "IC17" in Table 4 and "IC17 bivalent" in Table 5).

[0232] In one aspect, for bivalent antibodies, under the measurement conditions described herein, when the MFI value of IC17 is taken as 0% and the MFI value of DQN0139bb (WO2018 / 155692) is taken as 100%, the MFI value of the antibody having "substantially no binding activity" to a specific antigen is 5% or less, preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, and even more preferably 1% or less.

[0233] In one aspect, for bispecific antibodies, under the measurement conditions described herein, when the MFI value of the IC17 bivalent antibody is taken as 0% and the MFI value of DQN0139bb / / IC17 is taken as 100%, the MFI value of the antibody having "substantially no binding activity" to a specific antigen is 2% or less, more preferably 1% or less.

[0234] The antibody has binding activity against HLA-DQ2.5 complexed with a gluten peptide as described herein. Herein, the complex formed between an HLA-DQ2.5 molecule and a gluten peptide is referred to as a "complex formed by HLA-DQ2.5 and a gluten peptide," an "HLA-DQ2.5 / gluten peptide complex," or an "HLA-DQ2.5 / gluten peptide." This can be rephrased as, for example, "HLA-DQ2.5 loaded with a gluten peptide," "gluten peptide-loaded HLA-DQ2.5," "gluten peptide-bound HLA-DQ2.5," "HLA-DQ2.5 in complex with a gluten peptide," and "HLA-DQ2.5 complexed with a gluten peptide." The above language (e.g., “a complex formed by HLA-DQ2.5 and ... [peptide]”) also applies to peptides such as a 33-mer gliadin peptide, a 26-mer gliadin peptide, a 14-mer 1 peptide, an α1 gliadin peptide, an α1b gliadin peptide, an α2 gliadin peptide, an α3 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, a γ4b gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a secalin 1 peptide, a secalin 2 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, a hepatitis B virus peptide, a BC hordein peptide, a thyroid peroxidase peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, and the like.

[0235] The gluten peptide is preferably a gliadin peptide. The gliadin peptide is preferably a 33-mer gliadin peptide, a 26-mer gliadin peptide, a 14-mer peptide, an α1 gliadin peptide, an α1b gliadin peptide, an α2 gliadin peptide, an α3 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, a γ4b gliadin peptide, an ω1 gliadin peptide, or an ω2 gliadin peptide.

[0236] In another aspect, the gluten peptide is preferably selected from the group consisting of: BC hordein peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secalin 1 peptide, secalin 2 peptide.

[0237] Meanwhile, as used herein, "unrelated" peptides include those peptides that have been reported to be presentable on HLA-DQ2.5 but are unrelated to the present invention, i.e., peptides that are not the target gluten peptides described above. For example, unrelated peptides include, but are not limited to, CLIP peptides, hepatitis B virus (HBV) peptides, Salmonella peptides, thyroid peroxidase (TPO) peptides, and Mycobacterium bovis peptides.

[0238] These features are preferred to prevent any substantial inhibitory effects of HLA-DQ2.5 on these non-target MHC class II molecules and in complex with unrelated peptides, and to improve antibody PK in celiac disease patients.

[0239] *The characteristic of "binding activity" can be defined, for example, as described in the FACS results described herein. Under the measurement conditions described herein, an antibody having "binding activity" to a specific antigen has an MFI (mean fluorescence intensity) value of 300% or more, preferably 500% or more, and more preferably 1000% or more of the MFI value of the negative control (for example, herein, "IC17" in Table 4 and "IC17 bivalent" in Table 5).

[0240] On the one hand, for bivalent antibodies, under the measurement conditions described herein, when the MFI value of IC17 is regarded as 0% and the MFI value of DQN0139bb is regarded as 100%, the MFI value of the antibody having "binding activity" to a specific antigen is 7.5% or higher, preferably 10% or higher, and more preferably 20% or higher.

[0241] On the one hand, for bispecific antibodies, under the measurement conditions described herein, when the MFI value of the IC17 bivalent antibody is taken as 0% and the MFI value of DQN0139bb / / IC17 is taken as 100%, the MFI value of the antibody having "binding activity" to a specific antigen is 3% or higher, preferably 6% or higher, preferably 10% or higher, and more preferably 20% or higher.

[0242] When specificity of binding is specifically mentioned, "binding activity" can be rephrased as "specific binding activity".

[0243] *Anti-HLA-DQ2.5 antibodies of the present invention have a 5×10 -7 M or less, preferably 4×10 -7 M or less, preferably 3×10 -7 M or less, preferably 2×10 -7 M or less, preferably 1×10 -7 M or less, preferably 9×10 -8 M or less, preferably 8×10 -8M or less, preferably 7×10 -8 M or lower, preferably 6×10 -8 M or less, preferably 5×10 -8 M or less, preferably 4×10 -8 M or less, preferably 3×10 - 8 M or less, preferably 2×10 -8 M or less, preferably 1×10 -8 M or less, preferably 9×10 -9 M or less, preferably 8×10 -9 M or less, preferably 7×10 -9 M or less, preferably 6×10 -9 M or less, preferably 5×10 -9 M or less, preferably 4×10 -9 M or less, preferably 3×10 -9 M or less, preferably 2×10 -9 The dissociation constant (Kd) is M or lower.

[0244] In some embodiments, the antigen binding molecules / domains of the present invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, or all of the following complexes (1) to (20):

[0245] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0246] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0247] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0248] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0249] (5) a complex formed by HLA-DQ2.5 and γ2 gliadin peptide;

[0250] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0251] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0252] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0253] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0254] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0255] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0256] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0257] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0258] (14) complex formed by HLA-DQ2.5 and avenin 3 peptide;

[0259] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0260] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0261] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0262] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0263] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0264] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0265] In some embodiments, the antigen binding molecules / domains of the invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, or all of the following complexes:

[0266] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0267] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0268] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0269] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0270] (5) a complex formed by HLA-DQ2.5 and γ2 gliadin peptide;

[0271] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0272] (7) complex formed by HLA-DQ2.5 and species 2 gliadin peptide;

[0273] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0274] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0275] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0276] In some embodiments, the antigen binding molecules / domains of the invention have binding activity against one, two, three, four, or all of the following complexes:

[0277] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0278] (5) a complex formed by HLA-DQ2.5 and γ2 gliadin peptide;

[0279] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0280] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0281] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0282] In some embodiments, the antigen binding molecules / domains of the invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, or all of the following complexes:

[0283] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0284] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0285] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0286] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0287] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0288] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0289] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0290] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0291] (14) complex formed by HLA-DQ2.5 and avenin 3 peptide;

[0292] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0293] (17) a complex formed by HLA-DQ2.5 and secalin 1 peptide; and

[0294] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide.

[0295] In some embodiments, the antigen binding molecules / domains of the invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, or all of the following complexes:

[0296] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0297] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0298] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0299] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0300] (5) a complex formed by HLA-DQ2.5 and γ2 gliadin peptide;

[0301] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0302] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0303] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0304] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0305] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0306] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0307] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0308] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0309] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0310] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0311] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0312] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0313] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0314] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0315] In some embodiments, the antigen binding molecules / domains of the invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, or all of the following complexes:

[0316] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0317] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0318] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0319] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0320] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0321] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0322] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0323] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0324] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0325] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0326] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0327] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0328] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0329] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0330] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0331] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0332] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0333] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0334] In some embodiments, the antigen binding molecules / domains of the invention have binding activity against (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all of the following complexes:

[0335] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0336] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0337] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0338] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0339] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0340] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0341] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0342] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0343] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0344] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0345] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0346] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0347] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0348] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0349] In some embodiments, the antigen binding molecules / domains of the invention have binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, or all of the following complexes:

[0350] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0351] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0352] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0353] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0354] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0355] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0356] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0357] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0358] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0359] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0360] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0361] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0362] (14) complex formed by HLA-DQ2.5 and avenin 3 peptide;

[0363] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0364] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0365] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0366] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0367] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0368] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0369] In some embodiments, the antigen binding molecules / domains of the invention have binding activity against (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all of the following complexes:

[0370] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0371] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0372] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0373] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0374] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0375] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0376] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0377] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0378] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0379] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0380] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0381] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0382] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0383] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0384] In some embodiments, the antigen binding molecules / domains of the invention have substantially no binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, or all of the following complexes (1) to (20):

[0385] (1) A complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide;

[0386] (2) a complex formed by HLA-DQ2.5 and α1 gliadin peptide;

[0387] (3) a complex formed by HLA-DQ2.5 and α2 gliadin peptide;

[0388] (4) a complex formed by HLA-DQ2.5 and γ1 gliadin peptide;

[0389] (5) a complex formed by HLA-DQ2.5 and γ2 gliadin peptide;

[0390] (6) complex formed by HLA-DQ2.5 and ω1 gliadin peptide;

[0391] (7) complex formed by HLA-DQ2.5 and ω2 gliadin peptide;

[0392] (8) complex formed by HLA-DQ2.5 and BC hordein peptide;

[0393] (9) complex formed by HLA-DQ2.5 and α3 gliadin peptide;

[0394] (10) complex formed by HLA-DQ2.5 and α1b gliadin peptide;

[0395] (11) complex formed by HLA-DQ2.5 and γ4b gliadin peptide;

[0396] (12) complex formed by HLA-DQ2.5 and avenin 1 peptide;

[0397] (13) complex formed by HLA-DQ2.5 and avenin 2 peptide;

[0398] (14) complex formed by HLA-DQ2.5 and avenin 3 peptide;

[0399] (15) complex formed by HLA-DQ2.5 and hordein 1 peptide;

[0400] (16) complex formed by HLA-DQ2.5 and hordein 2 peptide;

[0401] (17) complex formed by HLA-DQ2.5 and secalin 1 peptide;

[0402] (18) Complex formed by HLA-DQ2.5 and secalin 2 peptide;

[0403] (19) a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; and

[0404] (20) Complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0405] In some embodiments, the antigen binding molecules / domains of the invention have substantially no binding activity to (at least) one, two, three, four, five, six, or all of the following (a) to (g):

[0406] (a) Complex formed by HLA-DQ2.5 and CLIP peptide;

[0407] (b) Complex formed by HLA-DQ2.5 and hepatitis B virus (HBV) peptide;

[0408] (c) Complex formed by HLA-DQ2.5 and Salmonella peptide;

[0409] (d) complex formed by HLA-DQ2.5 and thyroid peroxidase (TPO) peptide;

[0410] (e) complex formed by HLA-DQ2.5 and Mycobacterium bovis peptide;

[0411] (f) HLA-DQ2.5 positive PBMC B cells; and

[0412] (g) Ba / F3 cells expressing HLA-DQ2.5.

[0413] In some embodiments, the antigen-binding molecules / domains of the present invention have stronger binding activity to (at least) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, or all of the complexes of (1) to (20) above compared to (at least) one, two, three, four, five, six, or all of the complexes of (a) to (g) above.

[0414] The antibodies have neutralizing activity against the binding of a complex formed by HLA-DQ2.5 and a gluten peptide to the TCR. In other words, the antibodies block the binding of the HLA-DQ2.5 / gluten peptide complex to the TCR. This binding occurs in the presence of a gluten peptide, i.e., when HLA-DQ2.5 is bound by a gluten peptide or forms a complex with a gluten peptide. The gluten peptide is preferably any of the gliadin peptides described herein. The antibodies block the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells.

[0415] More preferably, the antibody blocks at least one, two, three, four, five, six, seven or all of the following: the interaction between the HLA-DQ2.5 / 33-mer gliadin peptide complex and the HLA-DQ2.5 / 33-mer gliadin peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / 26-mer gliadin peptide complex and the HLA-DQ2.5 / 26-mer gliadin peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / 14-mer gliadin peptide complex and the HLA-DQ2.5 / 16-mer gliadin peptide-restricted CD4+ T cells, The interaction between the HLA-DQ2.5 / α1 gliadin peptide complex and HLA-DQ2.5 / α1 gliadin peptide restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / α1b gliadin peptide complex and HLA-DQ2.5 / α1b gliadin peptide restricted CD4+ T cells, and the interaction between the HLA-DQ2.5 / α2 gliadin peptide complex and HLA-DQ2.5 / α1b gliadin peptide restricted CD4+ T cells. The interaction between the compound and HLA-DQ2.5 / α2 gliadin peptide restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / α3 gliadin peptide complex and HLA-DQ2.5 / α3 gliadin peptide restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / γ1 gliadin peptide complex and HLA-DQ2.5 / γ1 gliadin peptide restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / γ2 gliadin peptide complex and HLA -DQ2.5 / γ2 gliadin peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / γ4b gliadin peptide complex and HLA-DQ2.5 / γ4b gliadin peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / ω1 gliadin peptide complex and HLA-DQ2.5 / ω1 gliadin peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / ω2 gliadin peptide complex and HLA-DQ 2.5 / ω2 hordein peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / BC hordein peptide complex and HLA-DQ2.5 / BC hordein peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / avenin 1 peptide complex and HLA-DQ2.5 / avenin 1 peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / avenin 2 peptide complex and HLA-DQ2.5 / avenin 2 peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / avenin 3 peptide complex and HLA-DQ2.5 / avenin 3 peptide-restricted CD4+ T cells, and the interaction between the HLA-DQ2.5 / hordein 1 peptide complex and HLA-DQ2.5 / hordein 1 peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / hordein 2 peptide complex and HLA-DQ2.5 / hordein 2 peptide-restricted CD4+ T cells, the interaction between the HLA-DQ2.5 / secalin 1 peptide complex and HLA-DQ2.5 / secalin 1 peptide-restricted CD4+ T cells, and the interaction between the HLA-DQ2.5 / secalin 2 peptide complex and HLA-DQ2.5 / secalin 2 peptide-restricted CD4+ T cells.

[0416] Blocking of the interaction can be achieved by blocking the above-mentioned binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR.

[0417] *The characteristic of "neutralizing activity" can be defined, for example, as described herein. Under the measurement conditions described herein, an antibody having "neutralizing activity" can neutralize the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR by 95% or more, preferably 97% or more, and more preferably 99% or more at an antibody concentration of 1 microgram (μg) / mL.

[0418] The antibodies of the present invention may have substantially no binding activity (do not substantially bind) to one or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. In other words, the antibodies may have no specific / significant binding activity (do not specifically / significantly bind) to one or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. The phrase "substantially no binding activity" and similar expressions are defined elsewhere herein.

[0419] Preferably, the anti-HLA-DQ2.5 antibody (antigen-binding molecule) of the present invention has specific binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide, but has substantially no binding activity to HLA-DQ2.5 in the form of a complex with an irrelevant peptide or HLA-DQ2.5 in the form of a complex not with a peptide.

[0420] In some embodiments, the antigen binding molecules of the present invention have binding activity to at least one, two, three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA- a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0421] In some embodiments, the antigen-binding molecules of the present invention have binding activity to at least one, two, three, four, five, six, seven, eight or all of the following: a complex peptide formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen-binding molecules bind to HLA-DQ2.5-positive PBMCs. One or both of the B cells and the HLA-DQ2.5-expressing Ba / F3 cells have substantially no binding activity.

[0422] In some embodiments, the antigen binding molecules of the present invention have binding activity to at least one, two, three, four, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide, wherein the antigen binding molecules have substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5.

[0423] In some embodiments, the antigen binding molecules of the present invention have binding activity to at least one, two, three, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide, wherein the antigen binding molecules have substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5.

[0424] In some embodiments, the antigen-binding molecules of the present invention have binding activity to at least three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2 .5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and HLA-DQ2.5-expressing Ba / F3 cells.

[0425] In some embodiments, the antigen binding molecules of the present invention have binding activity to at least three, four, five, six, seven, eight or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen binding molecules are active against HLA-DQ2.5-positive PBMCs. One or both of the B cells and the HLA-DQ2.5-expressing Ba / F3 cells have substantially no binding activity.

[0426] In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DQ2.5-positive PBMC B cells. In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DQ2.5-expressing Ba / F3 cells. In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. Here, the absence of binding to HLA-DQ2.5-positive PBMC B cells and / or Ba / F3 cells expressing HLA-DQ2.5 means that the antigen-binding molecule has substantially no binding activity to HLA-DQ2.5 in a form that is not complexed with a gluten peptide or in a form complexed with an unrelated peptide.

[0427] In some embodiments, the antigen binding molecules of the invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide, wherein the antigen binding molecules have substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0428] In some embodiments, the antigen binding molecules of the invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide, wherein the antigen binding molecules have substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0429] In some embodiments, the antigen binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; and a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide, wherein the antigen binding molecules have substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0430] In some embodiments, the antigen binding molecules of the present invention have binding activity to a complex formed by HLA-DQ2.5 and an immunodominant peptide associated with celiac disease.

[0431] In some embodiments, the antigen binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and an immunodominant peptide associated with celiac disease; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; and a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide.

[0432] In some embodiments, the antigen binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an BC hordein peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 ... complex formed by HLA-DQ2.5 and α1b gliadin peptide; complex formed by HLA-DQ2.5 and γ4b gliadin peptide; complex formed by HLA-DQ2.5 and avenin 1 peptide; complex formed by HLA-DQ2.5 and avenin 2 peptide; complex formed by HLA-DQ2.5 and hordein 1 peptide; complex formed by HLA-DQ2.5 and hordein 2 peptide; complex formed by HLA-DQ2.5 and secalin 1 peptide; complex formed by HLA-DQ2.5 and secalin 2 peptide; complex formed by HLA-DQ2.5 and 14-mer 1 peptide; and complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

[0433] In some embodiments, the antigen binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide, wherein the antigen binding molecules have substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; HLA-DQ2.5-positive PBMC B cells; and Ba / F3 cells expressing HLA-DQ2.5.

[0434] In some embodiments, the antigen binding molecules of the present invention block the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. In this case, the gluten peptide is a peptide in the complex that binds to any of the above-mentioned antigen binding molecules.

[0435] In some embodiments, the gluten peptide is the following:

[0436] [1] One, two, three, four, five, six, seven, eight or all of α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, BC hordein peptide, α1b gliadin peptide and γ4a gliadin peptide;

[0437] [2] α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide and α1b gliadin peptide;

[0438] [3] α2-gliadin peptide, ω2-gliadin peptide, γ1-gliadin peptide, γ2-gliadin peptide, BC-gliadin peptide, α1b-gliadin peptide, and γ4a-gliadin peptide;

[0439] [4] α2 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide and BC hordein peptide;

[0440] [5] α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC gliadin peptide, α1b gliadin peptide, γ4a gliadin peptide and γ2 gliadin peptide;

[0441] [5a] α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC gliadin peptide, α1b gliadin peptide, and γ4a gliadin peptide;

[0442] [6] α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, BC hordein peptide and α1b gliadin peptide.

[0443] In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DQ8. In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DQ2.2, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, or HLA-DQ7.3. In some embodiments, the antigen-binding molecules of the present invention have substantially no binding activity to HLA-DR or HLA-DP.

[0444] In some embodiments, the antigen-binding molecules of the present invention have enhanced binding activity against a complex formed by HLA-DQ2.5 and a gluten peptide. In this case, the gluten peptide can be any of the gluten peptides described above. The degree of enhancement can be determined by comparing the binding activity against a complex formed by HLA-DQ2.5 and an unrelated peptide, or against cells lacking the target complex (e.g., HLA-DQ2.5-positive PBMC B cells and / or Ba / F3 cells expressing HLA-DQ2.5).

[0445] In some embodiments, the antigen binding molecules of the present invention have stronger binding activity to at least one, two, three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCB cells compared to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a Hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCB cells. a complex formed by a hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide.

[0446] In some embodiments, the antigen binding molecules of the invention have stronger binding activity for at least one, two, three, four, five, six, seven, eight, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCB cells compared to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a Hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCB cells. Complex formed by HLA-DQ2.5 and BC hordein peptide; complex formed by HLA-DQ2.5 and γ1 gliadin peptide; complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; complex formed by HLA-DQ2.5 and 14-mer 1 peptide; complex formed by HLA-DQ2.5 and 33-mer gliadin peptide; complex formed by HLA-DQ2.5 and ω2 gliadin peptide; complex formed by HLA-DQ2.5 and α1 gliadin peptide; complex formed by HLA-DQ2.5 and α2 gliadin peptide; and complex formed by HLA-DQ2.5 and ω1 gliadin peptide.

[0447] In some embodiments, the antigen binding molecules of the present invention have binding activity to at least one, two, three, four, five, six, seven, eight, nine, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA- A complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity against at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells, wherein the antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. In this case, the gluten peptide is a peptide in the complex bound to any of the above-mentioned antigen-binding molecules.

[0448] In some embodiments, the antigen-binding molecules of the present invention have binding activity to at least one, two, three, four, five, six, seven, eight, or all of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide. The present invention also provides a method for treating a gluten-restricted cell carcinoma (PCC) in which the antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. The method further provides a method for treating a gluten-restricted cell carcinoma (PCC) in which the antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. In this case, the gluten peptide is a peptide in the complex bound to any of the above-mentioned antigen-binding molecules.

[0449] In one aspect, the present invention provides an anti-HLA-DQ2.5 antibody comprising at least one, two, three, four, five or six HVRs (CDRs) selected from the following: (a) HVR-H1 (HCDR1) comprising an amino acid sequence of any one of SEQ ID NOs: 2, 6, 10 and 14; (b) HVR-H2 (HCDR2) comprising an amino acid sequence of any one of SEQ ID NOs: 3, 7, 11 and 15; (c) HVR-H3 (HCDR3) comprising an amino acid sequence of any one of SEQ ID NOs: 4, 8, 12 and 16; (d) HVR-L1 (LCDR1) comprising an amino acid sequence of any one of SEQ ID NOs: 18, 22, 26 and 30; (e) HVR-L2 (LCDR2) comprising an amino acid sequence of any one of SEQ ID NOs: 19, 23, 27 and 31; and (f) HVR-L3 (LCDR3) comprising an amino acid sequence of SEQ ID NOs: Any one of amino acid sequences of NO: 20, 24, 28 and 32.

[0450] In one aspect, the present invention provides an antibody comprising at least one, two, or all three of a VH HVR (HCDR) sequence selected from (a) HVR-H1 (HCDR) comprising any one of SEQ ID NOs: 2, 6, 10, and 14; (b) HVR-H2 (HCDR2) comprising any one of SEQ ID NOs: 3, 7, 11, and 15; and (c) HVR-H3 (HCDR3) comprising any one of SEQ ID NOs: 4, 8, 12, and 16.

[0451] In another aspect, the present invention provides an antibody comprising at least one, two, or all three of a VL HVR (LCDR) sequence selected from (a) HVR-L1 (LCDR1) comprising any one of the amino acid sequences of SEQ ID NOs: 18, 22, 26, and 30; (b) HVR-L2 (LCDR2) comprising any one of the amino acid sequences of SEQ ID NOs: 19, 23, 27, and 31; and (c) HVR-L3 (LCDR3) comprising any one of the amino acid sequences of SEQ ID NOs: 20, 24, 28, and 32.

[0452] In another aspect, an antibody of the present invention comprises (a) a VH domain and (b) a VL domain; wherein the VH domain comprises at least one or two or all three of a VH HVR (HCDR) sequence selected from (i) HVR-H1 (HCDR1) comprising any one of SEQ ID NOs: 2, 6, 10 and 14, (ii) HVR-H2 (HCDR2) comprising any one of SEQ ID NOs: 3, 7, 11 and 15, and (iii) HVR-H3 (HCDR3) comprising any one of SEQ ID NOs: 4, 8, 12 and 16; and wherein the VL domain comprises at least one or two or all three of a VL HVR (LCDR) sequence selected from (i) HVR-L1 (LCDR1) comprising any one of SEQ ID NOs: 3, 7, 11 and 15. NO: any one of the amino acid sequences of SEQ ID NO: 18, 22, 26 and 30, (ii) HVR-L2 (LCDR2) comprising any one of the amino acid sequences of SEQ ID NO: 19, 23, 27 and 31, and (c) HVR-L3 (LCDR3) comprising any one of the amino acid sequences of SEQ ID NO: 20, 24, 28 and 32.

[0453] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 (HCDR1) comprising any one of the amino acid sequences of SEQ ID NOs: 2, 6, 10 and 14; (b) HVR-H2 (HCDR2) comprising any one of the amino acid sequences of SEQ ID NOs: 3, 7, 11 and 15; (c) HVR-H3 (HCDR3) comprising any one of the amino acid sequences of SEQ ID NOs: 4, 8, 12 and 16; (d) HVR-L1 (LCDR1) comprising any one of the amino acid sequences of SEQ ID NOs: 18, 22, 26 and 30; (e) HVR-L2 (LCDR2) comprising any one of the amino acid sequences of SEQ ID NOs: 19, 23, 27 and 31; and (f) HVR-L3 (LCDR3) comprising any one of the amino acid sequences selected from SEQ ID NOs: 20, 24, 28 and 32.

[0454] In another aspect, the sequence ID numbers (SEQ ID NOs) of the VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the antibodies of the invention are as follows:

[0455] [Table 1]

[0456]

[0457] In certain embodiments, the antigen-binding molecule of the present invention is any one of the following (1) to (5):

[0458] (1) An antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0459] (2) An antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0460] (3) An antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0461] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of any one of (1) to (3);

[0462] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0463] In some embodiments, the antigen binding molecules of the invention are bispecific antigen binding molecules.

[0464] In some embodiments, the bispecific antigen binding molecules of the invention are bispecific antibodies.

[0465] In certain embodiments, any one or more amino acids of an anti-HLA-DQ2.5 antibody as provided above are substituted at any HVR position.

[0466] In certain embodiments, the substitutions are conservative substitutions, as provided herein.

[0467] In any of the above embodiments, the anti-HLA-DQ2.5 antibody is humanized. In one embodiment, the anti-HLA-DQ2.5 antibody comprises the HVRs as described in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-HLA-DQ2.5 antibody comprises the HVRs as described in any of the above embodiments and further comprises a FR1, FR2, FR3, or FR4 sequence as described herein.

[0468] In another aspect, an anti-HLA-DQ2.5 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences of SEQ ID NOs: 1, 5, 9, and 13. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-HLA-DQ2.5 antibody comprising such a sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in any of SEQ ID NOs: 1, 5, 9, and 13. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-HLA-DQ2.5 antibody comprises the following sequence: a VH sequence of any one of SEQ ID NOs: 1, 5, 9, and 13, or a sequence comprising a post-translational modification thereof. In a specific embodiment, the VH comprises one, two, or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 6, 10, and 14; (b) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 7, 11, and 15; and (c) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 8, 12, and 16. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0469] In another aspect, anti-HLA-DQ2.5 antibodies are provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 17, 21, 25, and 29. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HLA-DQ2.5 antibody comprising that sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in any one of SEQ ID NOs: 17, 21, 25, and 29. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., within the FR). Optionally, the anti-HLA-DQ2.5 antibody comprises the following sequence: a VL sequence of any one of SEQ ID NOs: 17, 21, 25, and 29, or a sequence comprising a post-translational modification thereof. In specific embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 18, 22, 26, and 30; (b) HVR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 19, 23, 27, and 31; and (c) HVR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 20, 24, 28, and 32. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0470] In another aspect, an anti-HLA-DQ2.5 antibody is provided, wherein the antibody comprises a VH as described in any of the embodiments provided above, and a VL as described in any of the embodiments provided above. In one embodiment, the antibody comprises the following sequence: a VH sequence of any one of SEQ ID NOs: 1, 5, 9, and 13, or a sequence comprising a post-translational modification thereof, and a VL sequence of any one of SEQ ID NOs: 17, 21, 25, and 29, or a sequence comprising a post-translational modification thereof. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0471] In other aspects, the present invention provides an antibody that binds to the same epitope as the anti-HLA-DQ2.5 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as any of the antibodies described herein. In certain embodiments, antibodies are provided that bind to an epitope within a fragment of HLA-DQ2.5 consisting of approximately 8-17 amino acids, or within a complex formed by HLA-DQ2.5 and a gluten peptide. In this case, the gluten peptide can be any of the gluten peptides described herein.

[0472] In another aspect, the present invention provides antibodies that compete with another antibody for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide. For example, in certain embodiments, antibodies are provided that compete with any of the antibodies described herein for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide. In this case, the gluten peptide can be any of the gluten peptides described herein.

[0473] In further aspects of the invention, the anti-HLA-DQ2.5 antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-HLA-DQ2.5 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0474] In further aspects, the anti-HLA-DQ2.5 antibody according to any of the above embodiments may incorporate any of the features described below, alone or in combination:

[0475] 1. Antibody affinity

[0476] In certain embodiments, the antibodies provided herein have an MFR of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or smaller, such as 10 - 8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0477] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed using a Fab form of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by titrating the antibody with a minimal concentration of ( 125 I) Fab was equilibrated with labeled antigen, and the bound antigen was then captured using an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23° C.) for two to five hours. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 The antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate has dried, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and the plate was placed on the TOPCOUNT TM The counts were performed on a gamma counter (Packard) for tens of minutes. The concentration of each Fab that resulted in less than or equal to 20% of maximal binding was selected for competitive binding assays.

[0478] According to another embodiment, Kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, at 25 ° C, an immobilized antigen CM5 chip is used to perform a determination using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIAcore, Inc., Piscataway, NJ) with ~ 10 response units (RU). In one embodiment, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIACORE) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / minute to obtain about 10 response units (RU) of coupled protein. After the antigen is injected, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into a TWEEN-2000 PBS solution with 0.05% polysorbate 20 at 25°C at a flow rate of approximately 25 μl / min. TM ) surfactant in PBS (PBST). The association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2). on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) was calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the above surface plasmon resonance assay exceeds 10 6 M -1 s -1 , the binding rate can be determined by fluorescence quenching techniques, such as in a spectrometer such as a spectrophotometer equipped with a flow block (Aviv Instruments) or an 8000-series SLM-AMINCO with a stirred cuvette. TM The increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS at 25°C, pH 7.2 was measured in a spectrophotometer (ThermoSpectronic) in the presence of increasing concentrations of antigen (excitation = 295 nM; emission = 340 nM, 16 nM bandpass).

[0479] 2. Antibody fragments

[0480] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that comprise salvage receptor binding epitope residues and have increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0481] Diabodies are antibody fragments with two antigen-binding sites. Diabodies can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Hudson et al., Nat. Med. 9:129-134 (2003) also describes triabodies and tetrabodies.

[0482] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

[0483] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0484] 3. Chimeric and humanized antibodies

[0485] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the type or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0486] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR (e.g., CDR) (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies optionally also include at least part of the human constant region. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0487] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, FrontFront.Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall′Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing “directed selection” methods for FR shuffling).

[0488] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al. J. Immunol., 151: 262). 3 (1993)); human mature (somatic) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0489] 4. Human Antibodies

[0490] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0491] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or they are present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE TMTechnology; U.S. Patent No. 5,770,429, which describes HUMAB (registered trademark) technology; U.S. Patent No. 7,041,870, which describes KM MOUSE (registered trademark) technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes VELOCIMOUSE (registered trademark) technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0492] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the preparation of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies prepared via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the preparation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Current Immunology, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0493] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. This variable domain sequence can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

[0494] 5. Antibodies from the library

[0495] The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired one or more activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, eds., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0496] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self antigens as well as self antigens without the need for any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and accomplishing rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0497] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0498] a) Glycosylation variants

[0499] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites in antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0500] When an antibody comprises an Fc region, the carbohydrate attached thereto can be changed. Natural antibodies produced by mammalian cells typically comprise branched, bipartite oligosaccharides, which are generally attached to the Asn297 of the CH2 domain of the Fc region via N-links. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the bipartite oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0501] In one embodiment, an antibody variant is provided that has a carbohydrate structure lacking fucose attached to the Fc region (directly or indirectly). For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complexes, heteroplexes, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 can also be located about + / - 3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300. Such fucosylated variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of disclosures concerning "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. BioL. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0502] Further provided are antibody variants having bisected oligosaccharides, for example, wherein the bisected oligosaccharides attached to the antibody Fc region are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0503] b) Fc region variants

[0504] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0505] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0506] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0507] Fc region

[0508] As used herein, the term "Fc region" or "Fc domain" is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0509] Fc receptors

[0510] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of these receptors and alternatively spliced forms of the receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For example, FcRs are reviewed in Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab Clin. Med. 126:330-41 (1995). Additional FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0511] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0512] In vivo human FcRn binding and the plasma half-life of human FcRn high-affinity binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0513] Fcγ receptors

[0514] Fcγ receptor refers to a receptor capable of binding to the Fc domain of a monoclonal IgG1, IgG2, IgG3 or IgG4 antibody, and includes all members belonging to the protein family substantially encoded by the Fcγ receptor gene. In humans, the family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all unidentified human Fcγ receptors, Fcγ receptor isoforms, and allotypes thereof. However, Fcγ receptors are not limited to these examples. Without limitation, Fcγ receptors include those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors can be from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified mouse Fcγ receptors, Fcγ receptor isoforms, and allotypes thereof. Such preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16). The polynucleotide sequence and amino acid sequence of FcγRI are shown in SEQ ID NOs: 80 (NM_000566.3) and 74 (NP_000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIA are shown in SEQ ID NOs: 81 (BC020823.1) and 75 (AAH20823.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIB are shown in SEQ ID NOs: 82 (BC146678.1) and 76 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIA are shown in SEQ ID NOs: 83 (BC033678.1) and 77 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIB are shown in SEQ ID NOs: 84 (BC128562.1) and 78 (AAI28563.1), respectively (RefSeq accession numbers are shown in each parentheses).In addition to the FACS and ELISA formats described above, whether Fcγ receptors have binding activity for the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be assessed by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), the surface plasmon resonance (SPR)-based BIACORE method, and others (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0515] At the same time, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to the Fc domain of an antibody to form an Fc / Fc ligand complex. The molecule can be from any organism. The binding of an Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fcγ receptors, Fcα receptors, Fcβ receptors, FcRn, C1q and C3, mannan binding lectin, mannose receptor, Staphylococcal protein A, Staphylococcal protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to Fcγ receptors. Fc ligands also include unidentified molecules that bind to Fc.

[0516] Fcγ receptor binding activity

[0517] Impaired binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and / or FcγRIIIB can be assessed by using the above-mentioned FACS and ELISA formats as well as ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the surface plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0518] ALPHA screening is performed using two types of beads (beads) based on the principle described below using ALPHA technology: donor beads and acceptor beads. Luminescent signals are detected only when the molecules connected to the donor beads interact biologically with the molecules connected to the acceptor beads and when the two beads are very close. Under the excitation of the laser beam, the photosensitizer in the donor beads converts the oxygen around the beads into excited singlet oxygen. When singlet oxygen diffuses around the donor beads and reaches the acceptor beads located nearby, it induces a chemiluminescent reaction in the acceptor beads. This reaction ultimately leads to light emission. If the molecule connected to the donor beads does not interact with the molecule connected to the acceptor beads, the singlet oxygen generated by the donor beads will not reach the acceptor beads and no chemiluminescent reaction will occur.

[0519] In some embodiments, the present invention relates to the antigen binding molecules of Fcγ receptors.For example, the antigen binding molecules or antibodies of biotin labeling are fixed on donor beads, and the Fcγ receptors of glutathione S-transferase (GST) labeling are fixed on acceptor beads. In the absence of the antigen binding molecules or antibodies comprising competitive mutant Fc domains, Fcγ receptors interact with the antigen binding molecules or antibodies comprising wild-type Fc domains, and the result induces a signal of 520 to 620nm. The antigen binding molecules or antibodies with unlabeled mutant Fc domains compete with the antigen binding molecules or antibodies comprising wild-type Fc domains for the interaction with Fcγ receptors. Relative binding affinity can be determined by the fluorescence reduction caused by quantitative competition. It is known that the method for biotinylating antigen binding molecules or antibodies (such as antibodies) using sulfo-NHS-biotin etc. is known. The suitable method for GST tag to be added to Fcγ receptors includes the following method: the polypeptide encoding Fcγ receptors is fused with GST frame, the cell expression fusion gene of the vector carrying the gene is used to introduce, and then glutathione column is used for purification. The induced signal can preferably be analyzed, for example, by fitting to a one-site competition model based on nonlinear regression analysis using software such as GRAPHPADPRISM (GraphPad; San Diego).

[0520] One of the substances that observe their interaction is fixed on the gold thin layer of sensor chip as a ligand. When light irradiates the back side of the sensor chip, so that total reflection occurs at the interface between the gold thin layer and the glass, the intensity of the reflected light can be partially weakened (SPR signal) at a certain site. Another substance used to observe their interaction is injected into the surface of the sensor chip as an analyte. When the analyte is bound to the ligand, the mass of the fixed ligand molecule increases. This will change the refractive index of the solvent on the sensor chip surface. The change in refractive index causes the position shift of the SPR signal (on the contrary, dissociation moves the signal back to the original position). In the Biacore system, the above-mentioned displacement (i.e., the mass change of the sensor chip surface) is plotted on the vertical axis, so that the change in mass over time is displayed as measurement data (sensor graph). Kinetic parameters (binding rate constant (ka) and dissociation rate constant (kd)) are determined by the curve of the sensor graph, and affinity (KD) is determined by the ratio between these two constants. In the BIACORE method, inhibition assay is preferably used. An example of such an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0521] Fc region with reduced Fcγ receptor binding activity

[0522] As used herein, “reduced Fcγ receptor binding activity” means, for example, that the competitive activity of the test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less or 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less, compared to the competitive activity of a control antigen-binding molecule or antibody based on the above-mentioned analysis method.

[0523] In the present invention, SG181 can be used as an Fc receptor-silencing Fc, which reduces Fc binding to Fcγ receptors. In some embodiments, SG181.S3n (SEQ ID NO: 33) and SG181.S3p (SEQ ID NO: 34) can be used as heavy chain constant region sequences. These heavy chain constant region sequences can be included in the antigen-binding molecules or antibodies of the present invention to reduce Fcγ receptor binding.

[0524] Antigen-binding molecules or antibodies comprising the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be appropriately used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in SEQ ID NO: 62 (A added to the N-terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 63 (A added to the N-terminus of RefSeq Accession No. AAB59393.1), SEQ ID NO: 64 (A added to the N-terminus of RefSeq Accession No. CAA27268.1), and SEQ ID NO: 65 (A added to the N-terminus of RefSeq Accession No. AAB59394.1). In addition, when an antigen-binding molecule or antibody comprising an Fc domain mutant of an antibody of a specific isotype is used as a test substance, the effect of the mutant mutation on Fcγ receptor binding activity is assessed using an antigen-binding molecule or antibody comprising an Fc domain of the same isotype as a control. As described above, antigen-binding molecules or antibodies comprising Fc domain mutants whose Fc receptor-binding activity has been determined to be reduced are suitably prepared.

[0525] Such known mutants include, for example, mutants having deletions of amino acids 231A-238S (EU numbering) (WO2009 / 011941), as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V and L235A (Blood (2007) 109, 1185-1192).

[0526] In some embodiments, the present invention relates to an antigen-binding molecule or an antibody comprising an Fc domain having at least one amino acid whose amino acid is selected from the group consisting of amino acids at the following amino acid positions (e.g., substitutions) in the amino acids forming the Fc domains of a specific isotype antibody: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The isotype of the antibody from which the Fc domain originates is not particularly limited, and suitable Fc domains derived from monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be used. Preferably, an Fc domain derived from an IgG1 antibody is used.

[0527] Preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain having any of the substitutions shown below in the amino acids forming the Fc domain of an IgG1 antibody, the positions of the substitutions being designated according to EU numbering (each number represents the position of an amino acid residue in EU; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution):

[0528] (a) L234F, L235E, P331S;

[0529] (b) C226S, C229S, P238S;

[0530] (c) C226S, C229S; or

[0531] (d)C226S, C229S, E233P, L234V, L235A;

[0532] and those having an Fc domain having a deletion of the amino acid sequence at positions 231 to 238.

[0533] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any of the following substitutions in the amino acids forming the Fc domain of an IgG2 antibody, where the positions of the substitutions are designated according to EU numbering:

[0534] (e) H268Q, V309L, A330S and P331S;

[0535] (f) V234A;

[0536] (g) G237A;

[0537] (h) V234A and G237A;

[0538] (i) A235E and G237A; or

[0539] (j) V234A, A235E and G237A. Each number represents the position of the amino acid residue in EU numbering; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0540] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any of the following substitutions in the amino acids forming the Fc domain of an IgG3 antibody, where the positions of the substitutions are designated according to EU numbering:

[0541] (k)F241A;

[0542] (l) D265A; or

[0543] (m) V264A. Each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0544] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any of the following substitutions in the amino acids forming the Fc domain of an IgG4 antibody, where the positions of the substitutions are designated according to EU numbering:

[0545] (n) L235A, G237A, and E318A;

[0546] (o) L235E; or

[0547] (p) F234A and L235A. Each number represents the position of the amino acid residue in EU numbering; the single-letter amino acid symbol before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0548] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which any of the amino acids at positions 233, 234, 235, 236, 237, 327, 330 or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is substituted with the amino acid at the corresponding position in EU numbering in the corresponding IgG2 or IgG4.

[0549] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain, in which any one or more amino acids at positions 234, 235, and 297 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody are substituted with other amino acids. The type of amino acid after substitution is not particularly limited; however, particularly preferred are antigen-binding molecules or antibodies comprising an Fc domain in which any one or more amino acids at positions 234, 235, and 297 are substituted with alanine.

[0550] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which the amino acid at position 265 (EU numbering) among the amino acids forming the Fc domain of an IgG1 antibody is substituted with another amino acid. The type of amino acid after the substitution is not particularly limited; however, particularly preferred are antigen-binding molecules or antibodies comprising an Fc domain in which the amino acid at position 265 is substituted with alanine.

[0551] c) Cysteine-engineered antibody variants

[0552] In certain embodiments, it may be desirable to prepare cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced by cysteine residues. In specific embodiments, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine engineered antibodies can be generated as described in, for example, U.S. Patent No. 7,521,541.

[0553] d) Antibody derivatives

[0554] In certain embodiments, provided herein is an antibody that can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable parts for antibody derivatization include but are not limited to water-soluble polymers. The limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in production due to its stability in water. Polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if attached more than one polymer, they can be identical or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0555] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in proximity to the antibody-non-protein moiety.

[0556] B. Recombinant Methods and Compositions

[0557] Antibodies can be prepared using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-HLA-DQ2.5 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method for preparing an anti-HLA-DQ2.5 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0558] For recombinant production of anti-HLA-DQ2.5 antibodies, nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0559] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be prepared in bacteria, particularly when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also, Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli) after expression, antibodies can be separated from the bacterial cell paste as soluble fractions and can be further purified.

[0560] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0561] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0562] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0563] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40 (COS-7)-transformed monkey kidney CV1 cell line; human embryonic kidney cell line (293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); TRI cells, as described, for example, in Mather et al., Annals of 44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0564] C. Determination

[0565] The anti-HLA-DQ2.5 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0566] Binding and other assays

[0567] In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, and the like.

[0568] In another aspect, competition assays can be used to identify antibodies that compete with, for example, any of the above-described antibodies for binding to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex). In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the above-described antibodies. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0569] In an exemplary competition assay, immobilized HLA-DQ2.5 (or an HLA-DQ2.5 / gluten peptide complex) is incubated in a solution containing a first labeled antibody that binds to HLA-DQ2.5 (or an HLA-DQ2.5 / gluten peptide complex) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to HLA-DQ2.5 (or an HLA-DQ2.5 / gluten peptide complex). The second antibody can be present in hybridoma supernatant. As a control, immobilized HLA-DQ2.5 (or an HLA-DQ2.5 / gluten peptide complex) is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the primary antibody to HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex), excess unbound antibody is removed, and the amount of label associated with the immobilized HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex) is measured. If the amount of label associated with the immobilized HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex) is significantly reduced in the test sample relative to the control sample, this indicates that the secondary antibody is competing with the primary antibody for binding to HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex). See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0570] Animals, such as rabbits, mice, rats and other animals suitable for immunity, are immunized with antigens (e.g., HLA-DQ2.5 or HLA-DQ2.5 / gluten peptide complexes). Any method can be used to prepare the antigen as a recombinant protein, for example, as mentioned herein. Antibody-containing samples, such as blood and spleen, are collected from the immunized animals. For B cell selection, for example, biotinylated antigens are prepared, and antigen-bound B cells are bound by the biotinylated antigen, and the cells are subjected to cell sorting and culture for selection. The specific binding of cells to antigens can be evaluated by any suitable method such as the ELISA method. This method can also be used to assess the lack of cross-reactivity to antigens of no interest. In order to isolate or determine the sequence of the selected antibody, for example, RNA is purified from the cells, and DNA encoding the antibody region is prepared by reverse transcription and PCR amplification of the RNA. In addition, the cloned antibody gene can be expressed in suitable cells, and the antibody can be purified from the culture supernatant for further analysis.

[0571] To test whether an anti-HLA-DQ2.5 antibody binds to an antigen of interest (e.g., a complex formed by HLA-DQ2.5 and a gluten peptide (e.g., those described herein)), any method for assessing binding can be used. For example, when using a FACS-based cell sorting method, cells expressing the antigen are incubated with the tested antibody, and then a suitable secondary antibody directed against the tested antibody (i.e., primary antibody) is added and incubated. Binding between the antigen and the tested antibody is detected by FACS analysis using, for example, a chromogenic / fluorescent label (e.g., as described herein) attached to the secondary antibody. Alternatively, any of the measurement methods mentioned in "1. Antibody Affinity" of this specification can be used. For example, measurement of Kd by BIACORE surface plasmon resonance assay can be used to assess binding between the tested antibody and the antigen of interest described herein.

[0572] In certain embodiments, the methods of the present invention further comprise: testing whether the antibody has neutralizing activity against binding between anti-HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR (or interaction between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and HLA-DQ2.5-restricted CD4+ T cells); and selecting antibodies with neutralizing activity. These steps can be performed in the presence of gluten peptides such as those described herein, i.e., using peptide-bound HLA-DQ2.5. Neutralizing activity can be assessed, for example, as described herein. Briefly, beads, such as yellow beads coated with streptavidin, are suitably prepared, and soluble HLA-DQ bound to the peptide is added to the beads for immobilization on a plate. The plate is washed and blocked, and the antibody is added thereto and incubated. When evaluating the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR, for example, D2 TCR tetramer-PE can be added and incubated. Based on the chromogenic / fluorescent labeling of the TCR bound by HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex), the binding between the two can be evaluated.

[0573] Multispecific antigen-binding molecules / antibodies

[0574] In the context of the present invention, the term "multispecific antibody (antigen binding molecule)" refers to an antibody that can specifically bind to different types of epitopes. More specifically, a multispecific antibody is an antibody that is specific for at least two different types of epitopes, and in addition to antibodies that recognize different antigens, also includes antibodies that recognize different epitopes on the same antigen. For example, when an antigen is a heterologous receptor, a multispecific antibody can recognize the different domains that constitute the heterologous receptor; or, when an antigen is a monomer, a multispecific antibody recognizes multiple sites on a monomeric antigen. Typically, such molecules bind to two antigens or epitopes ("bispecific antibodies (bispecific antibodies)"; used in this specification with the same meaning as "bispecific antibodies (dual-specific antibodies)"), but they may even have specificity (for example, three or more types of antigens) for more antigens or epitopes. In this article, terms such as "bispecific" and "multispecific" refer to the specificity of an antigen binding domain / region being different from the specificity of another antigen binding domain / region. That is, the term refers to the presence of two or more specificities in an antigen binding molecule. For example, in a "bispecific" antibody, the first antigen-binding domain can bind to a first group of complexes formed by HLA-DQ2.5 and gluten peptides, and the second antigen-binding domain can bind to a second group of complexes formed by HLA-DQ2.5 and gluten peptides. The members of the two groups (i.e., complexes) may overlap but may not be identical. That is, some complexes may be included in both groups. Terms such as "bispecific" and "multispecific" can cover this situation. The same applies to the first and second groups of complexes that are not bound by the first / second antigen-binding domains.

[0575] A multispecific antibody may comprise at least two antigen-binding domains. A bispecific antibody may comprise a first antigen-binding domain and a second antigen-binding domain. In the present invention, preferably, the bispecific antibody comprises a first antigen-binding domain that binds to one or more complexes formed by HLA-DQ2.5 and a gluten peptide and a second antigen-binding domain that binds to one or more complexes formed by HLA-DQ2.5 and a gluten peptide. In this respect, preferably, at least one gluten peptide in the complex bound by the first antigen-binding domain is different from at least one gluten peptide in the complex bound by the second antigen-binding domain. In other words, the members of the gluten peptide in the complex bound by the first antigen-binding domain and the members of the gluten peptide in the complex bound by the second antigen-binding domain may overlap but are not identical. The gluten peptide in the complex bound by the first / second antigen-binding domain may be selected from any gluten peptide described herein. Preferably, the first / second antigen-binding domain is capable of binding to one type of gluten peptide, or two or more types of gluten peptides.

[0576] In some embodiments, the antigen binding molecules of the present invention comprise at least two antigen binding domains, wherein any one of the antigen binding domains has binding activity to one or more complexes formed between HLA-DQ2.5 and an immunodominant peptide associated with celiac disease, wherein any one of the antigen binding domains has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell. In some embodiments, the antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0577] In some embodiments, the antigen-binding molecules of the present invention comprise at least two antigen-binding domains, wherein any one of the antigen-binding domains has binding activity against all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide , wherein any one of the antigen-binding domains has substantially no binding activity against at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell. In some embodiments, the antigen-binding molecule is a bispecific or multispecific antigen-binding molecule.

[0578] In some embodiments, the antigen-binding molecules of the present invention comprise at least two antigen-binding domains, wherein any one of the antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide. In some embodiments, the antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0579] In some embodiments, the antigen-binding molecules of the present invention comprise at least two antigen-binding domains, wherein each of the above antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and an α1 gliadin peptide. In some embodiments, the antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0580] In some embodiments, the antigen-binding molecules of the present invention comprise at least two antigen-binding domains, wherein each of the above antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide. complexes formed with BC hordein peptides; complexes formed with HLA-DQ2.5 and γ1 gliadin peptides; complexes formed with HLA-DQ2.5 and γ2 gliadin peptides; complexes formed with HLA-DQ2.5 and 26-mer gliadin peptides; complexes formed with HLA-DQ2.5 and 14-mer 1 complexes formed with peptides; complexes formed with HLA-DQ2.5 and α3 gliadin peptide; complexes formed with HLA-DQ2.5 and avenin 1 peptide; complexes formed with HLA-DQ2.5 and avenin 2 peptide; complexes formed with HLA-DQ2.5 and avenin 3 peptide; complexes formed with HLA-DQ2.5 and hordein 1 peptide; complexes formed with HLA-DQ2.5 and hordein 2 peptide; and complexes formed with HLA-DQ2.5 and γ4b gliadin peptide In some embodiments, the antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0581] In some embodiments, the antigen-binding molecules of the present invention comprise at least two antigen-binding domains, wherein each of the above antigen-binding domains has binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; complex formed by HLA-DQ2.5 and ω2 gliadin peptide; complex formed by HLA-DQ2.5 and secalin 1 peptide; complex formed by HLA-DQ2.5 and secalin 2 peptide; complex formed by HLA-DQ2.5 and BC hordein peptide; complex formed by HLA-DQ2.5 and γ1 gliadin peptide; complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; complex formed by HLA-DQ2.5 and 14-mer 1 peptide; complex formed by HLA- complex formed by HLA-DQ2.5 and α3 gliadin peptide; complex formed by HLA-DQ2.5 and avenin 1 peptide; complex formed by HLA-DQ2.5 and avenin 2 peptide; complex formed by HLA-DQ2.5 and avenin 3 peptide; complex formed by HLA-DQ2.5 and hordein 1 peptide; complex formed by HLA-DQ2.5 and hordein 2 peptide; and complex formed by HLA-DQ2.5 and γ4b gliadin peptide, any of which The antigen binding domain has no binding activity against at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell. In some embodiments, the antigen binding molecule is a bispecific or multispecific antigen binding molecule.

[0582] In some embodiments, the antigen-binding molecules of the present invention comprise a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain has binding activity to one or more complexes formed by HLA-DQ2.5 and gluten peptides, wherein the second antigen-binding domain has binding activity to one or more complexes formed by HLA-DQ2.5 and gluten peptides, wherein at least one gluten peptide in the complex bound by the first antigen-binding domain is different from at least one gluten peptide bound by the second antigen-binding domain.

[0583] In some embodiments, the antigen-binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide.

[0584] In some embodiments, the antigen-binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide.

[0585] In some embodiments, the antigen-binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; and a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide. The antigen-binding molecule comprises a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell.

[0586] In some embodiments, the antigen-binding molecules of the present invention have binding activity to all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; and a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide. complex, wherein the antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell.

[0587] In some embodiments, the antigen-binding molecules of the present invention comprise a first antigen-binding domain that has binding activity to a complex formed by HLA-DQ2.5 and a first gluten peptide and a second antigen-binding domain that has binding activity to a complex formed by HLA-DQ2.5 and a second gluten peptide, wherein the antigen-binding molecule has binding activity to at least two or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2b gliadin peptide; a complex formed by HLA-DQ2.5 and an α3b gliadin peptide. Complex formed by HLA-DQ2.5 and α2 gliadin peptide; complex formed by HLA-DQ2.5 and ω1 gliadin peptide; complex formed by HLA-DQ2.5 and ω2 gliadin peptide; complex formed by HLA-DQ2.5 and secalin 1 peptide; complex formed by HLA-DQ2.5 and secalin 2 peptide; complex formed by HLA-DQ2.5 and BC hordein peptide; complex formed by HLA-DQ2.5 and γ1 gliadin peptide; complex formed by HLA-DQ2.5 and γ2 gliadin peptide complexes formed by HLA-DQ2.5 and a 26-mer gliadin peptide; complexes formed by HLA-DQ2.5 and a 14-mer 1 peptide; complexes formed by HLA-DQ2.5 and an α3 gliadin peptide; complexes formed by HLA-DQ2.5 and an avenin 1 peptide; complexes formed by HLA-DQ2.5 and an avenin 2 peptide; complexes formed by HLA-DQ2.5 and an avenin 3 peptide; complexes formed by HLA-DQ2.5 and a hordein 1 peptide; and complexes formed by HLA-DQ2.5 and a γ4 b. A complex formed with a gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell.

[0588] In some embodiments, the antigen-binding molecule of the present invention comprises a first antigen-binding domain that has binding activity to a complex formed by HLA-DQ2.5 and a first gluten peptide and a second antigen-binding domain that has binding activity to a complex formed by HLA-DQ2.5 and a second gluten peptide, wherein the antigen-binding molecule has binding activity to at least two or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 Complex formed with α2 gliadin peptide; complex formed with ω1 gliadin peptide; complex formed with ω2 gliadin peptide; complex formed with HLA-DQ2.5 and secalin 1 peptide; complex formed with HLA-DQ2.5 and secalin 2 peptide; complex formed with HLA-DQ2.5 and BC hordein peptide; complex formed with HLA-DQ2.5 and γ1 gliadin peptide; complex formed with HLA-DQ2.5 and 26-mer gliadin peptide complexes of HLA-DQ2.5 and 14mer 1 peptide; complexes of HLA-DQ2.5 and α3 gliadin peptide; complexes of HLA-DQ2.5 and avenin 1 peptide; complexes of HLA-DQ2.5 and avenin 2 peptide; complexes of HLA-DQ2.5 and avenin 3 peptide; complexes of HLA-DQ2.5 and hordein 1 peptide; complexes of HLA-DQ2.5 and hordein 2 peptide; and complexes of HLA-DQ2.5 and γ4b gliadin peptide. The antigen-binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells.

[0589] In some embodiments, the antigen-binding molecules of the present invention comprise a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide, wherein the second antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide complexes formed; complexes formed by HLA-DQ2.5 and 14-mer 1 peptide; complexes formed by HLA-DQ2.5 and 33-mer gliadin peptide; complexes formed by HLA-DQ2.5 and α3 gliadin peptide; complexes formed by HLA-DQ2,5 and avenin 1 peptide; complexes formed by HLA-DQ2.5 and avenin 2 peptide; complexes formed by HLA-DQ2.5 and avenin 3 peptide; complexes formed by HLA-DQ2.5 and hordein 1 peptide; complexes formed by HLA-DQ2.5 and hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide, wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell. In some embodiments, the complex bound by the first antigen binding domain and the complex bound by the second antigen binding domain are different from each other.

[0590] In some embodiments, the antigen-binding molecule of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide. a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide, wherein the second antigen-binding domain has binding activity to at least one or more of the following: a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide; a complex formed by HLA-D complex formed by HLA-DQ2.5 and a 14-mer 1 peptide; complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; complex formed by HLA-DQ2.5 and α3 gliadin peptide; complex formed by HLA-DQ2.5 and avenin 1 peptide; complex formed by HLA-DQ2.5 and avenin 2 peptide; complex formed by HLA-DQ2.5 and avenin 3 peptide; complex formed by HLA-DQ2.5 and hordein 1 peptide; complex formed by HLA-DQ2.5 and hordein 2 peptide; and complex formed by HLA-D Q2.5 and γ4b gliadin peptide, wherein the antigen binding molecule has substantially no binding activity to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and CLIP peptide; a complex formed by HLA-DQ2.5 and Salmonella peptide; a complex formed by HLA-DQ2.5 and Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and thyroid peroxidase peptide; and an HLA-DQ2.5-positive PBMC B cell. In some embodiments, the complex bound by the first antigen binding domain and the complex bound by the second antigen binding domain are different from each other.

[0591] In some embodiments, the antigen binding molecules of the present invention block the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide restricted CD4+ T cells. In this case, the gluten peptide is a peptide in the complex bound by any of the above-mentioned antigen binding molecules / domains.

[0592] In some embodiments, the antigen binding molecules of the present invention have substantially no binding activity to HLA-DQ2.2, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, or HLA-DQ7.3. In some embodiments, the antigen binding molecules of the present invention have substantially no binding activity to HLA-DR or HLA-DP.

[0593] In some embodiments, the antigen binding molecules of the present invention have substantially no binding activity to HLA-DQ8.

[0594] In some embodiments, the antigen-binding molecules of the present invention have enhanced binding activity against a complex formed by HLA-DQ2.5 and a gluten peptide. In this case, the gluten peptide can be any of the gluten peptides described above. The degree of enhancement can be determined by comparing the binding activity against a complex formed by HLA-DQ2.5 and an unrelated peptide, or against cells lacking the target complex (e.g., HLA-DQ2.5-positive PBMC B cells and / or Ba / F3 cells expressing HLA-DQ2.5).

[0595] In some embodiments, the antigen binding molecules of the present invention have stronger binding activity for at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells compared to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a Hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMC B cells. complex formed by HLA-DQ2.5 and α1b gliadin peptide; complex formed by HLA-DQ2.5 and α2 gliadin peptide; complex formed by HLA-DQ2.5 and ω1 gliadin peptide; complex formed by HLA-DQ2.5 and ω2 gliadin peptide; complex formed by HLA-DQ2.5 and secalin 1 peptide; complex formed by HLA-DQ2.5 and secalin 2 peptide; complex formed by HLA-DQ2.5 and BC hordein peptide; complex formed by HLA-DQ2.5 and γ1 gliadin peptide; complex formed by HLA-DQ2.5 and γ complex formed by HLA-DQ2.5 and a hordein 1 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; a complex formed by HLA-DQ2.5 and a hordein 2 peptide; and a complex formed by HLA-DQ2.5 and a γ4b gliadin peptide.

[0596] In some embodiments, the PLGA2 antibody is compared to at least one, two, three, four, five, or all of the following: a complex formed by HLA-DQ2.5 and a CLIP peptide; a complex formed by HLA-DQ2.5 and a Salmonella peptide; a complex formed by HLA-DQ2.5 and a Mycobacterium bovis peptide; a complex formed by HLA-DQ2.5 and a hepatitis B virus peptide; a complex formed by HLA-DQ2.5 and a thyroid peroxidase peptide; and HLA-DQ2.5-positive PBMCs. B cells, the antigen-binding molecules of the present invention have stronger binding activity to at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, or all of the following: a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a secalin 1 peptide; a complex formed by HLA-DQ2.5 and a secalin 2 peptide; - Complex formed by HLA-DQ2.5 and BC hordein peptide; complex formed by HLA-DQ2.5 and γ1 gliadin peptide; complex formed by HLA-DQ2.5 and 26-mer gliadin peptide; complex formed by HLA-DQ2.5 and 14-mer 1 peptide; complex formed by HLA-DQ2.5 and α3 gliadin peptide; complex formed by HLA-DQ2.5 and avenin 1 peptide; complex formed by HLA-DQ2.5 and avenin 2 peptide; complex formed by HLA-DQ2.5 and avenin 3 peptide; complex formed by HLA-DQ2.5 and hordein 1 peptide; complex formed by HLA-DQ2.5 and hordein 2 peptide; and complex formed by HLA-DQ2.5 and γ4b gliadin peptide.

[0597] The bispecific antibodies of the present invention comprise the heavy and light chains of a first half antibody and the heavy and light chains of a second half antibody. In some embodiments, the bispecific antibodies comprise the VH and VL of the first half antibody and the VH and VL of the second half antibody. In some embodiments, the bispecific antibodies comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first half antibody and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second half antibody.

[0598] In some embodiments, the first half antibody is derived from DQN0344xx. In some embodiments, the second half antibody is derived from DQN0385ee or DQN0429cc. The sequences of the VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the (half) antibodies are described elsewhere herein, for example, in Table 1.

[0599] In some embodiments, the antigen-binding molecule of the present invention is any one of the following (1) to (5):

[0600] (1) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0601] (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0602] (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0603] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of any one of (1) to (3);

[0604] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0605] In some embodiments, the antigen binding molecules of the invention are bispecific antigen binding molecules.

[0606] In some embodiments, the bispecific antigen binding molecule is a bispecific antibody.

[0607] In some embodiments, the antigen binding molecule of the present invention is any one of the following (a) to (d):

[0608] (a) an antigen-binding molecule comprising the following (i) and (iii),

[0609] (b) an antigen-binding molecule comprising the following (ii) and (iii),

[0610] (c) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of (a) or (b),

[0611] (d) an antigen-binding molecule that competes with the antigen-binding molecule of (a) or (b) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide,

[0612] (i) a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0613] (ii) a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0614] (iii) HCDR1 sequence of SEQ ID NO: 10, HCDR2 sequence of SEQ ID NO: 11, HCDR3 sequence of SEQ ID NO: 12, LCDR1 sequence of SEQ ID NO: 26, LCDR2 sequence of SEQ ID NO: 27, and LCDR3 sequence of SEQ ID NO: 28.

[0615] In some embodiments, the present invention provides nucleic acids encoding the antigen binding molecules of the present invention.

[0616] In some embodiments, the present invention provides a vector into which the above-mentioned nucleic acid is introduced.

[0617] In some embodiments, the present invention provides a cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0618] In some embodiments, the present invention provides methods for producing antigen-binding molecules by culturing the above-mentioned cells.

[0619] In view of this disclosure and the technical knowledge in the art, the nucleic acids, vectors, cells and methods can be appropriately prepared / implemented.

[0620] In some embodiments, the present invention provides an antigen-binding molecule of any one of the following (1) to (5):

[0621] (1) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0622] (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0623] (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28;

[0624] (4) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of any one of (1) to (3);

[0625] (5) An antigen-binding molecule that competes with the antigen-binding molecule of any one of (1) to (3) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide.

[0626] In some embodiments, the antigen binding molecule of the present invention is any one of the following (a) to (d):

[0627] (a) an antigen-binding molecule comprising the following (i) and (iii),

[0628] (b) an antigen-binding molecule comprising the following (ii) and (iii),

[0629] (c) an antigen-binding molecule that binds to the same epitope as the antigen-binding molecule of (a) or (b),

[0630] (d) an antigen-binding molecule that competes with the antigen-binding molecule of (a) or (b) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide,

[0631] (i) a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20;

[0632] (ii) a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24;

[0633] (iii) HCDR1 sequence of SEQ ID NO: 10, HCDR2 sequence of SEQ ID NO: 11, HCDR3 sequence of SEQ ID NO: 12, LCDR1 sequence of SEQ ID NO: 26, LCDR2 sequence of SEQ ID NO: 27, and LCDR3 sequence of SEQ ID NO: 28.

[0634] Other implementation options:

[0635] 1) Immunoconjugates

[0636] The invention also provides immunoconjugates comprising an anti-HLA-DQ2.5 antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0637] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235). B1); auristatin, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines, such as daunorubicin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortaxel; trichothecenes; and CC1065.

[0638] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthus proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0639] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as Tc-99m or 123 I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0640] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl))hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0641] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linkers including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0642] 2) Pharmaceutical preparations

[0643] Pharmaceutical formulations of anti-HLA-DQ2.5 antibodies as described herein are prepared by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than (about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGPs are combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0644] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0645] The formulations herein may also contain more than one active ingredient, as necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a pharmaceutical composition that can be combined with an anti-HLA-DQ2.5 antibody. Such active ingredients are suitably combined in amounts effective for their intended purpose.

[0646] The active ingredient can be encapsulated in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, for example, prepared by coacervation techniques or by interfacial polymerization in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0647] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0648] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0649] 3) Treatment methods and compositions

[0650] Any of the anti-HLA-DQ2.5 antibodies provided herein can be used in therapeutic methods. In one aspect, an anti-HLA-DQ2.5 antibody for use as a medicament is provided. In a further aspect, an anti-HLA-DQ2.5 antibody for use in treating celiac disease is provided. In certain embodiments, an anti-HLA-DQ2.5 antibody for use in a therapeutic method is provided. In certain embodiments, the present invention provides an anti-HLA-DQ2.5 antibody for use in a method of treating an individual with celiac disease, the method comprising administering to the individual an effective amount of the anti-HLA-DQ2.5 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0651] In another aspect, the present invention provides use of an anti-HLA-DQ2.5 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat celiac disease. In another embodiment, the medicament is used in a method of treating celiac disease, the method comprising administering an effective amount of the medicament to a subject suffering from celiac disease. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0652] In another aspect, the present invention provides a method for treating celiac disease. In one embodiment, the method comprises administering to an individual suffering from celiac disease an effective amount of an anti-HLA-DQ2.5 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above embodiments can be a human.

[0653] In another aspect, the present invention provides pharmaceutical formulations comprising any of the anti-HLA-DQ2.5 antibodies provided herein, e.g., for use in any of the above-described methods for treating celiac disease. In one embodiment, the pharmaceutical formulation comprises any of the anti-HLA-DQ2.5 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-HLA-DQ2.5 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0654] The antibodies of the present invention can be used alone or in combination with other agents in treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is any agent suitable for co-administration and available to those skilled in the art.

[0655] Such combination therapies mentioned above include both combined administration (wherein the two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the anti-HLA-DQ2.5 antibody and administration of the additional therapeutic agent(s) occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0656] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is required) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulse infusions.

[0657] Two or more antibodies of the present invention (i.e., two or more therapeutic agents of the present invention) can be administered during the course of treatment. They can be administered separately or simultaneously. They can be administered concomitantly. In concomitant administration, two or more antibodies can be administered simultaneously or separately. In some cases, a certain antibody / agent can be administered first; and symptoms can be monitored; and based on the symptoms, if necessary, another antibody / agent can be further administered. Alternatively, two or more antibodies of the present invention can be included in a combination drug / agent. Such a combination drug / agent can be administered as described herein. The dose / dosage of each antibody included can be appropriately determined as described herein.

[0658] The antibodies of the present invention will be formulated, dosed, and used in a manner consistent with good medical practice. Factors considered in this regard include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of administration, the administration time arrangement, and other factors known to the physician. The antibody need not but is optionally formulated with one or more medicaments currently used to prevent or treat the condition in question. The effective amount of such other medicaments depends on the amount of antibody present in the preparation, the type of condition or treatment, and other factors discussed above. These are typically used in the same dosage and route of administration as described herein, or about 1 to 99% of the dosage described herein, or in any dosage and any route determined to be suitable empirically / clinically.

[0659] For the prevention or treatment of a disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is used for prevention or treatment purposes, previous treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient in a one-time or series of treatments. Depending on the type and severity of the disease, an antibody of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be an initial candidate dose for administration to the patient, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the patient's condition, treatment will typically continue until desired disease symptom suppression occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about twenty, or for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of such therapy is easily monitored by conventional techniques and assays.

[0660] It will be understood that any of the above-described formulations or treatment methods can be performed using the immunoconjugates of the invention in place of or in addition to the anti-HLA-DQ2.5 antibodies.

[0661] 4) Products

[0662] In another aspect of the present invention, a product containing materials that can be used to treat, prevent and / or diagnose the above-mentioned conditions is provided. The product includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition, which itself or in combination with another composition that is effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container having a composition contained therein, wherein the composition includes an antibody of the present invention; and (b) a second container having a composition contained therein, wherein the composition includes another cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0663] It will be understood that any of the above-described articles of manufacture may include an immunoconjugate of the invention instead of or in addition to an anti-HLA-DQ2.5 antibody.

[0664] 5) Methods of using antigen-binding molecules

[0665] The antigen binding molecules of the present disclosure can be combined with various previously existing technologies for medical purposes. Non-limiting examples of technologies that can be combined with the antigen binding molecules of the present disclosure include methods for integrating nucleic acids encoding antigen binding molecules into organisms and directly expressing antigen binding molecules using viral vectors, etc. Examples of such viral vectors include but are not limited to adenovirus. Alternatively, nucleic acids encoding antigen binding molecules can be directly integrated into organisms by methods such as electroporation or direct administration of nucleic acids, without the use of viral vectors. Alternatively, cells genetically modified to secrete / express antigen binding molecules can be applied to organisms, and antigen binding molecules can be continuously secreted in vivo.

[0666] Although the present invention will be described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0667] Example

[0668] The following are examples of compositions of the present invention. It will be understood that various other embodiments may be practiced, given the general description provided above.

[0669] Example 1

[0670] Recombinant protein expression and purification

[0671] 1.1. Expression and Purification of Recombinant HLA-DQ2.5 / 33-Mer Gliadin Peptide Complex, HLA-DQ8 / Gliadin Peptide Complex, HLA-DQ5.1 / DBY Peptide Complex, HLA-DQ2.2 / CLIP Peptide Complex, HLA-DQ7.5 / CLIP Peptide Complex, HLA-DQ2.5 / γ2 Gliadin Peptide Complex, and HLA-DQ2.5 / BC Hordein Peptide Complex

[0672] Expression and purification of recombinant HLA-DQ2.5 / 33-mer gliadin peptide complex:

[0673] The sequences used for expression and purification were: HLA-DQA1*0501 (Protein Database Accession No. 4OZG) and HLA-DQB1*0201 (Protein Database Accession No. 4OZG), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0501 has a C47S mutation, a GGGG linker (SEQ ID NO: 38), and a c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33), as well as a Flag-Tag at the C-terminus of HLA-DQA1*0501. HLA-DQB1*0201 has a 33-mer gliadin peptide sequence: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 39), and a factor X cleavage linker (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1;63(Pt 12):1021-1025.), a GGGGG linker (SEQ ID NO: 40) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33.), a GGGGG linker (SEQ ID NO: 40), and a BAP sequence (BMC Biotechnol. 2008;8:41) at the N-terminus of HLA-DQB1*0201. His-tag. Recombinant HLA-DQ2.5 / 33-mer gliadin peptide complex was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher). Conditioned medium expressing the HLA-DQ2.5 / 33-mer gliadin peptide complex was incubated with an immobilized metal affinity chromatography (IMAC) resin and then eluted with imidazole. Fractions containing the HLA-DQ2.5 / 33-mer gliadin peptide complex were collected and subsequently passed through a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1x PBS. Fractions containing the HLA-DQ2.5 / 33-mer gliadin peptide complex were then combined and stored at -80°C. The purified HLA-DQ2.5 / 33-mer gliadin peptide complex was biotinylated using BirA (Avidity).

[0674] Expression and Purification of Recombinant HLA-DQ8 / Gliadin Peptide Complexes:

[0675] The sequences used for expression and purification were: HLA-DQA1*0301 (Protein Database Accession No. 4GG6) and HLA-DQB1*0302 (Protein Database Accession No. 4GG6), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0301 has a SSADLVPRGGGGG linker (SEQ ID NO: 41) and a c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33), and a Flag-Tag at the C-terminus of HLA-DQA1*0301. HLA-DQB1*0302 has a gliadin peptide sequence: QQYPSGEGSFQPSQENPQ (SEQ ID NO: 42), and a factor X cleavage linker (Acta Crystallogr SectF Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt 12): 1021-1025.), a SSADLVPRGGGGG linker (SEQ ID NO: 43) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), a GGGGG linker (SEQ ID NO: 40), and a BAP sequence (BMC Biotechnol. 2008; 8: 41) at the N-terminus of HLA-DQB1*0302, and an 8x His-tag at the C-terminus of HLA-DQB1*0302. Recombinant HLA-DQ8 / gliadin peptide was transiently expressed using the FreeStyle293-F cell line. Conditioned medium expressing the HLA-DQ8 / gliadin peptide complex was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ8 / gliadin peptide complex were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ8 / gliadin peptide complex were then pooled and stored at -80°C.

[0676] Expression and purification of recombinant HLA-DQ5.1 / DBY peptide complex:

[0677] The sequences used for expression and purification were: HLA-DQA1*0101 (IMGT / HLA Accession No. HLA00601) and HLA-DQB1*0501 (IMGT / HLA Accession No. HLA00638), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0101 has the C30Y mutation. HLA-DQA1*0101 has the SSADLVPRGGGGG linker (SEQ ID NO: 41) and the c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33) and a Flag-tag at the C-terminus of HLA-DQA1*0101. HLA-DQB1*0501 has a DBY peptide sequence: ATGSNCPPHIENFSDIDMGE (SEQ ID NO: 44), and a factor X cleavage linker (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt 12): 1021-1025.), a SSADLVPRGGGGG linker (SEQ ID NO: 43) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), a GGGGG linker (SEQ ID NO: 40), and a BAP sequence (BMCBiotechnol. 2008; 8: 41) at the N-terminus of HLA-DQB1*0501, and an 8x His-tag at the C-terminus of HLA-DQB1*0501. Recombinant HLA-DQ5.1 / DBY peptide complexes were transiently expressed using the FreeStev 293-F cell line. Conditioned medium expressing the HLA-DQ5.1 / DBY peptide complexes was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ5.1 / DBY peptide complexes were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ5.1 / DBY peptide complexes were pooled and stored at -80°C. The purified HLA-DQ5.1 / DBY peptide was biotinylated using BirA.

[0678] Expression and purification of recombinant HLA-DQ2.2 / CLIP peptide complex:

[0679] The sequences used for expression and purification were: HLA-DQA1*0201 (IMGT / HLA Accession No. HLA00607) and HLA-DQB1*0202 (IMGT / HLA Accession No. HLA00623), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0201 has a SSADLVPRGGGGG linker (SEQ ID NO: 41) and a c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33) and a Flag-Tag at the C-terminus of HLA-DQA1*0201. HLA-DQB1*0202 has a CLIP peptide sequence: KLPKPPKPVSKMRMATPLLMQALPMGALP (SEQ ID NO: 45), and a factor X cleavage linker (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt12): 1021-1025.), a SSADLVPRGGGGG linker (SEQ ID NO: 43) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), a GGGGG linker (SEQ ID NO: 40), and a BAP sequence (BMCBiotechnol. 2008; 8: 41) at the N-terminus of HLA-DQB1*0202, and an 8x His tag at the C-terminus of HLA-DQB1*0202. Recombinant HLA-DQ2.2 / CLIP peptide complexes were transiently expressed using the FreeStyle293-F cell line. Conditioned medium expressing the HLA-DQ2.2 / CLIP peptide complexes was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ2.2 / CLIP peptide complexes were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ2.2 / CLIP peptide complexes were pooled and stored at -80°C.

[0680] Expression and purification of recombinant HLA-DQ7.5 / CLIP peptide complex:

[0681] The sequences used for expression and purification were: HLA-DQA1*0505 (IMGT / HLA Accession No. HLA00619) and HLA-DQB1*0301 (IMGT / HLA Accession No. HLA00625), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0505 has the C66S mutation. HLA-DQA1*0505 has the SSADLVPRGGGGG linker (SEQ ID NO: 41) and the cfos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33) and a Flag-Tag at the C-terminus of HLA-DQA1*0505. HLA-DQB1*0301 has the CLIP peptide sequence: KLPKPPKPVSKMRMATPLLMQALPMGALP (SEQ ID NO: 45), and a factor X cleavage linker (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt12): 1021-1025.), SSADLVPRGGGGG linker (SEQ ID NO: 43) and c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), GGGGG linker (SEQ ID NO: 40), and BAP sequence (BMCBiotechnol. 2008; 8: 41) at the N-terminus of HLA-DQB1*0301, and an 8x His-tag at the C-terminus of HLA-DQB1*0301. Recombinant HLA-DQ7.5 / CLIP peptide complexes were transiently expressed using the FreeStyle293-F cell line. Conditioned medium expressing the HLA-DQ7.5 / CLIP peptide complexes was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ7.5 / CLIP peptide complexes were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ7.5 / CLIP peptide complexes were pooled and stored at -80°C.

[0682] Expression and Purification of Recombinant HLA-DQ2.5 / γ2 Gliadin Peptide Complex:

[0683] The sequences used for expression and purification were: HLA-DQA1*0501 (Protein Database Accession No. 4OZG) and HLA-DQB1*0201 (Protein Database Accession No. 4OZG), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0501 has a C47S mutation, a 3C protease-cleavable linker: LEVLFQGP (SEQ ID NO: 46) and a GGGG linker (SEQ ID NO: 38), a c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33), and a Flag-tag at the C-terminus of HLA-DQA1*0501. HLA-DQB1*0201 has a γ2 gliadin peptide sequence: IIQPEQPAQLP (SEQ ID NO: 47) and a factor X cleavage linker at the N-terminus of HLA-DQB1*0201 (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt 12): 1021-1025.), a 3C protease cleavage linker: LEVLFQGP (SEQ ID NO: 46) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), a GGGGG linker (SEQ ID NO: 40) and a BAP sequence (BMCBiotechnol. 2008; 8: 41), and an 8x His-tag at the C-terminus of HLA-DQB1*0201. Recombinant HLA-DQ2.5 / γ2 gliadin peptide complexes were transiently expressed using the FreeStyle293-F cell line. Conditioned medium expressing the HLA-DQ2.5 / γ2 gliadin peptide complexes was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ2.5 / γ2 gliadin peptide complexes were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ2.5 / γ2 gliadin peptide complexes were pooled and stored at -80°C.

[0684] Expression and purification of recombinant HLA-DQ2.5 / BC hordein peptide complex:

[0685] The sequences used for expression and purification were: HLA-DQA1*0501 (Protein Database Accession No. 4OZG) and HLA-DQB1*0201 (Protein Database Accession No. 4OZG), both of which have the CAMPATH-1H signal sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 37). HLA-DQA1*0501 has a C47S mutation, a 3C protease-cleavable linker: LEVLFQGP (SEQ ID NO: 46) and a GGGG linker (SEQ ID NO: 38), and a c-fos leucine zipper sequence (PNAS, 1998 Sep 29;95(20):11828-33), and a Flag-Tag at the C-terminus of HLA-DQA1*0505. HLA-DQB1*0201 has a BC hordein peptide sequence: EPEQPIPEQPQPYPQQP (SEQ ID NO: 48) and a factor X cleavage linker at the N-terminus of HLA-DQB1*0201 (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1; 63(Pt 12): 1021-1025.), a 3C protease cleavage linker: LEVLFQGP (SEQ ID NO: 46) and a c-jun leucine zipper sequence (PNAS, 1998 Sep 29; 95(20): 11828-33), a GGGGG linker (SEQ ID NO: 40) and a BAP sequence (BMC Biotechnol. 2008; 8: 41), and an 8x His-tag at the C-terminus of HLA-DQB1*0201. Recombinant HLA-DQ2.5 / BC hordein peptide complexes were transiently expressed using the FreeStyle293-F cell line. Conditioned medium expressing the HLA-DQ2.5 / BC hordein peptide complexes was incubated with IMAC resin and then eluted with imidazole. Fractions containing the HLA-DQ2.5 / BC hordein peptide complexes were collected and subsequently passed through a Superdex 200 gel filtration column equilibrated with 1x PBS. Fractions containing the HLA-DQ2.5 / BC hordein peptide complexes were pooled and stored at -80°C.

[0686] Example 2

[0687] 2.1 Establishment of the J.RT3-T3.5 cell line expressing D2 TCR

[0688] The D2 TCR α chain cDNA (SEQ ID NO: 97) was inserted into the expression vector pCXND3 (WO2008 / 156083). The D2 TCR β chain cDNA (SEQ ID NO: 49) was inserted into the expression vector pCXZD1 (US2009 / 0324589). Linearized D2 TCR α chain (pCXND3) and D2 TCR β chain (pCXZD1) (1500 ng each) were simultaneously introduced into the J,RT3-T3,5 cell line by electroporation (LONZA, 4D-Nucleofector X). The transfected cells were then cultured in medium containing geneticin and bleomycin (Zeocin) and sorted using AriaIII (Becton Dickinson) to obtain a high-expressing cell population. Single-cell cloning was then performed to obtain cells that highly expressed the desired D2 TCR molecule.

[0689] 2.2 Establishment of Ba / F3 cell lines expressing HLA-DQ2.5, HLA-DQ2.2, HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DR, and HLA-DP

[0690] HLA-DQA1*0501cDNA (IMGT / HLA Accession No. HLA00613), HLA-DQA1*0201cDNA (IMGT / HLA Accession No. HLA00607), HLA-DQA1*0505cDNA (IMGT / HLA Accession No. HLA00619), HLA-DQA1*0301cDNA (IMGT / HLA Accession No. HLA00608), HLA-DQA1*0101cDNA (IMGT / HLA Accession No. HLA00601), HLA-DQA1*0103cDNA (IMGT / HLA Accession No. HLA00604), HLA-DQA1*0303cDNA (IMGT / HLA Accession No. HLA00611), and HLA-DQA1*0101cDNA (IMGT / HLA Accession No. HLA00613). cDNA (GenBank Accession No. NM_019111.4) or HLA DPA1*0103 cDNA (IMGT / HLA Accession No. HLA00499) was inserted into the expression vector pCXND3 (WO2008 / 156083). HLA-DQB1*0201 cDNA (IMGT / HLA Accession No. HLA00622), HLA-DQB1*0202 cDNA (IMGT / HLA Accession No. HLA00623), HLA-DQB1*0301 cDNA (IMGT / HLA Accession No. HLA00625), HLA-DQB1*0302 cDNA (IMGT / HLA Accession No. HLA00627), HLA-DQB1*0501 cDNA (IMGT / HLA Accession No. HLA00628), and HLA-DQB1*0602 cDNA (IMGT / HLA Accession No. HLA00629) were inserted into the expression vector pCXND3 (WO2008 / 156083). DNA (IMGT / HLA Accession No. HLA00638), HLA-DQB1*0603 cDNA (IMGT / HLA Accession No. HLA00647), HLA-DRB1*0301 cDNA (IMGT / HLA Accession No. HLA00671), or HLA-DPB1*0401 cDNA (IMGT / HLA Accession No. HLA00521) was inserted into the expression vector pCXZD1 (US / 20090324589).

[0691] Each linearized HLA-DQA1*0501-pCXND3 and HLADQB1*0201-pCXZD1, as well as each linearized HLA-DQA1*0201-pCXND3 and HLA-DQB1*0202-pCXZD1, HLA-DQA1*0505-pCXND3 and HLADQB1*0301-pCXZD1, HLA-DQA1*0301-pCXND3 and HLADQB1*0302-pCXZD1, HLA-DQA1*0101-pCXND3 and HLADQB HLA-DQA1*0501-pCXZD1, HLA-DQA1*0103-pCXND3, HLADQB1*0603-pCXZD1, HLA-DQA1*0303-pCXND3, HLADQB1*0301-pCXZD1, HLA-DRA1*0101-pCXND3, HLADRB1*0301-pCXZD1, HLA-DPA1*0103-pCXND3, and HLADPB1*0401-pCXZD1 were simultaneously introduced into the mouse IL-3-dependent pro-B cell line Ba / F3. The transfected cells were then cultured in a medium containing geneticin and zeocin. The cultured and expanded cells were then examined for HLA molecule expression, confirming high HLA expression. This procedure was performed to obtain cells that highly expressed the desired HLA molecules. Each established cell line was named Ba / F3-HLA-DQ2.5 (HLA-DQA1*0501, HLADQBl*0201), Ba / F3-HLA-DQ2.2 (HLA-DQA1*0201, HLA-DQB1*0202), Ba / F3-HLA-DQ7.5 (HLA-DQA1*0505, HLA-DQB1*0301), Ba / F3-HLA-DQ8 (HLA-DQA1*0301, HLADQB1*0302), and Ba / F3-HLA-DQ5.1 (HLA-DQA1*0101, HLA-DQB1*0501), Ba / F3-HLA-DQ6.3 (HLA-DQA1*0103, HLA-DQB1*0603), Ba / F3-HLA-DQ7.3 (HLA-DQA1*03 03, HLADQB1*0301), Ba / F3-HLA-DR (HLA-DRA1*0101, HLA-DRB1*0301), and Ba / F3-HLA-DP (HLA-DPA1*0103, HLA-DPB1*0401).

[0692] 2.3 Expression of HLA-DQ2.5 / CLIP peptide, HLA-DQ2.5 / hepatitis B virus peptide, HLA-DQ2.5 / Salmonella peptide, HLA-DQ2.5 / thyroid peroxidase peptide, HLA-DQ2.5 / Mycobacterium bovis peptide, HLA-DQ2.5 / α1 gliadin peptide, HLA-DQ2.5 / α2 gliadin peptide, HLA-DQ2.5 / γ1 gliadin peptide, HLA-DQ2.5 / γ2 gliadin peptide, HLA-DQ2.5 / ω1 gliadin peptide, HLA-DQ2.5 / ω2 gliadin peptide, HLA-DQ2.5 / BC hordein peptide, HLA-DQ2.5 / α3 gliadin peptide Establishment of Ba / F3 cell lines expressing HLA-DQ2.5 / α1b gliadin peptide, HLA-DQ2.5 / γ4b gliadin peptide, HLA-DQ2.5 / avenin 1 peptide, HLA-DQ2.5 / avenin 2 peptide, HLA-DQ2.5 / avenin 3 peptide, HLA-DQ2.5 / hordein 1 peptide, HLA-DQ2.5 / hordein 2 peptide, HLA-DQ2.5 / secalin 1 peptide, HLA-DQ2.5 / secalin 2 peptide, HLA-DQ2.5 / 14mer 1 peptide, HLA-DQ2.5 / 33mer gliadin peptide, and HLA-DQ2.5 / 26mer gliadin peptide

[0693] HLA-DQA1*0501 cDNA (IMGT / HLA Accession No. HLA00613) was inserted into the expression vector pCXND3 (WO2008 / 156083). HLA-DQB1*0201 cDNA (IMGT / HLA Accession No. HLA00622) was inserted into the expression vector pCXZD1 (US / 20090324589). The HLA-DQB1*0201 used in the HLA-DQ2.5 / each peptide complex had the each peptide sequence and a Factor X cleavage linker at the N-terminus of HLA-DQB1*0201: (Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Dec 1;63(Pt 12):1021-1025). Specifically, the peptide sequences are as follows: KLPKPPKPVSKMRMATPLLMQALPMGALP (SEQ ID NO: 45) for the CLIP peptide sequence, PDRVHFASPLHVAWR (SEQ ID NO: 50) for the hepatitis B virus peptide sequence, MMAWRMMRY (SEQ ID NO: 51) for the Salmonella peptide sequence, YIDVWLGGLAENFLPY (SEQ ID NO: 52) for the thyroid peroxidase peptide sequence, KPLLIIAEDVEGEY (SEQ ID NO: 53) for the Mycobacterium bovis peptide sequence, QPFPQPELPYP (SEQ ID NO: 54) for the α1 gliadin peptide sequence, FPQPELPYPQP (SEQ ID NO: 55) for the α2 gliadin peptide sequence, QPQQSFPEQQQ (SEQ ID NO: 56) for the γ1 gliadin peptide sequence, GIIQPEQPAQLP (SEQ ID NO: 57) for the α2 gliadin peptide sequence. NO:57} for the γ2 gliadin peptide sequence, QPFPQPEQPFP (SEQ ID NO:58) for the ω1 gliadin peptide sequence, FPQPEQPFPWQ (SEQ ID NO:59) for the ω2 gliadin peptide sequence, PQQPIPEQPQPYPQQP (SEQ ID NO:60) for the BC hordein peptide sequence, PFRPEQPYPQP (SEQ ID NO:61) for the α3 gliadin peptide sequence, LPYPQPELPYP (SEQ ID NO:62) for the α1b gliadin peptide sequence, FPQPEQEFPQP (SEQ ID NO:63) for the γ4b gliadin peptide sequence, QPYPEQEEPFV (SEQ ID NO:64) for the avenin 1 peptide sequence, QPYPEQEQPFV (SEQ ID NO:65) for the avenin 2 peptide sequence,QPYPEQEQPIL (SEQ ID NO: 66) for the avenin 3 peptide sequence, PQQPFPQPEQPFRQ (SEQ ID NO: 67) for the hordein 1 peptide sequence, QEFPQPEQPFPQQP (SEQ ID NO: 68) for the hordein 2 peptide sequence, PEQPFPQPEQPFPQ (SEQ ID NO: 69) for the secalin 1 peptide sequence, QPFPQPEQPFPQSQ (SEQ ID NO: 70) for the secalin 2 peptide sequence, PQQQTLQPEQPAQLP (SEQ ID NO: 71) for the 14-mer 1 peptide sequence, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 39) for the 33-mer gliadin peptide sequence, FLQPEQPFPEQPEQPYPEQPEQPFPQ (SEQ ID NO: NO: 72) for the 26-mer gliadin peptide sequence.

[0694] Each linearized HLA-DQA1*0501-pCXND3 and HLA-DQB1*0201 / each peptide-pCXZD1 were simultaneously introduced into the mouse IL-3-dependent pro-B cell-derived cell line Ba / F3 by electroporation (LONZA, 4D-Nucleofector X). The transfected cells were then cultured in a medium containing geneticin and bleomycin (Zeocin). The cultured and expanded cells were then examined for HLA-DQ2.5 molecule expression, confirming high HLA-DQ2.5 expression. Each established cell line was named: Ba / F3-HLA-DQ2.5 / CLIP (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / CLIP peptide), Ba / F3-HLA-DQ2.5 / HBV (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / hepatitis B virus peptide), Ba / F3-HLA-DQ2.5 / Salmonella (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / Salmonella). A-DQBl*0201 for HLADQ2.5 / Salmonella peptide), Ba / F3-HLA-DQ2.5 / TPO (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / thyroid peroxidase peptide), Ba / F3-HLA-DQ2.5 / Mycobacterium bovis (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / Mycobacterium bovis peptide), Ba / F3-HLA-DQ2.5 / α 1 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / α 1 gliadin peptide), Ba / F3-HLA-DQ2.5 / α 2 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / α 2 gliadin peptide), Ba / F3-HLA-DQ2.5 / γ 1 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / γ 1 gliadin peptide), Ba / F3-HLA-DQ2.5 / γ 2 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / γ 2 gliadin peptide), Ba / F3-HLA-DQ2.5 / ω1 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ 2.5 / ω1 gliadin peptide), Ba / F3-HLA-DQ2.5 / ω2 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / ω2 gliadin peptide), Ba / F3-HLA-DQ2.5 / BC hordein (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / BC hordein peptide), Ba / F3-HLA-DQ2.5 / α 3 gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / α 3 gliadin peptide), Ba / F3-HLA-DQ2.5 / α 1b gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLADQ2.5 / α 1b gliadin peptide), Ba / F3-HLA-DQ2.5 / γ 4b gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / γ4b gliadin peptide), Ba / F3-HLA-DQ2.5 / avenin 1 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / avenin 1 peptide), Ba / F3-HLA-DQ2.5 / avenin 2 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / avenin 2 peptide), Ba / F3-HLA-DQ2.5 / avenin 3 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / avenin 3 peptide), Ba / F3-HLA-DQ2.5 / hordein 1 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / hordein 1 peptide), Ba / F3-HLA-DQ2.5 / hordein 2 (HLA-DQA1*0501, HLA-DQB 1*0201 for HLA-DQ2.5 / hordein 2 peptide), Ba / F3-HLA-DQ2.5 / secalin 1 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / secalin 1 peptide), Ba / F3-HLA-DQ2.5 / secalin 2 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / secalin 2 peptide), Ba / F3-HLA-DQ2.5 / secalin 1 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / secalin 2 peptide). 4-mer 1 (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / 14-mer 1 peptide), Ba / F3-HLA-DQ2.5 / 33-mer gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / 33-mer gliadin peptide), and Ba / F3-HLA-DQ2.5 / 26-mer gliadin (HLA-DQA1*0501, HLA-DQB1*0201 for HLA-DQ2.5 / 26-mer gliadin peptide).

[0695] Example 3

[0696] Generation of anti-DQ2.5 antibodies

[0697] Anti-DQ2.5 antibodies were prepared, selected, and assayed as follows:

[0698] NZW rabbits were intradermally immunized with an HLA-DQ2.5 / 33-mer gliadin peptide complex. Four repeated doses were administered over a 2-month period, followed by blood and spleen collection. For B cell selection, biotinylated HLA-DQ5.1 / DBY peptide complex, biotinylated HLA-DQ8 / gliadin peptide complex, and Alexa Fluor 488-labeled HLA-DQ2.5 / 33-mer gliadin peptide complex were prepared. B cells capable of binding to HLA-DQ2.5 but not HLA-DQ5.1 or HLA-DQ8 were stained with the above-labeled proteins, sorted using a cell sorter, and then plated and cultured according to the procedures described in WO2016098356A1. After culture, B cell culture supernatants were collected for further analysis, and B cell pellets were cryopreserved.

[0699] Specific binding to the HLA-DQ2.5 / 33-mer gliadin peptide complex was evaluated, and non-cross-reactivity to the HLA-DQ5.1 / DBY peptide complex and the HLA-DQ8 / gliadin peptide complex was confirmed by ELISA using B cell culture supernatants. The results showed that the 336B cell line exhibited specific binding to the HLA-DQ2.5 / 33-mer gliadin peptide complex.

[0700] To evaluate cross-reactivity to HLA-DQ2.2 / CLIP peptide complexes and HLA-DQ7.5 / CLIP peptide complexes, ELISA was performed using the supernatants of the selected 336B cells. In addition, neutralization activity was examined by a neutralization assay using the supernatants of the selected 336B cells.

[0701] The neutralization assay procedure was identical to the AlphaLISA neutralization assay (HLA-DQ2.5 / 33-mer gliadin peptide-D2 TCR) described below. B cells with high neutralization activity were preferred and selected for cloning.

[0702] RNA of 180B cell lines with the desired binding specificity was purified from frozen cell pellets using the ZR-96Quick-RNA kit (ZYMO RESEARCH, Cat No. R1053). These were named DQN0377-0464. The DNA encoding the heavy chain variable region of the antibody in the selected cell line was amplified by reverse transcription PCR and recombined with the DNA encoding the F1332m heavy chain constant region (SEQ ID NO: 73) (WO2018 / 155692). The DNA encoding the light chain variable region of the antibody was also amplified by reverse transcription PCR and recombined with the DNA encoding the hk0MC light chain constant region (SEQ ID NO: 74) (WO2018 / 155692). The cloned antibodies were expressed in FreestyleTM293-F cells (Invitrogen) and purified from the culture supernatant. Through further evaluation described below, two clones (DQN0385ee, DQN0429cc) were selected based on binding ability, specificity and functionality. DQN0344xx (WO2019 / 069993) was also used. DQN0139bb was used as a control. The sequence ID numbers for the VH, VL, HCDR, and LCDR of these antibodies are listed in Table 1 above. The sequences of FR1 to FR4 and CDR1 to CDR3 of the heavy and light chains of these antibodies are shown in Table 2.

[0703] [Table 2]

[0704]

[0705] Example 4

[0706] Generation of bispecific antibodies:

[0707] Bispecific antibodies were generated that exhibit cross-reactivity with various HLA-DQ2.5 / gluten peptide complexes. To generate bispecific antibodies, six multi-gluten peptide-selective HLA-DQ2.5 bivalent antibodies (DQN0344Hx-SG181.S3n, DQN0385He-SG181.S3n, DQN0429Hc-SG181.S3n, DQN0139Hb-SG181.S3n, p, DQN0385He-SG181.S3p, and DQN0429Hc-SG181.S3p) and a negative control antibody (IC17HdK-SG181.S3p) were used. SG181 is an Fcγ receptor-silencing Fc that reduces Fc binding to Fcγ receptors. cDNA encoding the antibody, comprising the variable regions and human IgG1 constant regions, was synthesized and cloned into a standard mammalian expression vector. Each bivalent antibody was transiently transfected and expressed using the Expi293 expression system (Thermo Fisher Scientific). Culture supernatants were harvested and antibodies were purified from the supernatant using MabSelect SuRe pcc affinity chromatography (GE Healthcare) and subsequently using the gel permeation chromatography of Superdex200 (GE Healthcare). In order to produce bispecific antibodies, seven bivalent antibodies of purification were subjected to Fab arm exchange technology (as described in WO2015 / 046467). Six bispecific antibodies were then produced and named as DQN0344xx / / IC17, DQN0385ee / / IC17, DQN0429cc / / IC17, DQN0344xx / / DQN0385ee, DQN0344xx / / DQN0429cc, and DQN0139bb / / IC17. The summary and sequence of bispecific antibodies are shown in Table 3. SG181.S3n (SEQ ID NO: 33) and SG181.S3p (SEQ ID NO: 34) are heavy chain constant region sequences, and kOMC (SEQ ID NO: 35) and SK1 (SEQ ID NO: 36) are light chain constant region sequences.

[0708] A summary and sequences of the bispecific antibodies are shown in Table 3 below.

[0709] [Table 3]

[0710]

[0711] Example 5

[0712] Antibody binding analysis to HLA:

[0713] Figures 1 to 12Shown is the binding of each anti-HLA-DQ antibody to a panel of HLA-DQ expressing Ba / F3 cell lines in complex with several peptides, as determined by FACS. The anti-HLA-DQ antibodies were tested for binding to the following: Ba / F3-HLA-DQ2.5, Ba / F3-HLA-DQ2.2, Ba / F3-HLA-DQ7.5, Ba / F3-HLA-DQ8, Ba / F3-HLA-DQ5.1, Ba / F3-HLA-DQ6.3, Ba / F3-HLA-DQ7.3, Ba / F3-HLA-DR, Ba / F3-HLA-DP, Ba / F3-HLA-DQ2.5 / CLIP, Ba / F3-HLA-DQ8. LA-DQ2.5 / HBV, Ba / F3-HLA-DQ2.5 / Salmonella, Ba / F3-HLA-DQ2.5 / TPO, Ba / F3-HLA-DQ2.5 / Mycobacterium bovis, Ba / F3-HLA-DQ2.5 / α1 gliadin, Ba / F3-HLA-DQ2.5 / α2 gliadin, Ba / F3-HLA-DQ2.5 / γ1 gliadin, Ba / F3-HLA-DQ2.5 / γ2 gliadin, Ba / F3-HLA-DQ2 .5 / ω1 gliadin, Ba / F3-HLA-DQ2.5 / ω2 gliadin, Ba / F3-HLA-DQ2.5 / BC hordein, Ba / F3-HLA-DQ2.5 / α3 gliadin, Ba / F3-HLA-DQ2.5 / α1b gliadin, Ba / F3-HLA-DQ2.5 / γ4b gliadin, Ba / F3-HLA-DQ2.5 / avenin 1, Ba / F3-HLA-DQ2.5 / avenin 2, Ba / F3-HLA-DQ2.5 LA-DQ2.5 / avenin 3, Ba / F3-HLA-DQ2.5 / hordein 1, Ba / F3-HLA-DQ2.5 / hordein 2, Ba / F3-HLA-DQ2.5 / secalin 1, Ba / F3-HLA-DQ2.5 / secalin 2, Ba / F3-HLA-DQ2.5 / 14-mer 1, Ba / F3-HLA-DQ2.5 / 33-mer gliadin, Ba / F3-HLA-DQ2.5 / 26-mer gliadin. 5 μg / mL of each anti-HLA-DQ antibody was incubated with each cell line at room temperature for 30 minutes and washed with FACS buffer (2% FBS, 2 mM EDTA in PBS). Then goat F(ab')2 anti-human IgG, mouse ads-PE (SouthernBiotech, Cat. 2043-09) was added and incubated at 4 degrees Celsius for 20 minutes, followed by washing with FACS buffer.Data were acquired on an LSRFortessa X-20 (Becton Dickinson) and analyzed using FlowJo software (TreeStar) and Microsoft Office Excel 2013. The % MFI of the bispecific antibody was determined by assuming the MFI value of IC17 to be 0% and the MFI value of DQN0139bb / IC17 to be 100%. The % MFI of the bivalent antibody was determined by assuming the MFI value of IC17 to be 0% and the MFI value of DQN0139bb to be 100%.

[0714] Figure 1 and Figure 7 DQN0344xx and DQN0344xx / / IC17 showed binding activity against HLA-DQ2.5 only when the peptides formed a complex with gluten-derived peptides, specifically 33-mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, α3 gliadin peptide, α1b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, secalin 1 peptide, and secalin 2 peptide. On the other hand, DQN0344xx and DQN0344xx / / IC17 showed substantially no binding activity against HLA-DQ2.5 when the peptides formed a complex with peptides unrelated to gluten peptides.

[0715] Figure 2 and Figure 8 The results showed that DQN0385ee and DQN0385ee / / IC17 had binding activity against HLA-DQ2.5 only when HLA-DQ2.5 formed complexes with 33-mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, γ1 gliadin, γ2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, hordein 1 peptide, hordein 2 peptide, secalin 1 peptide, secalin 2 peptide, 14-mer 1 peptide, and 26-mer gliadin peptide. On the other hand, DQN0385ee and DQN0385ee / / IC17 had substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 formed a complex with a peptide unrelated to gluten peptides.

[0716] Figure 3 and Figure 9DQN0429cc and DQN0429cc / / IC17 showed binding activity against HLA-DQ2.5 only when HLA-DQ2.5 formed complexes with a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α1b gliadin peptide, a γ4b gliadin peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, a 14-mer 1 peptide, and a 26-mer gliadin peptide. On the other hand, DQN0429cc and DQN0429cc / / IC17 showed essentially no binding activity against HLA-DQ2.5 when HLA-DQ2.5 formed complexes with peptides unrelated to gluten peptides.

[0717] Figure 4 It was shown that DQN0344xx / / DQN0385ee had binding activity against HLA-DQ2.5 only when HLA-DQ2.5 formed a complex with a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalein 1 peptide, a secalein 2 peptide, a 14-mer 1 peptide, and a 26-mer hordein peptide. On the other hand, DQN0344xx / / DQN0385ee has substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 forms a complex with a peptide unrelated to gluten peptides.

[0718] Figure 5 It was shown that DQN0344xx / / DQN0429cc had binding activity against HLA-DQ2.5 only when HLA-DQ2.5 formed a complex with 33-mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, γ1 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secalin 1 peptide, secalin 2 peptide, 14-mer 1 peptide, and 26-mer gliadin peptide. On the other hand, DQN0344xx / / DQN0429cc has substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 forms a complex with a peptide unrelated to gluten peptides.

[0719] Figure 6 and Figure 10 DQN0139bb and DQN0139bb / / IC17 were shown to have binding activity against HLA-DQ2.5 in complex with or without any peptide.

[0720] Figure 11 Analysis of IC17 binding to complexes formed by HLA-DQ2.5 and gluten-derived peptides or unrelated peptides is shown. IC17 had essentially no binding activity to the tested complexes.

[0721] Figure 12 This analysis shows antibody binding to HLA molecules, such as HLA-DQ5.1, HLA-DQ6.3, HLA-DR, and HLA-DP. The four lanes, from left to right, show the results for HLA-DQ5.1, HLA-DQ6.3, HLA-DR, and HLA-DP, respectively. DQN0344xx / / IC17, DQN0385ee / / IC17, DQN0429cc / / IC17, DQN0344xx / / DQN0429cc, DQN0344xx, DQN0385ee, and DQN0429cc showed essentially no binding activity to the tested HLA molecules.

[0722] Example 6

[0723] Analysis of antibody binding to HLA-DQ2.5+ PBMC B cells:

[0724] Figure 13 and 14Binding of anti-HLA-DQ antibodies to HLA-DQ2.5-positive PBMC B cells, as determined by FACS, is shown. PBMCs were incubated with 20 μg / mL of each anti-HLA-DQ antibody at room temperature for 30 minutes in the presence of human FcR blocking reagent (Miltenyi Biotech, Cat. 130-059-901) and washed with FACS buffer (2% FBS, 2 mM EDTA in PBS). Pacific Blue™ anti-human CD19 antibody mouse IgG1k (Biolegend, Cat. 2043-09) and AlexaFluor 555-labeled anti-human IgG Fc antibody (Reference Examples 1-3) were then added, incubated at 4°C for 30 minutes, and washed with FACS buffer. Data were acquired on an LSR Fortessa X-20 (Becton Dickinson) and analyzed using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad). The % MFI of the bivalent antibody was determined when the MFI value of IC17 was taken as 0% and the MFI value of DQN0139bb / IC17 was taken as 100%. The % MFI of the bivalent antibody was determined when the MFI value of IC17 was taken as 0% and the MFI value of DQN0139bb was taken as 100%.

[0725] Figure 13 It was shown that DQN0139bb / IC17 had binding activity against HLA-DQ2.5 positive PBMC B cells, while DQN0344xx / IC17, DQN0385ee / IC17, DQN0429cc / IC17,

[0726] DQN0344xx / DQN0385ee and DQN0344xx / DQN0429cc have essentially no binding activity to cells.

[0727] Figure 14 DQN0139bb showed binding activity to HLA-DQ2.5-positive PBMC B cells, while DQN0344xx, DQN0385ee, and DQN0429cc had essentially no binding activity to the cells.

[0728] Figure 15 and Figure 16The above results are summarized as follows. DQN0139bb and DQN0139bb / / IC17 have binding activity against HLA-DQ2.5 in the form of a complex with or without any peptide, whereas DQN0344xx and DQN0344xx / / IC17 have binding activity against HLA-DQ2.5 only when HLA-DQ2.5 forms a complex with gluten-derived peptides, specifically 33-mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, α3 gliadin peptide, α1b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, secalin 1 peptide, and secalin 2 peptide. On the other hand, DQN0344xx and DQN0344xx / / IC17 had substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 formed a complex with a peptide unrelated to gluten peptides.

[0729] DQN0385ee and DQN0385ee / / IC17 have binding activity against HLA-DQ2.5 only when HLA-DQ2.5 forms a complex with a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, a 14-mer 1 peptide, and a 26-mer gliadin peptide. On the other hand, DQN0385ee and DQN0385ee / / IC17 had substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 formed a complex with a peptide unrelated to gluten peptides.

[0730] DQN0344xx / / DQN0385ee has binding activity against HLA-DQ2.5 only when HLA-DQ2.5 forms a complex with a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalein 1 peptide, a secalein 2 peptide, a 14-mer 1 peptide, and a 26-mer hordein peptide. On the other hand, DQN0344xx / / DQN0385ee has substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 forms a complex with a peptide unrelated to gluten peptides.

[0731] DQN0429cc and DQN0429cc / / IC17 showed binding activity against HLA-DQ2.5 only when HLA-DQ2.5 formed a complex with a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α1b gliadin peptide, a γ4b gliadin peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, a 14-mer 1 peptide, and a 26-mer gliadin peptide. On the other hand, DQN0429cc and DQN0429cc / / IC17 showed essentially no binding activity against HLA-DQ2.5 when HLA-DQ2.5 formed a complex with a peptide unrelated to gluten peptides.

[0732] DQN0344xx / / DQN0429cc has binding activity against HLA-DQ2.5 only when HLA-DQ2.5 forms a complex with 33-mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, γ1 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secalin 1 peptide, secalin 2 peptide, 14-mer 1 peptide, and 26-mer gliadin peptide. On the other hand, DQN0344xx / / DQN0429cc has substantially no binding activity to HLA-DQ2.5 when HLA-DQ2.5 forms a complex with a peptide unrelated to gluten peptides.

[0733] Figure 15 and 16The numerical data are shown in Table 4 and Table 5 respectively.

[0734] [Table 4]

[0735]

[0736] [Table 5]

[0737]

[0738] Example 7

[0739] Cell-based neutralization assay

[0740] Cell-based neutralization activity was confirmed. Lymphoblastoid cell lines (ECACC, IHW9088) transformed by Epstein-Barr virus (EBV) with HLA-DQ2.5 were distributed in 96-well plates (Corning, 3799). Then, a chemically synthesized 33-mer gliadin peptide (Genscript, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 39)) and serially diluted anti-HLA-DQ antibodies and J.RT3-T3.5 cells expressing D2 TCR were added and cultured overnight at 37°C, 5% CO2. The final concentration of the 33-mer gliadin peptide was 200 μg / mL, and the concentration of IHW9088 was 3.0x10 4 cells / well, 1.0x10 J.RT-T3.5 cells expressing D2 TCR 5 cells / well, and the final assay volume was 100 μl / well. After overnight culture, the cells were harvested and washed with FACS buffer (2% FBS, 2mM EDTA in PBS). 40-fold diluted APC anti-human CD20 antibody (Biolegend, 103112) and 40-fold diluted Brilliant Violet 421 anti-human CD69 antibody (Biolegend, 410930) were then added and incubated at 4°C for 30 minutes, washed and resuspended with FACS buffer. Data acquisition was performed on an LSR Fortessa (BectonDickinson), and then analyzed using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad) to determine the neutralizing activity of anti-HLA-DQ antibodies on the activation of J.RT-T3.5 cells expressing D2 TCR. CD69 expression on J.RT-T3.5 cells was used as an activation marker. As Figure 17 and 18 As shown, all tested anti-HLADQ2.5 antibodies inhibited the activation of T cells expressing the D2 TCR induced by the 33-mer gliadin peptide.

[0741] Example 8

[0742] The affinity of anti-HLA-DQ2.5 antibodies for binding to human HLA-DQ2.5 / 33-mer gliadin peptide complex, HLA-DQ2.5 / γ2 gliadin peptide complex, and HLA-DQ2.5 / BC hordein peptide complex at pH 7.4 was determined at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized onto all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). All antibodies and analytes were prepared in ACES (pH 7.4) containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. Each antibody was captured on the sensor surface via the anti-human Fc. The target antibody capture level was 200 resonance units (RU). Recombinant human HLA-DQ2.5 / 33-mer gliadin peptide complexes and HLA-DQ2.5 / γ2 gliadin peptide complexes, prepared by two-fold serial dilution, were injected at 50 to 800 nM, followed by dissociation. Recombinant human HLA-DQ2.5 / BC hordein peptide complexes, prepared by two-fold serial dilution, were injected at 25 to 400 nM, followed by dissociation. The sensor surface was regenerated with 3 M MgCl2 after each cycle. Binding affinity was determined by processing the data using Biacore T200 evaluation software (GE Healthcare) and fitting them to a 1:1 binding model.

[0743] The binding affinities of the anti-HLA-DQ2.5 antibodies to the human HLA-DQ2.5 / 33-mer gliadin peptide complex, the HLA-DQ2.5 / γ2 gliadin peptide complex, and the HLA-DQ2.5 / BC hordein peptide complex are shown in Table 6.

[0744] [Table 6]

[0745]

[0746] Example 9

[0747] 9.1 Establishment of TCR KO Jurkat NFAT-Luc cell line

[0748] A ribonucleoprotein (RNP) complex consisting of Cas9 and a single guide RNA targeting the TCR constant region (Blood. 2018; 131: 311-22.) was introduced into the NFAT-RE-luc2 Jurkat cell line (Promega corporation, CS176401) by electroporation (LONZA, Nucleofector 2b). All single guide RNAs for the TCR α chain and TCR β chain were mixed and introduced simultaneously. The RNP-introduced cells were cultured in a medium containing hygromycin B and then single-cell cloned using FACS Aria III (Becton, Dickinson and Company). The TCR α chain and TCR β chain sequences were then examined and Jurkat NFAT-Luc-derived clones in which the TCR α chain and TCR β chain were knocked out were identified. The established clone was named TCR KO Jurkat NFAT-Luc.

[0749] 9.2 Establishment of a TCR KO Jurkat NFAT-Luc Cell Line Expressing a DQ2.5 / Gluten Peptide-Restricted TCR

[0750] The amino acid sequence information of DQ2.5 / α1 gliadin-restricted TCR (TCC ID: 387.9), DQ2.5 / α1b gliadin-restricted TCR (TCC ID: 370.2.25), DQ2.5 / ω1 gliadin-restricted TCR (TCC ID: 442P.C.21), DQ2.5 / ω2 gliadin-restricted TCR (TCC ID: 578.42), DQ2.5 / γ1 gliadin-restricted TCR (TCC ID: 820.27), DQ2.5 / γ2 gliadin-restricted TCR (TCC ID: 430.1.41), and DQ2.5 / γ4a gliadin-restricted TCR (TCC ID: 430.1.36) were obtained from the University of Oslo based on a material transfer agreement. The amino acid sequence information of the DQ2.5 / α2 gliadin-restricted TCR (D2 TCR) was obtained from Nat Struct Mol Biol 2014; 21: 480-8, and the amino acid sequence information of the DQ2.5 / BC hordein-restricted TCR (TCC ID: 1468.2) was obtained from Eur J Immunol. 2020; 50: 256-269.

[0751] Each TCR β chain sequence is connected to the corresponding TCR α chain sequence by a 2A self-cleaving peptide sequence (P2A, amino acid sequence: GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 93). Except for the DQ2.5 / γ2 gliadin-restricted TCR and the DQ2.5 / α2 gliadin-restricted TCR, all TCR α chains and TCR β chains have these native signal peptide sequences. The native signal sequence of the DQ2.5 / γ2 gliadin-restricted TCR is replaced by the Campath signal sequence (MGWSCIILFLVATATGVHS, SEQ ID NO: 37). The Campath signal sequence (MGWSCIILFLVATATGVHS, SEQ ID NO: 37) is also attached to the N-terminus of the DQ2.5 / α2 gliadin-restricted TCR α chain β chain. Each codon-optimized TCR β chain-P2A-TCR α chain cDNA was inserted into the expression vector pCXZD1 (US / 20090324589). For DQ2.5 / α1 gliadin restricted TCR, DQ2.5 / α2 gliadin restricted TCR (D2 TCR), DQ2.5 / ω1 gliadin restricted TCR, DQ2.5 / ω2 gliadin restricted TCR, DQ2.5 / γ1 gliadin restricted TCR, DQ2.5 / γ2 gliadin restricted TCR, DQ2.5 / BC hordein restricted TCR, each TCR β chain-P2A-TCR α chain-pCXZD1 was introduced into TCR KO Jurkat NFAT-Luc by electroporation (LONZA, 4D-Nucleofector). The transfected cells were then cultured in medium containing Zeocin and Hygromycin B, and the TCR-positive fraction (determined by staining with anti-TCRαβ antibody, Miltenyi Biotech) was single-cell cloned using FACS Aria III (Becton, Dickinson and Company).When the DQ2.5 / α1 gliadin-restricted TCR was introduced, the established clone was named α1 gliadin TCR Jurkat NFAT-Luc, when the DQ2.5 / ω1 gliadin-restricted TCR was introduced, the established clone was named ω1 gliadin TCR Jurkat NFAT-Luc, when the DQ2.5 / ω2 gliadin-restricted TCR was introduced, the established clone was named ω2 gliadin TCR Jurkat NFAT-Luc, when the DQ2.5 / γ1 gliadin-restricted TCR was introduced, the established clone was named γ1 gliadin TCR Jurkat NFAT-Luc, when the DQ2.5 / γ2 gliadin-restricted TCR was introduced, the established clone was named γ2 gliadin TCR Jurkat NFAT-Luc, and when the DQ2.5 / α2 gliadin-restricted TCR (D2) was introduced, the established clone was named D2 TCR Jurkat NFAT-Luc, and when the DQ2.5 / BC hordein restricted TCR was introduced, the established clone was named BC hordein TCRJurkat NFAT-Luc. For the DQ2.5 / α1b gliadin restricted TCR and the DQ2.5 / γ4a gliadin restricted TCR, each TCR β chain-P2A-TCR α chain-pCXZD1 was introduced into TCRKO Jurkat NFAT-Luc by electroporation (LONZA, 4D-Nucleofector). The transfected cells were then cultured in a medium containing bleomycin (Zeocin) and hygromycin B and directly used as a cell line for transient TCR expression. These transient TCR-expressing cell lines were named α1b gliadin TCR Jurkat NFAT-Luc when the DQ2.5 / α1b gliadin-restricted TCR was introduced, and γ4a gliadin TCR Jurkat NFAT-Luc when the DQ2.5 / γ4a gliadin-restricted TCR was introduced.

[0752] Example 10

[0753] Preparation of tissue transglutaminase-treated pepsin-trypsin-digested gliadin (tTG-PT gliadin)

[0754] 10 grams of gliadin (Sigma, G3375) are suspended in 100mL 0.2N HCl, and then the pH value is adjusted to pH 7.4 with 2M NaOH. Then 201mg of pepsin (Sigma, P7012) are added and stirred for 2 hours in a water bath set at 37°C. The pepsin-treated gliadin is then processed with 201mg of trypsin (Sigma, T0303), and stirred for 4 hours in a water bath set at 37°C. In order to inactivate pepsin and trypsin, the gliadin digested with pepsin-trypsin was incubated for 30 minutes at 98°C, then freeze-dried at -75°C.

[0755] Pepsin-trypsinized gliadin was reconstituted to 1 mg / mL with PBS. Tissue transglutaminase (Sigma, T5398) was reconstituted to 1 mg / mL with 1 mM CaCl2-PBS. 1 mg / mL pepsin-trypsinized gliadin was mixed with 1 mg / mL tissue transglutaminase in a 9:1 ratio and incubated at 37°C for 2 hours to prepare 0.9 mg / mL tTG-PT gliadin.

[0756] Example 11 11.1

[0758] The inhibitory effect of anti-HLA DQ antibodies on DQ2.5 / α1 gliadin peptide-dependent Jurkat T cell activation was demonstrated. An Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line expressing HLA-DQ2.5 (ECACC, IHW9023) was used as antigen-presenting cells.

[0759] A mixture of IHW9023 cells and tTG-PT gliadin was plated in a 96-well plate (Corning, 3799). Serially diluted anti-HLA-DQ antibodies and α1 gliadin TCR Jurkat NFAT-Luc were then added and incubated overnight at 37°C in 5% CO2. The final concentration of tTG2-PT gliadin was 100 μg / mL, and the concentration of IHW9023 cells was 8.0×10 4 Cells / well, α1-gliadin TCR Jurkat NFAT-Luc is 2.0×10 4Cells / well, the final assay volume was 100 μL / well. After overnight incubation, 50 μL of cultured cells were harvested and redistributed in an OptiPlate-96 (PerkinElmer, 6005299). 50 μL of Bio-Glo (Promega, G7491) was then added and incubated at room temperature for 10 minutes. Luminescence was measured using Envision (PerkinElmer) and analyzed using Outlook Excel 2013 (Microsoft) and GraphPad Prism software (GraphPad) to determine the inhibitory effect of anti-HLA DQ antibodies on DQ2.5 / α1 gliadin peptide-dependent Jurkat T cell activation. The percentage of inhibition of the anti-HLA DQ antibody was determined when the counts per second (CPS) of the wells in the absence of antibody antigen was taken as 100% and the CPS of the wells in the absence of antibody antigen was taken as 0%. IC50 values were determined using XLfit Excel plug-in software (IDBS).

[0760] like Figure 22 As shown in Table 7, DQN0344xx, DQN0139bb, DQN0344xx / / DQN0385ee, and DQN0344xx / / DQN0429cc inhibited DQ2.5 / α1 gliadin peptide-dependent Jurkat T cell activation in a dose-dependent manner. DQN0385xx also moderately inhibited DQ2.5 / α1 gliadin peptide-dependent Jurkat T cell activation in a dose-dependent manner, but an IC50 value was not determined. On the other hand, DQN0429cc did not inhibit DQ2.5 / α1 gliadin peptide-dependent Jurkat T cell activation even at the highest antibody concentration of 1000 ng / mL. 11.2

[0762] The inhibitory effect of anti-HLA DQ antibodies on DQ2.5 / α2 gliadin peptide-dependent Jurkat T cell activation was confirmed. IHW9023 cells were used as antigen-presenting cells.

[0763] A mixture of IHW9023 cells and tTG-PT gliadin was distributed in a 96-well plate (Corning, 3799). Serially diluted anti-HLA-DQ antibodies and D2 TCR Jurkat NFAT-Luc were then added and incubated overnight at 37°C, 5% CO2. The final concentration of tTG2-PT gliadin was 50 μg / mL and that of IHW9023 was 8.0 x 10 4 cells / well, D2 TCR JurkatNFAT-Luc is 2.0x10 4Cells were added per well, with a final assay volume of 100 μL / well. After overnight incubation, 50 μL of cultured cells were harvested and redistributed in an OptiPlate-96 (PerkinElmer, 6005299). 50 μL of Bio-Glo (Promega, G7491) was then added and incubated at room temperature for 10 minutes. Luminescence was measured using Envision (PerkinElmer) and analyzed using Microsoft Outlook Excel 2013 and GraphPad Prism software (GraphPad) to determine the inhibitory effect of anti-HLA DQ antibodies on DQ2.5 / α2 gliadin peptide-dependent Jurkat T cell activation. The percentage inhibition of anti-HLA DQ antibodies was determined by taking the counts per second (CPS) of the wells in the absence of antibody and antigen as 100% and the CPS of the wells in the absence of antibody and antigen as 0%. IC50 values were determined using XLfit Excel plug-in software (IDBS).

[0764] like Figure 23 As shown in Table 7, all tested anti-HLA DQ antibodies inhibited DQ2.5 / α2 gliad...

Claims

1. An antigen-binding molecule, which is any one of the following (1) to (3): (1) An antigen-binding molecule comprising (a) or (b) below: (a) a first half antibody comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28, and a second half antibody comprising the HCDR1 sequence of SEQ ID NO: 2, the HCDR2 sequence of SEQ ID NO: 3, the HCDR3 sequence of SEQ ID NO: 4, the LCDR1 sequence of SEQ ID NO: 18, the LCDR2 sequence of SEQ ID NO: 19, and the LCDR3 sequence of SEQ ID NO: 20; or (b) a first half antibody comprising a HCDR1 sequence of SEQ ID NO: 10, a HCDR2 sequence of SEQ ID NO: 11, a HCDR3 sequence of SEQ ID NO: 12, a LCDR1 sequence of SEQ ID NO: 26, a LCDR2 sequence of SEQ ID NO: 27, and a LCDR3 sequence of SEQ ID NO: 28, and A second half antibody comprising the HCDR1 sequence of SEQ ID NO: 6, the HCDR2 sequence of SEQ ID NO: 7, the HCDR3 sequence of SEQ ID NO: 8, the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; wherein the antigen binding molecule has binding activity to all of the following: a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and an α3 Complexes formed with gliadin peptides; complexes formed with HLA-DQ2.5 and α1b gliadin peptide; complexes formed with HLA-DQ2.5 and γ4b gliadin peptide; complexes formed with HLA-DQ2.5 and avenin 1 peptide; complexes formed with HLA-DQ2.5 and avenin 2 peptide; complexes formed with HLA-DQ2.5 and avenin 3 peptide; complexes formed with HLA-DQ2.5 and hordein 1 peptide; complexes formed with HLA-DQ2.5 and hordein 2 peptide; complexes formed with HLA-DQ2.5 and secalin 1 peptide; complexes formed with HLA-DQ2.5 and secalin 2 peptide; complexes formed with HLA-DQ2.5 and 14mer 1 and a complex formed by HLA-DQ2.5 and a 26-mer gliadin peptide, wherein the antigen-binding molecule has substantially no binding activity to one or both of HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5; (2) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 2, a HCDR2 sequence of SEQ ID NO: 3, a HCDR3 sequence of SEQ ID NO: 4, a LCDR1 sequence of SEQ ID NO: 18, a LCDR2 sequence of SEQ ID NO: 19, and a LCDR3 sequence of SEQ ID NO: 20; (3) an antigen-binding molecule comprising a HCDR1 sequence of SEQ ID NO: 6, a HCDR2 sequence of SEQ ID NO: 7, a HCDR3 sequence of SEQ ID NO: 8, a LCDR1 sequence of SEQ ID NO: 22, a LCDR2 sequence of SEQ ID NO: 23, and a LCDR3 sequence of SEQ ID NO: 24, wherein the antigen-binding molecules of (2) and (3) have binding activity to all of the following: a complex formed by HLA-DQ2.5 and a 33-mer gliadin peptide; a complex formed by HLA-DQ2.5 and an α1 gliadin peptide; a complex formed by HLA-DQ2.5 and an α2 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ1 gliadin peptide; a complex formed by HLA-DQ2.5 and a γ2 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω1 gliadin peptide; a complex formed by HLA-DQ2.5 and an ω2 gliadin peptide; a complex formed by HLA-DQ2.5 and a BC hordein peptide; a complex formed by HLA-DQ2.5 and an α3 gliadin peptide; a complex formed by HLA-DQ2.5 and an α1b gliadin peptide; a complex formed by HLA-DQ2.5 and a γ4b complex formed by HLA-DQ2.5 and a gliadin peptide; a complex formed by HLA-DQ2.5 and avenin 1 peptide; a complex formed by HLA-DQ2.5 and avenin 2 peptide; a complex formed by HLA-DQ2.5 and hordein 1 peptide; a complex formed by HLA-DQ2.5 and hordein 2 peptide; a complex formed by HLA-DQ2.5 and secalin 1 peptide; a complex formed by HLA-DQ2.5 and secalin 2 peptide; a complex formed by HLA-DQ2.5 and 14-mer 1 peptide; and a complex formed by HLA-DQ2.5 and 26-mer gliadin peptide.

2. The antigen-binding molecule according to claim 1, wherein the first half antibody of the antigen-binding molecule of (1) (a) comprises a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO: 25; and wherein the second half antibody of the antigen-binding molecule of (1) (a) comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO:

17.

3. The antigen-binding molecule according to claim 1, wherein the first half antibody of the antigen-binding molecule of (1) (b) comprises a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO: 25; and wherein the second half antibody of the antigen-binding molecule of (1) (b) comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO:

21. 4 . The antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule of (2) comprises a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO:

17.

5. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule of (3) comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO:

21. The antigen-binding molecule according to claim 1 , wherein The antigen-binding molecule of (1) is a bispecific antibody. The antigen-binding molecule according to claim 2 , wherein the antigen-binding molecule is a bispecific antibody.

8. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule of (2) or (3) is an antibody.

9. The antigen-binding molecule according to claim 4 or 5, wherein the antigen-binding molecule is an antibody. 10 . A nucleic acid encoding the antigen-binding molecule according to claim 1 . A vector into which the nucleic acid according to claim 10 is introduced.

12. A cell comprising the nucleic acid of claim 10 or the vector of claim 11.

13. A method for producing an antigen-binding molecule by culturing the cell of claim 12. 14 . An antigen-binding molecule obtainable by the method of claim 13 , which has binding activity against a complex formed by HLA-DQ2.5 and a gluten peptide.

15. Use of the antigen-binding molecule according to any one of claims 1 to 9 in the preparation of a medicament for treating celiac disease.

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