Anti-HLA-DQ2.5 antibody and its use for the treatment of celiac disease

Abstract

The present invention relates to anti-HLA-DQ2.5 antibodies and their use for the treatment of celiac disease. The present invention provides anti-HLA-DQ2.5 antibodies that have been modified. The anti-HLA-DQ2.5 antibodies of the invention have binding activity to complexes formed by HLA-DQ2.5 and a gluten peptide, but have essentially no binding activity to complexes formed by HLA-DQ2.5 and an unrelated peptide. Furthermore, the antibodies of the invention have been shown to have inhibitory effects on T cell activation by gluten peptides.

Description

[Description] [Title of invention] Anti-HLA-DQ2.5 antibody and its use for the treatment of celiac disease [Technical background]

[0001] The present invention relates to anti-HLA-DQ2.5 antibodies and their use for the treatment of celiac disease. [Background knowledge]

[0002] Celiac disease (CD) is an autoimmune disorder in which gluten ingestion causes damage to the small intestine in genetically susceptible patients (NPL 1 to 5). It is thought that approximately 1% of the Western population, or 8 million people in the United States and the European Union, suffers from celiac disease; however, no significant therapeutic advances have been made since the disease was first identified in the 1940s. Human leukocyte antigens (HLAs) belong to the major histocompatibility complex (MHC) class II, consisting of HLA-DR, HLA-DP, and HLA-DQ molecules, such as the HLA-DQ2.5 isoform (hereafter referred to as "HLA-DQ2.5"), which form heterodimers consisting of alpha and beta chains on the cell surface. The majority (more than 90%) of patients with celiac disease carry the HLA-DQ2.5 haplotype allele (NPL 6). The isoform is thought to have a stronger affinity for gluten peptides. Like other isoforms, HLA-DQ2.5 presents processed antigens derived from exogenous sources to the T cell receptor (TCR) on T cells. As a result of the digestion of gluten-rich foods such as bread in patients with celiac disease, immunogenic gluten peptides such as gliadin peptides (NPL 2) are formed. The peptides are transported across the small intestinal epithelium to the lamina propria and deamidated by tissue transglutaminases such as transglutaminase 2 (TG2). The 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 a variety of gastrointestinal disorders, nutritional deficiencies, and systemic symptoms (NPL8, 9, 10). A neutralizing anti-HLA DQ antibody has been reported to inhibit gluten peptide-dependent activation of T cells from celiac patients (NPL7). The current practical treatment for celiac disease is lifelong adherence to a gluten-free diet (GFD). However, in reality, it is difficult to completely eliminate gluten exposure even with a GFD. The tolerable dose of gluten for these patients is only about 10–50 mg / day (NPL 11). Cross-contamination can occur extensively in GFD production, and small amounts of gluten can trigger celiac disease symptoms even in patients who are well compliant with the GFD. If such a risk of unintended exposure to gluten exists, there is a need for adjunctive therapy for the GFD. [Citation list] [Non-patent sources]

[0003] [NPL 1] N Engl J Med 2007; 357:1731-1743 [NPL 2] J Biomed Sci. 2012; 19(1): 88 [NPL 3] N Engl J Med 2003; 348:2517-2524 [NPL 4] Gut 2003;52:960-965 [NPL 5] Dig Dis Sci 2004; 49:1479-1484 [NPL 6] Gastroenterology 2011; 141:610-620 [NPL 7] Gut 2005; 54:1217-1223 [NPL 8] Gastroenterology 2014; 146:1649-58 [NPL 9] Nutrients 2013 Oct 5(10): 3975-3992 [NPL 10] J Clin Invest. 2007; 117(1):41-49 [NPL 11] Am J Clin Nutr 2007; 85: 160-6 [Invention Summary] [Technical problem]

[0004] Under the aforementioned conditions requiring adjuvant therapy, the present invention provides anti-HLA-DQ2.5 antigen binding molecules. [Problem Solution]

[0005] The antigen-binding molecules of the present invention have been modified and can bind to two or more complexes formed by HLA-DQ2.5 and a gluten peptide.

[0006] Specifically, the present invention provides the following. [1] A multispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide; and (ii) a second antigen-binding moiety that has HLA-DQ2.5 binding activity in the form of a complex with a gluten peptide; in which the antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides, wherein at least one of the gluten peptides in the complexes bound by the first antigen-binding moiety is different from at least one of the gluten peptides in the complexes bound by the second antigen-binding moiety; and wherein the antigen-binding molecule has essentially no binding activity to one or both of an HLA-DQ2.5 positive PBMC B cell and a Ba / F3 cell expressing HLA-DQ2.5, in which the antigen-binding molecule has been humanized and wherein one or more amino acids in a heavy chain and / or a light chain of the first antigen-binding portion and / or the second antigen-binding portion of the multispecific antigen-binding molecule are altered. [1a] A multispecific antigen-binding molecule [1] wherein the antigen-binding molecule has essentially no binding activity to a Ba / F3 cell expressing HLA-DQ2.2. [1-1] The multispecific antigen-binding molecule of [1] or [1a] wherein one or more amino acids in a heavy chain and / or a light chain of the first antigen-binding portion and / or the second antigen-binding portion of the multispecific antigen-binding molecule are substituted. [1-2] Multispecific antigen-binding molecule [1-1] comprising at least one amino acid substitution in a variable region of the heavy chain; at least one amino acid substitution in a constant region of the heavy chain; at least one amino acid substitution in a variable region of the light chain; and at least one amino acid substitution in a constant region of the light chain. [2] The multispecific antigen-binding molecule of any of [1] to [1-2], wherein the gluten peptide is a predominant immunological peptide associated with celiac disease. [3] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide is selected from the group consisting of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2, and 26-mer gliadin peptide. [3-1] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide(s) is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19 or all of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2 and 26-mer gliadin peptide. [3-2] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide is selected from the group consisting of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2, and 26-mer gliadin peptide. [3-3] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide(s) is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18 or all of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, Hordin 2, secalin peptide 1, secalin peptide 2, and gliadin 26-mer peptide. [4] The multispecific antigen-binding molecule of any of [1] to [3-3] having substantially no binding activity to HLA-DQ2.5 in the form of a complex with an irrelevant peptide, wherein the irrelevant peptide is at least one peptide selected from the group consisting of: CLIP peptide, hepatitis B virus 1 peptide, Salmonella peptide, Mycobacterium bovis peptide, and thyroperoxidase peptide. [4-1] A multispecific antigen-binding molecule of any of [1] to [3-3] having essentially no binding activity to HLA-DQ2.5 in the form of complexes with unrelated peptides, wherein the unrelated peptides are: CLIP peptide, hepatitis B virus 1 peptide, Salmonella peptide, Mycobacterium bovis peptide and thyroperoxidase peptide. [5] A multispecific antigen-binding molecule of any of [1] to [4-1] which has essentially no binding activity to HLA-DP, HLA-DR, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DQ7.5 and HLA-DQ8. [6] The multispecific antigen-binding molecule of any of [1] to [5] that (i) blocks the interaction between an HLA-DQ2.5 / gluten peptide complex and a CD4+ T cell restricted by HLA-DQ2.5 / gluten peptide; and / or (ii) blocks the interaction between an HLA-DQ2.2 / gluten peptide complex and a CD4+ T cell restricted by HLA-DQ2.2 / gluten peptide. [6-2] Multispecific antigen-binding molecule [6] wherein the gluten peptide is selected from the group consisting of: alpha-1 gliadin peptide, alpha-1b gliadin peptide, alpha-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, gamma-3 gliadin peptide, gamma-4a gliadin peptide, gamma-4d gliadin peptide and hordein BC peptide. [7] The multispecific antigen-binding molecule of any one of [1] to [6-2], wherein the antigen-binding molecule has improved binding activity to the complex formed by HLA-DQ2.5 and gluten peptide compared to before said humanization and modification. [8] The multispecific antigen-binding molecule of any one of [1] to [7], wherein the antigen-binding molecule has increased cross-reactivity to gluten peptides compared to the aforementioned humanization and modification. [8-1] Multispecific antigen-binding molecule [8] in which the gluten peptides are omega-2 gliadin peptide, hordein BC peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, gamma-4a gliadin peptide and gamma-4d gliadin peptide. [9] The multispecific antigen-binding molecule of any one of [1] to [8-1], wherein one, two, three or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (d) below in the heavy chain and the light chain of the antigen-binding molecule are amino acid residues that electrostatically repel each other: (a) an amino acid residue in a heavy chain constant region (CH1) located at position 175 according to EU numbering and an amino acid residue in a light chain constant region (CL) located at position 131 according to Kabat numbering, (b) an amino acid residue in CH1 located at position 175 according to the European Union numbering system and an amino acid residue in CL located at position 160 according to the Kabat numbering system, (c) An amino acid residue in CH1 located at position 175 according to EU numbering and an amino acid residue in CL located at positions 131 and 160 according to Kabat numbering. (d) Amino acid residues in CH1 located at positions 147 and 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to Kabat numbering.

[10] A multispecific antigen-binding molecule [9] in which two or more amino acid residues that form the interface between a heavy chain variable region and a light chain variable region are amino acid residues that electrostatically repel each other.

[11] A multispecific antigen-binding molecule

[10] wherein the amino acid residues that electrostatically repel each other are one or two sets of amino acid residues selected from the group consisting of sets of amino acid residues (a) and (b) below: (a) An amino acid residue in the variable region of the heavy chain located at position 39 according to Kabat numbering and an amino acid residue in the variable region of the light chain located at position 38 according to Kabat numbering. (b) An amino acid residue in the variable region of the heavy chain located at position 45 according to Kabat numbering and an amino acid residue in the variable region of the light chain located at position 44 according to Kabat numbering.

[12] The multispecific antigen-binding molecule of any of [9] to

[11] , wherein the amino acid residues that electrostatically repel each other are selected from amino acid residues present in either of the following sets (X) or (Y): (X) Glutamic acid (E), Aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[13] The multispecific antigen-binding molecule of any of [9] to

[12] , further comprising an Fc domain that exhibits reduced binding affinity for the human Fc gamma receptor compared to a natural human IgG1 Fc domain.

[14] A multispecific antigen-binding molecule

[13] in which the Fc domain contains Arg at position 235 and Arg at position 236, In which amino acid positions are numbered according to the European Union numbering system.

[15] A multispecific antigen-binding molecule

[13] or

[14] in which the Fc domain consists of a first Fc region subunit and a second Fc region subunit that are capable of stable association.

[16] A multispecific antigen-binding molecule

[15] wherein the Fc domain comprises (e1) or (e2) of the following: (e1) The first subunit of the Fc region contains Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and the second subunit of the Fc region contains Cys at position 354 and Trp at position 366. (e2) The first subunit of the Fc region contains Glu at position 439 and the second subunit of the Fc region contains Lys at position 356, In which amino acid positions are numbered according to the European Union numbering system.

[17] The multispecific antigen-binding molecule of any of

[13] to

[16] , wherein the Fc domain exhibits a stronger binding affinity for human FcRn than the Fc domain of natural human IgG1.

[18] A multispecific antigen-binding molecule

[16] wherein the first and / or second subunit of the Fc region comprises Leu at position 428, Ala at position 434, Arg at position 438 and Glu at position 440, In which amino acid positions are numbered according to the European Union numbering system.

[19] The multispecific antigen-binding molecule of any one of [1] to [8], wherein the multispecific antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xii): (i) glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) leucine at position 428 (EU numbering) in the heavy chain constant region; (ix) Alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region. [19-1] Multispecific antigen-binding molecule

[19] which is a bispecific antibody comprising: A first heavy chain comprising lysine at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering), glutamic acid at position 439 (EU numbering), and glutamic acid at position 440 (EU numbering); A first light chain containing glutamic acid at position 131 (Kabat numbering) and glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising glutamic acid at position 147 (EU numbering), glutamic acid at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), lysine at position 356 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering) and glutamic acid at position 440 (EU numbering); and A second light chain contains lysine at position 131 (Kabat numbering) and lysine at position 160 (Kabat numbering). [19-2] Multispecific antigen-binding molecule [19-1] wherein: The first heavy chain additionally contains glutamic acid at position 419 (EU numbering) and proline at position 445 (EU numbering) and amino acid deletions at positions 446 and 447 (EU numbering); and The second heavy chain additionally contains lysine at position 196 (EU numbering), proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). [19-3] Multispecific antigen-binding molecule [19-1] or [19-2], wherein: The first heavy chain additionally contains glycine at position 16 (Kabat numbering), alanine at position 32 (Kabat numbering), lysine at position 61 (Kabat numbering), valine at position 35a (Kabat numbering), alanine at position 50 (Kabat numbering), glutamic acid at position 64 (Kabat numbering), threonine at position 73 (Kabat numbering), glutamic acid at position 95 (Kabat numbering), and valine at position 102 (Kabat numbering). The first light chain additionally contains glutamic acid at position 28 (Kabat numbering), tyrosine at position 55 (Kabat numbering), glutamic acid or tyrosine at position 56 (Kabat numbering), glutamic acid at position 92 (Kabat numbering), valine at position 94 (Kabat numbering), and alanine at position 95a (Kabat numbering). The second heavy chain additionally comprises glutamic acid at position 28 (Kabat numbering), alanine or glutamic acid at position 30 (Kabat numbering), glutamic acid at position 31 (Kabat numbering), tryptophan at position 32 (Kabat numbering), phenylalanine at position 34 (Kabat numbering), methionine at position 35 (Kabat numbering), serine at position 35a (Kabat numbering), serine at position 50 (Kabat numbering), glutamic acid or glycine at position 61 (Kabat numbering), glutamic acid at position 64 (Kabat numbering), and glutamic acid at position 65 (Kabat numbering); and The second light chain additionally contains threonine at position 25 (Kabat numbering), lysine at position 54 (Kabat numbering), glutamic acid at position 56 (Kabat numbering), leucine at position 67 (Kabat numbering), glutamine at position 79 (Kabat numbering), and lysine at position 94 (Kabat numbering). [19a] The multispecific antigen-binding molecule of any one of [1] to [19-3], wherein the multispecific antigen-binding molecule has essentially no binding activity to the gluten peptide itself.

[20] A multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion; wherein the first antigen-binding moiety comprises any of the following (a1) to (a3): (a1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (a2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (a3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (a1) or (a2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (a1) or (a2).

[21] Multispecific antigen-binding molecule

[20] , wherein the second antigen-binding moiety comprises any of the following (b1) to (b8): (b1) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (b2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b3) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b4) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b5) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b6) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b7) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (b8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (b1) to (b7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (b1) to (b7). [21-2] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the second antigen-binding moiety comprises any of the following (b1) to (b8): (b1) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (b2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b3) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b4) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b5) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b6) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b7) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (b8) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (b1) to (b7) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (b1) to (b7).

[22] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety comprises any of the following (c1) to (c3): (c1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (c2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (c3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (c1) or (c2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (c1) or (c2), wherein the second antigen-binding moiety comprises any of the following (d1) to (d8): (d1) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (d2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d3) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d4) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d5) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d6) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d7) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (d8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (d1) to (d7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (d1) to (d7). [22-2] A multispecific antigen-binding molecule comprising a first antigen-binding portion comprising first and second antibody variable regions and a second antigen-binding portion comprising third and fourth antibody variable regions, wherein the multispecific antigen-binding molecule comprises any of (1) to (15) below: (1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (3) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (4) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (5) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (6) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (7) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (8) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (9) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (10) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (11) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (12) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (13) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (14) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (15) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in any of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in any of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (1) to (14).

[23] The multispecific antigen-binding molecule of any one of

[20] to [22-2], wherein the antibody variable region in the first and / or second antigen-binding portion comprises human antibody frameworks or humanized antibody frameworks.

[24] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety comprises any of the following (e1) to (e3): (e1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; (e2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; and (e3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (e1) or (e2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (e1) or (e2).

[25] A multispecific antigen-binding molecule

[24] wherein the second antigen-binding moiety comprises any of the following (f1) to (f8): (f1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 98; (f2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 93 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f4) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 94 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f5) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f6) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 96 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f7) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 97 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (f1) to (f7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (f1) to (f7).

[26] A multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion; wherein the first antigen-binding moiety comprises any of the following (e1) to (e3): (e1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; (e2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; and (e3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (e1) or (e2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (e1) or (e2) and wherein the second antigen-binding portion comprises any of (f1) to (f8) below: (f1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 98; (f2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 93 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f4) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 94 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f5) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f6) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 96 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f7) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 97 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (f1) to (f7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (f1) to (f7). [26-2] A multispecific antigen-binding molecule comprising a first antigen-binding portion comprising the first and second variable regions of an antibody and a second antigen-binding portion comprising the third and fourth variable regions of an antibody, wherein the multispecific antigen-binding molecule comprises any of (1) to (15) below: (1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 98; (2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (3) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 93; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (4) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 94; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (5) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (6) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 96; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (7) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 97; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (8) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 98; (9) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (10) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 93; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (11) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 94; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (12) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (13) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 96; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (14) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 97; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; and (15) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in any of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in any of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence similarity to the fourth variable region of the antibody expressed in any one of (1) to (14).

[27] A multispecific antigen-binding molecule comprising a combination of two polypeptide chains selected from the group consisting of (A1) to (A3) below: (A1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43; (A2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46; and (A3) an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first heavy chain expressed in (A1) or (A2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first light chain expressed in (A1) or (A2). [27-2] A multispecific antigen-binding molecule comprising a combination of two polypeptide chains selected from the group consisting of (A1) to (A3) below: (A1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43; (A2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46; and (A3) an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first heavy chain expressed in (A1) or (A2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first light chain expressed in (A1) or (A2).

[28] A multispecific antigen-binding molecule

[27] or [27-2], further comprising a combination of two polypeptide chains selected from the group consisting of (B1) to (B8) below: (B1) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (B2) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B3) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B4) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B5) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B6) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B7) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; and (B8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second heavy chain expressed in any of (B1) to (B7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second light chain expressed in any of (B1) to (B7). [28-2] A multispecific antigen-binding molecule

[27] or [27-2], further comprising a combination of two polypeptide chains selected from the group consisting of (B1) to (B8) below: (B1) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (B2) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B3) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B4) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B5) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B6) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B7) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; and (B8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second heavy chain expressed in any of (B1) to (B7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second light chain expressed in any of (B1) to (B7).

[29] A multispecific antigen-binding molecule comprising a combination of four polypeptide chains selected from the group consisting of (1) to (15) below: (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (4) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (9) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (10) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (11) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (12) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (13) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (14) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; and (15) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first heavy chain expressed in any of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first light chain expressed in any of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second heavy chain expressed in any of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second light chain expressed in any of (1) to (14). [29-2] A multispecific antigen-binding molecule comprising a combination of four polypeptide chains selected from the group consisting of (1) to (15) below: (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (4) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (9) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (10) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (11) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (12) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (13) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (14) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; and (15) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first heavy chain expressed in any of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first light chain expressed in any of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second heavy chain expressed in any of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second light chain expressed in any of (1) to (14). [29a] A combination of any of (i) to (iii) below: (i) a multispecific antigen-binding molecule comprising the sequences set forth in any one of (a1) to (a3) ​​of

[20] and a multispecific antigen-binding molecule comprising the sequences set forth in any one of (b1) to (b8) of

[21] ; (ii) a multispecific antigen-binding molecule comprising the sequences set forth in any one of (e1) to (e3) of

[24] and a multispecific antigen-binding molecule comprising the sequences set forth in any one of (f1) to (f8) of

[25] ; and (iii) a multispecific antigen-binding molecule comprising the sequences set forth in any one of (A1) to (A3) of

[27] or [27-2] and a multispecific antigen-binding molecule comprising the sequences set forth in any one of (B1) to (B8) of

[28] or [28-2].

[30] A nucleic acid encoding a multispecific antigen-binding molecule of any of [1] to

[29] .

[31] A vector comprising nucleic acid

[30] .

[32] A cell containing nucleic acid

[30] or vector

[31] .

[33] A method for producing a multispecific antigen-binding molecule comprising culturing cells

[32] such that the multispecific antigen-binding molecule is produced.

[34] The method of

[33] , further comprising recovering the multispecific antigen-binding molecule from the cell culture.

[35] A pharmaceutical composition comprising the multispecific antigen-binding molecule of any of [1] to

[29] or a combination of [29a] and a pharmaceutically acceptable carrier.

[36] The compound

[35] which is a pharmaceutical composition for use in the treatment and / or prevention of celiac disease.

[37] Use of a multispecific antigen-binding molecule of any of [1] to

[29] or a combination of [29a] in the manufacture of a medicament.

[38] Use of

[37] wherein the medicament is a medicament for the treatment and / or prevention of celiac disease.

[39] A method for treating a subject with celiac disease comprises administering to the subject an effective amount of the multispecific antigen-binding molecules of any of [1] to

[29] or a combination of [29a].

[40] A kit for use in the treatment and / or prevention of celiac disease comprising at least one multispecific antigen-binding molecule of any of [1] to

[29] or a combination of [29a] and instructions for use. [Brief description of shapes]

[0007] [Figure 1-1] Figure 1-1 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg (μg) / mL and control DQN0139bb (DQN0139bb-SG181) (WO2018 / 155692) and IC17dK at 1 μg / mL). In the specified names of gluten peptides, "a", "g" and "w" mean "alpha", "gamma" and "omega", respectively. [Figure 1-2] Figure 1-2 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1270 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Fig. 1-3] Figure 1-3 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1352 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies tested at 0.05 μg / mL) and controls DQN0139bb and IC17dK tested at 1 μg / mL). [Figure 1-4] Figure 1-4 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1527 / L0605-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies tested at 0.05 μg / mL) and controls DQN0139bb and IC17dK tested at 1 μg / mL). [Figure 1-5] Figure 1-5 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1255 / L0605-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at a concentration of 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-6] Figure 1-6 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1270 / L0722-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-7] Figure 1-7 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1521 / L0605-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-8] Figure 1-8 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1270 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-9] Figure 1-9 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1352 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-10] Figure 1-10 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (all antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-11] Figure 1-11 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1521 / L0605-F6) against HLA class II-expressing Ba / F3 cell lines (antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) for HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Fig. 1-12] Figure 1-12 shows the binding results of anti-HLA-DQ antibody (species DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6) against HLA class II-expressing Ba / F3 cell lines (antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) for HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-13] Figure 1-13 shows the binding results of anti-HLA-DQ antibody (species DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6) against HLA class II-expressing Ba / F3 cell lines (antibodies were tested at 0.05 μg / mL and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) for HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-14] Figure 1-14 shows the results of binding of anti-HLA-DQ antibody (species) to HLA-DP, DR, DQ5.1, and DQ6.3 (all antibodies tested at 0.05 μg / mL and controls DQN0139bb and IC17dK at 1 μg / mL). [Figure 1-15] Figure 1-15 shows the results of DQN0139bb binding to HLA class II-expressing Ba / F3 cell lines (DQN0139bb control tested at 1 μg / mL). [Figure 1-16] Figure 1-16 shows the results of IC17dK against HLA class II-expressing Ba / F3 cell lines (control IC17dK was tested at 1 μg / mL). [Figure 2] Figure 2 shows the results of antibody binding to PBMC-derived CD19+ B cells (antibodies were tested at 0.05 μg / mL and DQN0139bb and IC17dK controls at 1 μg / mL; (#) for HLA-DQ2.5 and HLA-DQ2.5-CLIP, antibodies were tested at 0.313 μg / mL and DQN0139bb and IC17dK controls at 20 μg / mL). [Figure 3-1] Figure 3-1 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha-1 gliadin-dependent Jurkat T cell activation. [Figure 3-2] Figure 3-2 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha-2 gliadin-dependent Jurkat T cell activation. [Figure 3-3] Figure 3-3 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha 1b gliadin-dependent Jurkat T cell activation. [Figure 3-4] Figure 3-4 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / omega-1 gliadin-dependent Jurkat T cell activation. [Figure 3-5] Figure 3-5 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / omega-2 gliadin-dependent Jurkat T cell activation. [Figure 3-6] Figure 3-6 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / hordin BC-dependent Jurkat T cell activation. [Figure 3-7] Figure 3-7 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma-1 gliadin-dependent Jurkat T cell activation. [Figure 3-8] Figure 3-8 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma 2 gliadin-dependent Jurkat T cell activation. [Figure 3-9] Figure 3-9 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma 3 gliadin-dependent Jurkat T cell activation. [Figure 3-10] Figure 3-10 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma 4a gliadin-dependent Jurkat T cell activation. [Figure 4-1] Figure 4-1 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha-1 gliadin-dependent Jurkat T cell activation. [Figure 4-2] Figure 4-2 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha-2 gliadin-dependent Jurkat T cell activation. [Figure 4-3] Figure 4-3 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / alpha 1b gliadin-dependent Jurkat T cell activation. [Figure 4-4] Figure 4-4 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / omega-1 gliadin-dependent Jurkat T cell activation. [Figure 4-5] Figure 4-5 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / omega-2 gliadin-dependent Jurkat T cell activation. [Figure 4-6] Figure 4-6 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / hordin BC-dependent Jurkat T cell activation. [Figure 4-7] Figure 4-7 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma-1 gliadin-dependent activation of Jurkat T cells. [Figure 4-8] Figure 4-8 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma 2 gliadin-dependent activation of Jurkat T cells. [Figure 4-9] Figure 4-9 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma-3 gliadin-dependent activation of Jurkat T cells. [Figure 4-10] Figure 4-10 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.5 / gamma 4a gliadin-dependent activation of Jurkat T cells. [Figure 5-1] Figure 5-1 shows HLA-DQ2.2 / alpha 1a gliadin-dependent activation of Jurkat T cells mediated by gliadin 33mer. [Figure 5-2] Figure 5-2 shows HLA-DQ2.2 / alpha-2 gliadin-dependent activation of Jurkat T cells mediated by gliadin 33mer. [Figure 5-3] Figure 5-3 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.2 / alpha 1a gliadin-dependent activation of Jurkat T cells. [Figure 5-4] Figure 5-4 shows the inhibitory effect of anti-HLA DQ antibodies on HLA-DQ2.2 / alpha-2 gliadin-dependent Jurkat T cell activation. [Description of the incarnations]

[0008] The techniques and procedures described or referred to herein are generally well understood and are commonly used by those skilled in the art using conventional methodology, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (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 (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PERoberts, 1998) Plenum Press؛ Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons؛ Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.)؛ Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987)؛ PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994)؛ Current Protocols in Immunology (J.E. Coligan et al., eds., 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 J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0009] An "acceptor human framework" for purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) or a heavy chain variable domain (VH) 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 as that, or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations 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 acceptor human framework VL is identical in sequence to the human immunoglobulin VL framework sequence or the human consensus framework sequence.

[0010] "Affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "combined affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (Kd). Affinity can be measured by methods commonly known in the art, including those described herein. Illustrative and specific exemplary embodiments for measuring binding affinity are described below.

[0011] An affinity-matured antibody refers to an antibody with one or more changes in one or more hypervariable regions (HVRs), compared to the parent antibody that does not have such changes, such changes resulting in improved affinity of the antibody for the antigen.

[0012] The term "antigen-binding portion" or "antigen-binding domain" refers to a portion of an antibody that includes a region that specifically binds to part or all of an antigen and is complementary thereto. An antigen-binding portion / domain may be provided, for example, with one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen-binding portions / domains comprise both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0013] The term "anti-HLA-DQ2.5 antigen-binding molecule (antibody)" refers to an antigen-binding molecule (antibody) that is capable of binding to HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and a gluten peptide with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting HLA-DQ2.5. In one embodiment, the extent of binding of an anti-HLA-DQ2.5 antigen-binding molecule (antibody) to an unrelated antigen is less than about 10% of the binding of the antibody to HLA-DQ2.5 or the HLA-DQ2.5 peptide / gluten peptide complex, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody having "binding activity" to HLA-DQ2.5 or the HLA-DQ2.5 / gluten peptide complex has a dissociation constant (Kd) of 1 micromolar (microM) or less, 100 nanomolar or less, 10 nanomolar or less, 1 nanomolar or less, 0.1 nanomolar or less, 0.01 nanomolar or less, or 0.001 nanomolar or less (e.g., 8-10 molar or less, e.g., from 8-10 molar to 10-13 molar, e.g., from 9-10 molar to 10-13 molar).

[0014] The term "antigen-binding molecule" as used herein refers to any molecule that has an antigen-binding site or any molecule that has antigen-binding activity and may refer to molecules such as a peptide or protein of about five amino acids or more in length. A peptide and protein are not limited to those derived from a living organism and may, for example, be a polypeptide produced from an artificially designed sequence. They may also be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc. Scaffold molecules comprising a known stable structural structure such as an alpha / beta barrel as a scaffold in which a portion of the molecule is made to bind to the antigen, are also one embodiment of the antigen-binding molecule described herein. In some embodiments, the "antigen-binding molecule" is an antibody.The terms "antigen-binding molecule" and "antibody" are used broadly herein and encompass a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments of sufficient length to exhibit binding activity to the antigen of interest. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody.

[0015] An "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds an 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 (such as scFv); and multispecific antibodies that are composed of antibody fragments.

[0016] 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 competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0017] "Autoimmune disease" refers to a non-malignant disease or disorder that arises from and is directed against a person's own tissues. Autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, with the specific exception of B-cell lymphoma, 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 (such as atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as 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 deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes mellitus (e.g.Type I or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); leukocyte diapedesis diseases; central nervous system (CNS) inflammatory disorder; multiple organ damage syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenia syndrome; Bullous pemphigoid; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff man syndrome; Behçet's disease; giant cell arthritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0018] The term "celiac disease (celiac)" refers to an inherited autoimmune disease that is caused by damage to the small intestine caused by the consumption of gluten found in food. Symptoms of celiac disease include, but are not limited to, digestive disorders such as abdominal pain, diarrhea, and gastroesophageal reflux disease, vitamin deficiencies, mineral deficiencies, central nervous system (CNS) symptoms such as fatigue and depression, bone symptoms such as osteomalacia and osteoporosis, skin symptoms such as dermatitis, blood symptoms such as anemia and lymphocytopenia, and other symptoms such as infertility, hypogonadism, and failure to thrive and short stature in children.

[0019] 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.

[0020] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and a number of these may be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0021] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount that is effective at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

[0022] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, a C-terminal lysine (Lys447) or glycine-lysine (residues 446 to 447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the European Union numbering system, also called the European Union 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.

[0023] "Framework" or "FR" refers to the residues of the variable domain other than the residues of the hypervariable region (HVR). The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0024] The terms "full-length antibody", "intact antibody", and "complete antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to that of a natural antibody or has heavy chains containing an Fc region as defined herein.

[0025] Here, the term "gluten" refers collectively to a combination of storage proteins called prolamins found in wheat and other related grains. In the intestinal tract, gluten is broken down into so-called gluten peptides. Gluten peptides include, but are not limited to, gliadin from wheat, hordein from barley and seculin from rye, and avenin from oats.

[0026] In celiac disease, gluten peptides are antigenic peptides that are recognized by T cells that cause the disease. Meanwhile, immune dominance is a phenomenon in which the immune response is primarily stimulated by a relatively small number of antigenic peptides. Such antigenic peptides can be called “immune dominant peptides.” In celiac disease, such immune dominant peptides include, for example, alpha-1 gliadin (which may also be called “alpha-1a gliadin”) and alpha-2 gliadin (both of which are located in the 33mer gliadin sequence) and omega-1 gliadin, omega-2 gliadin, and hordein BC (five peptides in total) (Science Translational Medicine 21 Jul 2010: Vol. 2, Issue 41, pp. 41ra51). On the other hand, the dominant immune peptides include, but are not limited to, alpha-1 gliadin, alpha-2 gliadin, omega-1 gliadin, omega-2 gliadin, hordein BC, gamma-1 gliadin, and gamma-2 gliadin (a total of seven peptides). Herein, such dominant immune peptides may be referred to as "celiac disease-related dominant immune peptides."The types and total number of peptides are not specifically limited, as long as they are primarily related to celiac disease.

[0027] The phrase "substantially no binding activity," as used herein, refers to the activity of an antibody to bind to an antigen of no interest at a level of binding that includes nonspecific or background binding but does not include specific binding; in other words, such an antibody has "no specific / significant binding activity" toward the antigen of interest. Specificity can be measured by any method described in this specification or known in the art. The aforementioned level of nonspecific or background binding may be zero, or may not be zero but close to zero, or may be so low as to be technically negligible by those skilled in the art. For example, when a skilled person cannot detect or observe a significant (or relatively strong) signal for binding between the antibody and the antigen of interest in an appropriate binding assay, the antibody can be said to have "essentially no binding activity" or "no specific / significant binding activity" toward the antigen of interest.Alternatively, "substantially no binding activity" or "no specific / significant binding activity" can be rewritten as "does not specifically / significantly / substantially bind" (to an antigen of interest). Sometimes, the phrase "no binding activity" is essentially the same as the phrase "substantially no binding activity" or "no specific / significant binding activity" in the literature.

[0028] Here, "HLA-DR / DP" means "HLA-DR and HLA-DP" or "HLA-DR or HLA-DP". These HLAs are MHC class II molecules encoded by the corresponding haplotype alleles at the MHC class II locus in humans. "HLA-DQ" collectively refers to the HLA-DQ isoforms including HLA-DQ2.5, 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-DQ molecules include, but are not limited to, HLA-DQ molecules of known subtypes (isoforms) such as HLA-DQ2.2, 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 HLA-DR (DP) isoforms.

[0029] 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 original transformed cells and the progeny obtained therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have a similar function or biological activity as screened or selected in the original transformed cell are included herein.

[0030] "Human antibody" is an antibody that has an amino acid sequence that is consistent with an antibody produced by a human or a human cell or derived from a non-human source that utilizes a human antibody reservoir or other human antibody coding sequences. This definition of a human antibody specifically excludes a human antibody that contains non-human antigen-binding residues.

[0031] A "human consensus framework" is a framework that represents the most common amino acid residues in a set of human immunoglobulin VL or VH framework sequences. The term "human antibody framework" may also be used to refer to the framework. In general, the selection of a human immunoglobulin VL or VH sequence is from a subset of the variable domain sequences. In general, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is the kappa subgroup I, as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III, as in Kabat et al., supra.

[0032] A "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 essentially comprises at least one and usually two variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to non-human antibodies and all or substantially all of the FRs correspond to human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0033] The term "hypervariable region" or "HVR" as used herein refers to any of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contacts"). In general, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein are: (a) Highly variable 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)); (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)). (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 (d) a combination of (a), (b) and / or (c), including 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). In one embodiment, the HVR residues include those specified in the specification.]] Unless otherwise noted, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al.

[0034] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules.

[0035] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats). In certain embodiments, the individual or subject is a human.

[0036] In the present invention, a CLIP peptide may be used in conjunction with a suitable HLA-DQ molecule as mentioned above when assessing the binding of anti-HLA-DQ2.5 antibodies to these HLA-DQ molecules.

[0037] An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95 or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0038] "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 present in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

[0039] "Isolated nucleic acid encoding an anti-HLA-DQ2.5 molecule (antibody)" (also simply referred to as "nucleic acid encoding an anti-HLA-DQ2.5 molecule (antibody)") refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0040] The term "monoclonal antibody" as used herein refers to an antibody derived from a population of completely homogeneous antibodies, i.e. the individual antibodies constituting the population are identical and / or bind to the same epitope, except for variants of the antibody, e.g., containing naturally occurring mutations or those induced during the production of a monoclonal antibody, such variants being usually present in minor amounts. In contrast to polyclonal antibody preparations which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is a monoclonal antibody against a single determinant on an antigen; Therefore, the modifier "monoclonal" specifies that the specificity of the antibody is derived from a completely homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0041] "Naked antibody" refers to an antibody that has not been conjugated to a heterologous moiety or radiolabel. Naked antibody may be present in a pharmaceutical formulation.

[0042] "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or variable heavy chain domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or variable light chain domain, followed by a constant light domain (CL). The light chain of an antibody may be assigned to one of two types, kappa and lambda, based on the amino acid sequence of its constant domain.

[0043] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that is composed of a polymer of nucleotides. Each nucleotide is composed of a base, typically a purine or pyrimidine base (e.g. cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (e.g. deoxyribose or ribose), and a phosphate group. Often, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is usually indicated from 5' to 3'. Here, the term nucleic acid molecule includes deoxyribonucleic acid (DNA) including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. A nucleic acid molecule may 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 molecule described herein can contain naturally occurring or unnatural nucleotides. Examples of unnatural nucleotides include nucleotide bases modified with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules that are suitable as vectors for the direct expression of the antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. These DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to increase the stability of the RNA vector and / or expression of the encoded molecule, so that the mRNA can be injected into an individual to produce antibodies in vivo (see, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0044] "Percent (%) amino acid sequence similarity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence similarity and not including any conservative substitutions as part of the sequence similarity. Alignment for the purposes of determining percent amino acid sequence similarity can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in this knowledge can determine the appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum alignment across the full length of the sequences being compared.

[0045] The ALIGN-2 sequence comparison computer program was developed by Genentech Corporation and the source code is filed with the U.S. Copyright Office, Washington, D.C., 20559, under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not subject to change. In situations where ALIGN-2 is used for amino acid sequence comparison, the percentage of amino acid sequence similarity of a given amino acid sequence A to or against a given amino acid sequence B (which can be defined as a given amino acid sequence A that has a certain percentage of amino acid sequence similarity to or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues identified as identical matches by the ALIGN-2 sequence alignment program in alignments A and B of that program, and Y is the total number of amino acid residues in B. It is understood that where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the percent similarity of the amino acid sequence A to B will not be equal to the percent similarity of the amino acid sequence B to A. Unless specifically stated otherwise, all percent similarity values ​​of amino acid sequence used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0046] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a substance that is in a form that allows the biological activity of an active ingredient contained therein to be effective and does not contain any additional components that are unacceptably toxic to a person receiving the formulation / composition.

[0047] "Pharmaceutically acceptable carrier" refers to a substance in a pharmaceutical formulation / composition, other than an active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0048] The term "HLA-DQ2.5" as used herein refers to any naturally occurring HLA-DQ2.5 from any vertebrate source, including mammals such as mammals (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term includes "full-length" unprocessed HLA-DQ2.5 as well as any form of HLA-DQ2.5 resulting from processing in a cell. The term also includes naturally occurring variants of HLA-DQ2.5, e.g., splice variants or allelic variants. The amino acid sequence of the HLA-DQ2.5 sample is publicly available in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) accession code 4OZG and the IPD-IMGT / HLA database.

[0049] Here, “TCR” stands for “T cell receptor,” which is a membrane protein located on the surface of T cells (such as HLA-DQ2.5-restricted CD4 + T cells) that recognizes an antigen fragment (such as a gluten peptide) present on MHC molecules including HLA-DQ2.5.

[0050] As used herein, "treatment" (and its grammatical variations such as "treatment" or "treating") refers to clinical intervention in an attempt to alter the natural course of the subject and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition of the disease, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the rate of progression of a disease.

[0051] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have similar structures, with each domain consisting of four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by using a VH or VL domain from the antibody that binds to the antigen to screen a library of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0052] The term "vector" as used herein refers to a nucleic acid molecule that is capable of expressing another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid construct as well as a vector that is incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0053] Amino acid modifications An antigen-binding molecule (or antibody) of the invention may include one or more modifications. Such modifications include deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be made to achieve the final structure, provided that the final structure has the desired properties, e.g., antigen binding.

[0054] An antigen-binding molecule (or antibody) of the invention may include amino acid substitutions. Conservative substitutions are shown in Table 1-1 under the heading "Preferred Substitutions". More radical changes are presented in Table 1-1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for the desired activity, e.g., antigen binding.

[0055] [Table 1-1]

[0056] Here, as a term indicating an amino acid change, a term indicating a number before and after a specific position, one-letter or three-letter codes for the amino acids before and after the change, respectively, may be used appropriately. For example, the change N100bL or Asn100bLeu used when replacing an amino acid in the variable region of an antibody indicates the replacement of Asn at position 100b (according to Kabat numbering) with Leu; that is, the number indicates the position of the amino acid according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before the substitution, and the one-letter or three-letter amino acid code written before the number indicates the amino acid after the substitution.Similarly, the P238D or Pro238Asp change used when replacing an amino acid from the Fc region of an antibody constant region represents the replacement of Pro at position 238 (according to EU numbering) with Asp; that is, the number indicates the position of the amino acid according to EU numbering, the one-letter or three-letter amino acid code preceding the number indicates the amino acid before the substitution, and the one-letter or three-letter amino acid code preceding the number indicates the amino acid after the substitution.

[0057] Multispecific antigen-binding molecules / antibodies The term "antigen-binding molecule (antibody)" refers to an antigen-binding molecule (antibody) that specifically binds to more than one antigen (e.g., a peptide) or epitope. In some embodiments, the antigen-binding molecule (antibody) has at least a first antigen-binding portion / domain that can bind to one or more antigens (e.g., peptides) and a second antigen-binding portion / domain that can bind to one or more antigens (e.g., peptides). Some or all of the antigens bound by the first antigen-binding portion / domain may be different from some or all of the antigens bound by the second antigen-binding portion / domain. Alternatively, some of the antigens bound by the first antigen-binding portion / domain may be identical to some of the antigens bound by the second antigen-binding portion / domain.

[0058] In the context of the present invention, a "multispecific antigen-binding molecule (antibody)" may specifically bind to different types of antigens or epitopes. More specifically, multispecific antigen-binding molecules (antibodies) are those that have specificity for at least two different types of antigens or epitopes, and include molecules / antibodies that recognize different antigens as well as molecules / antibodies that recognize different epitopes on the same antigen. For example, such molecules typically bind to two antigens or epitopes ("bispecific antigen-binding molecules (antibodies)"; used herein to mean "bispecific antigen-binding molecules (antibodies)"), but may even have specificity for more antigens or epitopes (e.g., three or more antigens).

[0059] Here, terms such as "multispecific" and "bispecific" mean that the specificity of one antigen-binding domain / region is different from that of another antigen-binding domain / region; that is, these terms mean that two or more specificities are present in one antigen-binding molecule. For example, in a "bispecific" antigen-binding molecule (antibody), the first antigen-binding portion / domain may bind to a first group of complexes formed by HLA-DQ2.5 and a gluten peptide, and the second antigen-binding portion / domain may bind to a second group of complexes formed by HLA-DQ2.5 and a gluten peptide. The members (i.e., complexes) of the two groups may overlap but may not be identical; that is, some complexes may fall into both groups. Terms such as "multispecific" and "bispecific" can cover this situation. The same applies to the first and second groups of complexes that are not limited to the first / second antigen-binding portion / domain.

[0060] The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two distinct antigenic determinants. Examples of the preferred embodiment of the "multispecific antigen-binding molecule" of the present invention include multispecific antibodies. When an Fc region with reduced Fc gamma receptor binding activity is used as the Fc region of the multispecific antibody, an Fc region derived from a multispecific antibody may be suitably used. Bispecific antibodies are particularly preferred as the multispecific antibodies of the present invention. In this case, a bispecific antibody is an antibody that has two different specificities. Bispecific IgG antibodies can be secreted from a hybrid hybridoma (quadroma) produced by the fusion of two hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).

[0061] A multispecific antigen-binding molecule (antibody) may comprise at least two antigen-binding moieties / domains. A bispecific antigen-binding molecule (antibody) may comprise a first antigen-binding moietie and a second antigen-binding moietie. A bispecific antigen-binding molecule (or bispecific antibody) may comprise a first antigen-binding portion / domain and a second antigen-binding portion / domain. The first antigen-binding portion / domain may comprise a first variable region of the antibody and a second variable region of the antibody. The first variable region of the antibody is associated with the second variable region of the antibody. The association between the first variable region of the antibody and the second variable region of the antibody enables the binding of the first antigen-binding portion / domain to the first antigen / epitope. Similarly, the second antigen-binding portion / domain may comprise a third variable region of the antibody and a fourth variable region of the antibody. The third variable region of the antibody is associated with the fourth variable region of the antibody. The association between the third variable region of the antibody and the fourth variable region of the antibody enables the binding of the second antigen-binding portion / domain to the second antigen / epitope.In some embodiments, the first variable region of the antibody is a heavy chain (H chain) variable region (VH) (which may be referred to as the "first variable region of the heavy chain (H chain)") and the second variable region of the antibody is a light chain (L chain) variable region (VL) (which may be referred to as the "first variable region of the light chain (L chain) (VL)"). In some embodiments, the third variable region of the antibody is a heavy chain (H chain) variable region (VH) (which may be referred to as the "second variable region of the heavy chain (H chain)" (VH)) and the fourth variable region of the antibody is a light chain (L chain) variable region (VL) (which may be referred to as the "second variable region of the light chain (L chain) (VL)"). The first variable region of the heavy chain (H chain) (VH) is associated with the first variable region of the light chain (L chain) (VL) to bind to the first antigen / epitope. The second variable region of the heavy chain (H chain) (VH) is associated with the second variable region of the light chain (L chain) (VL) to bind to the second antigen / epitope.The association (alternatively referred to as "interaction") between variable regions (e.g., between VH and VL) relies on the structure (e.g., amino acid residues) at the VH / VL interface, which is known in the art. In the present invention, preferably an antigen-binding molecule (antibody) can bind to two or more gluten peptides (or complexes formed by HLA-DQ2.5 and gluten peptides). In some embodiments, a bispecific antigen-binding molecule (antibody) comprises a first antigen-binding portion / domain (comprising a first antibody variable region and a second antibody variable region (supra)) that binds to one or more complexes formed by HLA-DQ2.5 and a gluten peptide, and comprises a second antigen-binding portion / domain (comprising a third antibody variable region and a fourth antibody variable region (supra)) that binds to one or more complexes formed by HLA-DQ2.5 and a gluten peptide.In this context, preferably, at least one gluten peptide in the complexes bound by the first antigen-binding portion / domain is different from at least one gluten peptide in the complexes bound by the second antigen-binding portion / domain; in other words, the members of the gluten peptides in the complexes bound by the first antigen-binding portion / domain and the members of the gluten peptides in the complexes bound by the second antigen-binding portion / domain may overlap but are not entirely identical. The gluten peptides in the complexes bound by the first / second antigen-binding portion / domain may be selected from any gluten peptide described herein. Preferably, the first / second antigen-binding portion / domain is capable of binding to one type of gluten peptide, or two or more types of gluten peptides.

[0062] In the context of the present disclosure, for simplicity, the term "antibody" may be used instead of referring to "antigen-binding molecule." However, a skilled person will appreciate that the term "antibody" may be replaced with "antigen-binding molecule" as appropriate.

[0063] In one aspect, the invention is based in part on binding an anti-HLA-DQ2.5 antigen-binding molecule (antibody) to HLA-DQ2.5 that presents a gluten peptide to T cells. In certain embodiments, antibodies that bind to HLA-DQ2.5 are provided.

[0064] In one aspect, the invention provides antigen-binding molecules or antibodies that have binding activity to HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and a gluten peptide. In certain embodiments, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has the functions / properties listed below.

[0065] The anti-HLA-DQ2.5 molecule (antibody) has binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide (i.e., an HLA-DQ2.5 / gluten peptide complex). Preferably, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has specific binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide (i.e., an HLA-DQ2.5 / gluten peptide complex).

[0066] The anti-HLA-DQ2.5 antigen-binding molecule (antibody) has essentially no binding activity to an antigen to which it has no interest, such as HLA-DQ5.1 / DQ6.3 / DQ7.3 / DQ7.5 / DQ8 / DR / DP, i.e., the anti-HLA-DQ2.5 antigen-binding molecule (antibody) does not significantly bind to an antigen to which it has no interest. For example, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has no specific binding activity to HLA-DR / DP or significant binding activity to HLA-DR / DP; i.e., the antibody does not specifically bind to HLA-DR / DP or significantly binds to HLA-DR / DP. Similarly, the molecule (antibody) binding to the anti-HLA-DQ2.5 antigen has essentially no binding activity to the HLA-DQ molecule such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3 and HLA-DQ7.3, i.e., the molecule (antibody) binding to the anti-HLA-DQ2.5 antigen does not essentially bind to the HLA-DQ molecule such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3 and HLA-DQ7.3; in other words, the molecule (antibody) binding to the anti-HLA-DQ2.5 has no specific / significant binding activity to an HLA-DQ molecule such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3 and HLA-DQ7.3; that is, the molecule (antibody) binding to the anti-HLA-DQ2.5 antigen does not specifically / significantly bind to the HLA-DQ molecule such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3 and HLA-DQ7.3. To avoid any significant inhibitory effects on these non-target MHC class II molecules and to improve the PK of the antibody for patients with celiac disease who have HLA-DQ2.5, these properties ("substantially no binding activity") are preferred. For example, using the FACS results described herein, the property "substantially no binding activity" can be defined. An anti-HLA-DQ2.5 antigen-binding molecule (antibody) that has "substantially no binding activity" to a specific antigen may have an MFI (mean fluorescence intensity) value of 250% or less, preferably 200% or less, preferably 150% or less of the MFI value of the negative control under the measurement conditions described herein.

[0067] In one aspect, for a bispecific antigen-binding molecule (antibody), the anti-HLA-DQ2.5 antigen-binding molecule (antibody) that has "substantially no binding activity" to a specific antigen has an MFI value of 2% or less, preferably 1% or less, when the MFI value of IC17dK is considered to be zero percent and the MFI value of DQN0139bb is considered to be 100 percent under the measurement conditions described herein. DQN0139bb is disclosed in, for example, WO2018 / 155692.

[0068] The anti-HLA-DQ2.5 molecule (antibody) has binding activity to HLA-DQ2.5 in a complex with a gluten peptide 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 "HLA-DQ2.5 / gluten peptide". It may also be rewritten as, for example, "HLA-DQ2.5 loaded with gluten peptide", "HLA-DQ2.5 loaded with gluten peptide", "HLA-DQ2.5 bound by gluten peptide", "HLA-DQ2.5 in the form of a complex with a gluten peptide" and "a complex of HLA-DQ2.5 and a gluten peptide". The above statement (for example, "a complex formed by HLA-DQ2.5 and ...[peptide]") also for peptides such as 33-mer gliadin peptide, alpha-1 gliadin peptide (which may also be called "alpha-1a gliadin peptide"), alpha-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, seculin peptide 1, seculin peptide 2 and 26-mer gliadin peptide, 14-mer 1 peptide, CLIP peptide (hCLIP), hepatitis B virus 1 (HBV1) peptide, Salmonella peptide, Mycobacterium bovis (M. bovis) peptide, thyroperoxidase (TPO) peptide, etc. are applied.

[0069] Meanwhile, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has essentially no binding activity to an "irrelevant" peptide. Here, "irrelevant" peptides include those reported to be present in HLA-DQ2.5 but are not relevant to celiac disease or are not relevant to the present invention, that is, those not the gluten peptides of interest mentioned above. For example, the irrelevant peptides include CLIP peptide (hCLIP), hepatitis B virus 1 (HBV1) peptide, Salmonella peptide, Mycobacterium bovis (M. bovis) peptide, thyroperoxidase (TPO) peptide, etc. These properties (“essentially no binding activity”) are preferred to avoid any significant inhibitory effects on these non-target MHC class II and HLA-DQ2.5 molecules in the form of a complex with an unrelated peptide and to improve the PK of the antibody for patients with celiac disease. The "binding activity" property can be defined, for example, using the FACS results described herein. An anti-HLA-DQ2.5 antigen-binding molecule (antibody) having "binding activity" to a specific antigen may have an MFI (mean fluorescence intensity) value of 300% or higher, preferably 500% or higher, preferably 1000% or higher than the MFI value of the negative control under the measurement conditions described herein.

[0070] In one aspect, for a bispecific antigen-binding molecule (antibody), the anti-HLA-DQ2.5 molecule (antibody) having "binding activity" to a specific antigen has an MFI value that is 3% or higher, preferably 6% or higher, preferably 10% or higher, preferably 20% or higher when the MFI value of IC17dK is considered as zero percent and the MFI value of DQN0139bb is considered as 100% under the measurement conditions described herein.

[0071] When referring specifically to connection specificity, "connection activity" can be rewritten as "specific connection activity". The anti-HLA-DQ2.5 antigen-binding molecules (antibodies) of the invention have a dissociation constant (Kd) of 5 × 10-7M or less, preferably 4 × 10-7M or less, preferably 3 × 10-7M or less, preferably 2 × 10-7M or less, preferably 1 × 10-7M or less, preferably 9 × 10-8M or less, preferably 8 × 10-8M or less, preferably 7 × 10-8M or less, preferably 6 × 10-8M or less, preferably 5 × 10-8M or less, preferably 4 × 10-8M or less, preferably 3 × 10-8M or less, preferably 2 × 10-8M or less, preferably 1 × 10-8M or less, preferably 9 × 10-9M or less, preferably 8 × 10-9M or less, preferably 7 × 10-9M or less, preferably 7 × 10-9M or less, preferably 6 × 10-9M or less, preferably 5 × 10-9M or less, preferably 4 × 10-9M or less, preferably 3 × 10-9M or less, preferably 2 × 10-9M or less, for binding to one or more complexes formed by HLA-DQ2.5 and a gluten peptide as described herein.

[0072] A suitable multispecific antigen-binding molecule of the present invention comprises the following: (1) a portion / domain comprising an antibody variable region with binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide(s); (2) a portion / domain comprising an antibody variable region with binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide; and (3) A portion / domain comprising an Fc region with reduced binding activity to the above-mentioned Fc gamma receptor, without limitation to its structure. In the present invention, each of the above-mentioned domains can be directly linked by peptide bonds. For example, when using F(ab')2 as the domain containing an antibody variable region of (1) and (2) and these Fc regions as the domain containing an Fc region with reduced Fc gamma receptor binding activity of (3), polypeptides formed by linking the antibody variable region-containing domains of (1) and (2) and the Fc region-containing domain of (3) by peptide bonds form an antibody structure. Such antibodies can be produced by purifying from the culture medium of the above-mentioned hybridoma as well as by purifying antibodies from the culture medium of the desired host cells stably carrying polynucleotides encoding the antibody-forming polypeptides.

[0073] Examples of the preferred antibody H chain variable region present in the antibody variable region having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide include any of the antibody H chain variable regions described herein, or antibody H chain variable regions having CDR sequences whose amino acid sequences CDR1, CDR2 and CDR3 are identical to the amino acid sequences CDR1, CDR2 and CDR3 present in the H chain variable regions described herein, or antibody H chain variable regions that are functionally equivalent to the aforementioned variable regions.

[0074] Examples of a preferred antibody variable region having T-cell receptor complex binding activity in the present invention include antibody variable regions having HLA-DQ2.5 binding activity in the form of a complex(s) with gluten peptide(s). Examples of the antibody H chain variable region contained in such antibody variable regions include any of the antibody H chain variable regions described herein, or antibody H chain variable regions having CDR sequences in which the amino acid sequences of CDR1, CDR2 and CDR3 are identical to the amino acid sequences of CDR1, CDR2 and CDR3 contained in the H chain variable regions described herein, or antibody H chain variable regions that are functionally equivalent to the aforementioned variable regions.

[0075] In the present invention, the term "functionally equivalent" means that the binding affinities for an antigen are equivalent, or in other words, it means that the neutralizing activities against cells expressing HLA-DQ2.5 / gluten peptide (or tissues containing these cells) are equivalent when used as a multispecific antigen-binding molecule. The binding affinity and neutralizing activity can be measured as described herein. The cells used to measure activity may be the HLA-DQ2.5 / gluten peptide expressing cell of interest (or tissues containing these cells) and any suitable cell line may be used. In the case of antibody constant regions, this term may mean that the reductions in Fc gamma receptor binding activity are equivalent.

[0076] For example, an antibody H chain variable region that is functionally equivalent to the antibody H chain variable region described herein (i.e., the parent H chain variable region) means that this region, when combined with the antibody L chain variable region described herein that forms a pair with the parent H chain, has the same binding affinity, or alternatively, when used for a multispecific antigen-binding molecule, this region has the same neutralizing activity toward cells expressing HLA-DQ2.5 / gluten peptide (or tissue containing these cells).Furthermore, an antibody L chain variable region is functionally equivalent to the antibody L chain variable region described herein (i.e., the original L chain variable region) in the sense that this region has the same binding affinity when combined with the antibody H chain variable region that forms a pair with the original L chain, or alternatively, when used for a multispecific antigen-binding molecule, this region has the same neutralizing activity toward cells expressing HLA-DQ2.5 / gluten peptide (or tissue containing these cells).

[0077] The term "equivalent" does not necessarily mean the same degree of activity and the activity may be increased. In particular, for antigen binding affinity, examples include those in which the value (KD value / parent KD value) obtained by comparing with the binding affinity of the variable region of the antibody as a control (parent KD value) is 1.5 or less. The KD value / parent KD value is preferably 1.3 or less, more preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. While there is no lower limit, examples include 1-10, 2-10, 3-10, 4-10, 5-10, or 6-10. Specifically, in the present invention, the KD value / parent KD value is preferably 10-6 to 1.5 x 10-0, preferably 10-6 to 1.5 x 10-1, even more preferably 10-6 to 1.5 x 10-2, and even more preferably 10-6 to 1.5 x 10-3.

[0078] In relation to a portion / domain comprising an antibody variable region having binding activity to HLA-DQ2.5 / gluten peptide, the KD value relative to HLA-DQ2.5 / gluten peptide may be, for example, 2 x 10-8 M or less, 1 x 10-8 M or less, 9 x 10-9 M or less, 8 x 10-9 M or less, 7 x 10-9 M or less, 6 x 10-9 M or less, 5 x 10-9 M or less, 4 x 10-9 M or less, 3 x 10-9 M or less, 2 x 10-9 M or less, or 1 x 10-9 M or less.

[0079] In the present invention, antibody variable regions that are "functionally equivalent" are not limited to antibody H chain and / or antibody L chain variable regions that meet the above conditions. Examples of such antibody variable regions include regions generated by substituting, deleting, adding, and / or inserting one or more amino acids (e.g., 1, 2, 3, 4, 5, or 10 amino acids) in the amino acid sequences of the variable regions of Tables 1 to 3 listed above. A method for introducing one or more substitutions, deletions, additions, and / or insertions into an amino acid sequence is well known to those skilled in the art, which is a method for introducing mutations into proteins. For example, those skilled in the art can prepare variable regions that are functionally equivalent to antibody variable regions that have the above functions by appropriately introducing mutations into amino acid sequences using methods such as site-directed mutagenesis (Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and Nakagawa, M.(1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275؛ Zoller, M.J., and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500؛ Kramer, W., Drutsa, V., Jansen, H.W., Kramer, B., Pflugfelder, M., and Fritz, H.J. (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456؛ Kramer, W., and Fritz, H.J. (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367؛ و Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad. Sci. U S A. 82, 488-492).

[0080] When an amino acid residue changes, the amino acid preferentially mutates to a different amino acid that retains the properties of the amino acid's side chain. Examples of amino acid side chain properties include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), amino acids containing aliphatic side chains (G, A, V, L, I, and P), amino acids containing side chains containing hydroxyl groups (S, T, and Y), amino acids containing side chains containing sulfur atoms (C and M), amino acids containing side chains containing carboxylic acids and amides (D, N, E, and Q), amino acids containing basic side chains (R, K, and H), and amino acids containing aromatic side chains (H, F, Y, and W) (amino acids are indicated by one-letter codes in parentheses). Amino acid substitutions within each of these groups are called conservative substitutions.It has previously been shown that a polypeptide containing a modified amino acid sequence in which one or more amino acid residues in a given amino acid sequence are deleted, added, and / or replaced with other amino acids can retain the original biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA; (1984) 81: 5662-6; Zoller, MJ and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984) 224: 1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6409-13). The variable regions of the present invention containing such amino acid changes have an amino acid sequence similarity of at least 70%, preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, but more preferably at least 90% and most preferably at least 95% to the amino acid sequence of the CDR sequences, FR sequences or the entire variable regions of the variable region before modification.Here, sequence similarity is defined as the percentage of residues identical to those in the original amino acid sequence of the H chain variable region or the L chain variable region determined after the sequences are aligned, and gaps are inserted appropriately to maximize sequence similarity as necessary. Amino acid sequence similarity can be determined by the method described below.

[0081] Furthermore, a "functionally equivalent antibody variable region" can be obtained, for example, from nucleic acids that hybridize under stringent conditions with nucleic acids comprising a nucleotide sequence encoding the amino acid sequence of a variable region in Tables 1 to 3 listed above. Stringent hybridization conditions for isolating a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of a variable region are, for example, conditions of 6 M urea, 0.4% SDS, 0.5x SSC and 37°C, or hybridization conditions of equivalent stringency. Isolation of nucleic acids with much higher homology can be expected with more stringent conditions, for example, conditions of 6 M urea, 0.4% SDS, 0.1x SSC and 42°C. Post-hybridization wash conditions include, for example, washing using 0.5x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate at pH 7.0) and 0.1% SDS at 60°C, with a more preferred wash using 0.2x SSC and 0.1% SDS at 60°C, even more preferred wash using 0.2x SSC and 0.1% SDS at 62°C, even more preferably washing with 0.2x SSC and 0.1% SDS at 65°C, and more preferably washing with 0.1x SSC and 0.1% SDS at 65°C. The sequence of the isolated nucleic acids can be determined by known methods as described below. The overall nucleotide sequence identity of the isolated nucleic acid is at least 50% or greater, preferably 70% or greater, and more preferably 90% or greater (e.g., 95%, 96%, 97%, 98%, 99% or greater) sequence similarity.

[0082] Nucleic acids that hybridize under stringent conditions to a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of a variable region can also be isolated using the methods described above using hybridization techniques. Gene amplification methods such as polymerase chain reaction (PCR) which utilize primers synthesized based on nucleotide sequence information encoding the amino acid sequence of the variable region.

[0083] The similarity of one nucleotide sequence or amino acid sequence to another can be determined using the BLAST algorithm of Carlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7). Programs called BLASTN and BLASTX were developed based on this algorithm (Altschul et al., J. Mol. Biol. (1990) 215: 403-10). For analysis of nucleotide sequences by BLASTN based on BLAST, the parameters are set to, for example, score = 100 and word length = 12. On the other hand, the parameters used for analysis of amino acid sequences by BLASTX based on BLAST include, for example, score = 50 and word length = 3. When using the BLAST and Gapped BLAST programs, the default parameters for each program are used. Specific techniques for such analyses are known in the art (see the National Center for Biotechnology Information (NCBI) website, Base Local Alignment Search Tool (BLAST); http: / / www.ncbi.nlm.nih.gov).

[0084] The Fc region present in the multispecific antigen-binding molecule of the present invention is not limited as long as it is an Fc region with reduced binding activity to the Fc gamma receptor, examples of a preferred Fc region of the present invention include a combination of the Fc region portions described herein.

[0085] Examples of preferred multispecific antigen-binding molecules of the present invention include bispecific antibodies comprising a first variable region of an antibody having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide or peptides and a second variable region of an antibody having binding activity to HLA-DQ2.5 in the form of a complex(s) with gluten peptide(s). Examples of such bispecific antibodies include bispecific antibodies comprising the H and L chains described herein and bispecific antibodies that bind to an epitope that overlaps with an epitope bound by the above antibodies and that contain an Fc region with reduced binding activity to the Fc gamma receptor.

[0086] Whether an antibody recognizes an epitope that overlaps with an epitope recognized by another antibody can be confirmed by competition between two antibodies against the epitope. Competition between antibodies can be assessed by competitive binding assays using tools such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer (FRET) and fluorometric microassay technology (FMAT (registered trademark). The amount of an antibody bound to an antigen is indirectly related to the binding ability of a candidate competing antibody (a test antibody) that competitively binds to the overlapping epitope; in other words, as the amount or affinity of a test antibody for the overlapping epitope increases, the amount of antibody bound to the antigen decreases and the amount of test antibody bound to the antigen increases. Specifically, the antibody is appropriately labeled and the antibody to be evaluated is added to the antigen simultaneously, and the bound antibody is thus detected using the label.The amount of antibody bound to the antigen can be easily determined by labeling the antibody in advance. This label is not particularly limited, and the labeling method is selected according to the assay technique used. Specifically, the labeling method includes fluorescent labeling, radio labeling, enzyme labeling, and the like.

[0087] For example, fluorescently labeled antibody and unlabeled antibody or test antibody are simultaneously added to beads immobilized with HLA-DQ2.5 / gluten peptide, and the labeled antibody is detected by fluorometric microvolume assay technology.

[0088] Here, "antibody that binds to an overlapping epitope" refers to a test antibody that can reduce the amount of bound labeled antibody by at least 50% at a concentration that is typically 100-fold, preferably 80-fold, more preferably 50-fold, even more preferably 30-fold, and still more preferably 10-fold higher than the concentration at which the unlabeled antibody reduces the binding amount of the labeled antibody by 50% (IC50).

[0089] Multispecific antigen-binding molecules that have antigen-binding sites of antibodies that bind to epitopes that overlap with epitopes bound by the aforementioned antibodies can have excellent binding activity or neutralizing activity.

[0090] The multispecific antigen-binding molecules of the present invention are produced by a similar method to the production of the recombinant antibodies mentioned herein.

[0091] In certain embodiments, any one or more amino acids of an anti-HLA-DQ2.5 antigen-binding molecule (antibody) provided above are substituted in any of the constant and / or variable regions or domains of the heavy chain and / or light chain.

[0092] In certain embodiments, the substitutions are conservative substitutions as provided herein.

[0093] Human antibodies In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various 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).

[0094] Human antibodies may be produced by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are integrated extrachromosomally or randomly into the animal's chromosomes. In these transgenic mice, the endogenous immunoglobulin loci are generally 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 describing XENOMOUSETM technology; U.S. Patent No. 5,770,429 describing HUMAB (registered trademark) technology; U.S. Patent No. 7,041,870 describing KM MOUSE (registered trademark) technology; and U.S. Patent Application Publication No. US 2007 / 0061900 describing VELOCIMOUSE (registered trademark) technology. The human variable regions of intact antibodies produced by such animals may be further modified, for example, by fusion with a different human constant region.

[0095] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, for example, 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 produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 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).

[0096] Human antibodies may also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. This variable domain sequence may then be fused to a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0097] Chimeric and humanized antibodies In certain embodiments, an antibody provided herein is a chimeric antibody. Some 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 class or subclass of the parent antibody has been changed. Chimeric antibodies comprise antigen-binding fragments thereof.

[0098] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or binding affinity of the antibody.

[0099] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and, 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) binding); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "exfoliation"). Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR mixing"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR mixing).

[0100] Human framework regions that may be used for humanization include: framework regions selected using a "best fit" approach (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a specific subgroup of light 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., 151:2623 (1993)); human mature (sotically mutated) framework regions or human component 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)), but are not limited to these.

[0101] In each of the above embodiments, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is humanized. In one embodiment, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises HVRs as in any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises HVRs as in any of the above embodiments and further comprises the FR1, FR2, FR3, or FR4 sequence as shown herein. Herein, the "human framework" may also be referred to as a "humanized framework" which focuses on the fact that the antibody is humanized.

[0102] In some embodiments, a multispecific antigen-binding molecule of the invention comprises: (i) a first antigen-binding moiety having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide; and (ii) a second antigen-binding moiety that has HLA-DQ2.5 binding activity in the form of a complex with a gluten peptide; in which the antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides, wherein at least one of the gluten peptides in the complexes bound by the first antigen-binding moiety is different from at least one of the gluten peptides in the complexes bound by the second antigen-binding moiety; and wherein the antigen-binding molecule has essentially no binding activity to one or both of HLA-DQ2.5 positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5 or HLA-DQ2.2, in which the antigen-binding molecule has been humanized and wherein one or more amino acids in a heavy chain and / or light chain and / or variable region in the first antigen-binding portion and / or the second antigen-binding portion of the multispecific antigen-binding molecule are altered.

[0103] In some embodiments, in the multispecific antigen-binding molecule, one or more amino acids in a heavy chain and / or a light chain of the first antigen-binding portion and / or the second antigen-binding portion of the multispecific antigen-binding molecule are substituted. In some embodiments, the multispecific antigen-binding molecule comprises at least one amino acid substitution in a variable region of the heavy chain; at least one amino acid substitution in a constant region of the heavy chain; at least one amino acid substitution in a variable region of the light chain; and at least one amino acid substitution in a constant region of the light chain. In some embodiments, the gluten peptide is a predominant immunological peptide associated with celiac disease. In some embodiments, the gluten peptide is selected from the group consisting of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2, and 26-mer gliadin peptide. In some embodiments, the gluten peptide(s) are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19 or all of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, Secalin peptide 1, secalin peptide 2, and 26-mer peptide are gliadin. In some embodiments, the gluten peptide is selected from the group consisting of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2, and 26-mer gliadin peptide. In some embodiments, the gluten peptide(s) are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18 or all of: 33-mer gliadin peptide, alpha-1 gliadin peptide, alpha-2 gliadin peptide, gamma-1 gliadin peptide, omega-1 gliadin peptide, omega-2 gliadin peptide, hordein BC peptide, alpha-3 gliadin peptide, alpha-1b gliadin peptide, gamma-4a gliadin peptide, gamma-4b gliadin peptide, avenin peptide 1, avenin peptide 2, avenin peptide 3, hordein peptide 1, hordein peptide 2, secalin peptide 1, secalin peptide 2 and The 26-mer peptide is gliadin. In some embodiments, the multispecific antigen-binding molecule has essentially no binding activity to the gluten peptide(s) itself. In this context, the terms "self" and "self" refer to a state in which the gluten peptide(s) do not form a complex with HLA-DQ2.5. In some embodiments, the multispecific antigen-binding molecule has essentially no binding activity to HLA-DQ2.5 in the form of a complex with an irrelevant peptide, wherein the irrelevant peptide is at least one peptide selected from the group consisting of: CLIP peptide (hCLIP), hepatitis B virus 1 peptide, Salmonella peptide, Mycobacterium bovis peptide, and thyroperoxidase peptide. In some embodiments, the multispecific antigen-binding molecule has essentially no binding activity to HLA-DQ2.5 in the form of complexes with unrelated peptides, wherein the unrelated peptides are all of: CLIP peptide (hCLIP), hepatitis B virus 1 peptide, Salmonella peptide, Mycobacterium bovis peptide, and thyroperoxidase peptide. In some embodiments, the antigen-binding molecule has improved binding activity to the complex formed by HLA-DQ2.5 and gluten peptide compared to the prior humanization and modification. In this context, "improved binding activity" means that the antigen-binding molecule binds to the complex formed by HLA-DQ2.5 and gluten peptide more strongly than the prior antibody before the modifications, i.e., humanization and modification. In some embodiments, the antigen-binding molecule has improved cross-reactivity to gluten peptides compared to the previous humanization and modification. In some embodiments, the gluten peptides are omega-2 gliadin peptide, hordein BC peptide, gamma-1 gliadin peptide, gamma-2 gliadin peptide, gamma-4a gliadin peptide, and gamma-4d gliadin peptide. In this context, "improved cross-reactivity to gluten peptides" means that the antigen-binding molecule binds to or exhibits neutralizing activity against more gluten peptides than the previous antibody before the modifications, i.e., humanization and modification.

[0104] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a heavy chain variable domain (VH) sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to the amino acid sequence of the heavy chain variable domain (VH) sequence disclosed herein. In certain embodiments, a VH sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity includes substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference (i.e., parent) sequence, but an anti-HLA-DQ2.5 antigen-binding molecule (antibody) containing that sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids relative to the reference (i.e., parent) sequence are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a VH sequence disclosed herein or a sequence comprising a post-translational modification thereof.In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 disclosed herein, (b) HVR-H2 disclosed herein and (c) HVR-H3 disclosed herein. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0105] Amino acids in the amino acid sequences of the present invention may be post-translationally modified (for example, the conversion of an N-terminal glutamine to a pyroglutamic acid by pyroglutamylation is known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the amino acid sequences of the present invention.

[0106] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is provided, wherein the molecule (antibody) comprises a light chain variable domain (VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to the amino acid sequence of the light chain variable domain (VL) disclosed herein. In certain embodiments, a VL sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity includes substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference (i.e., parent) sequence, but an anti-HLA-DQ2.5 antigen-binding molecule (antibody) containing that sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids relative to the reference (i.e., parent) sequence are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a VL sequence disclosed herein or a sequence comprising a post-translational modification thereof.In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 disclosed herein, (b) HVR-L2 disclosed herein and (c) HVR-L3 disclosed herein. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0107] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is provided, wherein the molecule / antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the molecule / antibody comprises the VH sequence disclosed herein or a sequence comprising a post-translational modification thereof and comprises the VL sequence disclosed herein or a sequence comprising a post-translational modification thereof. Post-translational modifications include, but are not limited to, modifying a glutamine or glutamate at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0108] In another aspect, the invention provides an antigen-binding molecule (antibody) that binds to the same epitope as the anti-HLA-DQ2.5 antigen-binding molecule (antibody) provided herein. For example, in certain embodiments, a molecule / antibody is provided that binds to the same epitope as any of the molecules / antibodies described herein. In certain embodiments, a molecule / antibody is provided that binds to an epitope within a fragment of HLA-DQ2.5 consisting of about 8 to 17 amino acids or within a complex formed by HLA-DQ2.5 and a gluten peptide. In this context, the gluten peptide may be any of the gluten peptides described herein.

[0109] In another aspect, the invention provides an antigen-binding molecule (antibody) that competes with another antigen-binding molecule (antibody) for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide. For example, in certain embodiments, a molecule / antibody is provided that competes with any of the molecules / antibodies described herein for binding to HLA-DQ2.5 or a complex formed by HLA-DQ2.5 and a gluten peptide. In this context, the gluten peptide may be any of the gluten peptides described herein.

[0110] In another aspect of the invention, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) according to any of the above embodiments is a monoclonal antigen-binding molecule (antibody), including a chimeric, human or humanized antigen-binding molecule (antibody). In preferred embodiments, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) of the invention is a humanized antigen-binding molecule (antibody). In one embodiment, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody or another class or isotype of antibody as defined herein.

[0111] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) according to any of the above embodiments may have any of the features described below, alone or in combination:

[0112] Antibody affinity In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of 1 micromolar (microM) or less, 100 nanomolar or less, 10 nanomolar or less, 1 nanomolar or less, 0.1 nanomolar or less, 0.01 nanomolar or less, or 0.001 nanomolar or less (e.g., 8-10 M or less, e.g., from 8-10 M to 10-13 M, e.g., from 9-10 M to 10-13 M).

[0113] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with a Fab version of an antibody of interest and its antigen. For example, the binding affinity of solution Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing the bound antigen with a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 micrograms (μg) / mL of anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).In a non-absorbent plate (Nunc #269620), 100 picomolar or 26 picomolar [125I]-antigen was mixed with serial dilutions of a Fab of interest (e.g., as evaluated for the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest was then incubated overnight. However, incubation may be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is achieved. The mixtures were then transferred to the absorbent plate for incubation at room temperature (e.g., for one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plates were dry, 150 microliters (microliters) / well of scintillator (MICROSCINT-20TM; Packard) was added and the plates were counted on a TOPCOUNTTM gamma counter (Packard) for ten minutes. Concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for use in competitive binding assays.

[0114] In another embodiment, Kd was measured using the BIACORE (registered trademark) surface plasmon resonance method. For example, an assay was performed using a BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at approximately 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted with 10 mM sodium acetate, pH 4.8 to 5 μg / mL (approximately 0.2 μM) before injection at a flow rate of 5 μL / min to yield approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) was injected at 25°C at a flow rate of approximately 25 μL / min. Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model (BIACORE Evaluation Software (registered trademark) version 3.2) by simultaneous fitting of the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio of koff / kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 106M-1s-1 by the surface plasmon resonance method above, the association rate can be determined using the fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C from 20 nM of an anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured in a spectrophotometer, such as a stopped-flow spectrophotometer (Aviv Instruments) or a SLM-AMINCOTM 8000 Series spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0115] Antibody fragments In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0116] Diabodies are antibody fragments with two antigen-binding sites that may 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). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0117] Single-domain antibodies are antibody fragments that comprise 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, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. B1 6,248,516).

[0118] Antibody fragments can be made by various techniques, including, but not limited to, proteolytic digestion of an intact antibody as well as production in recombinant host cells (such as Escherichia coli or phage), as described herein.

[0119] Species of the Fc region In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, resulting in an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0120] Antibodies with an extended half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that enhance the binding of the Fc region to FcRn. These Fc variants include those that have been substituted at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 3237,376 380, 382, ​​413, 424 or 434, e.g., substitution of residue 434 of the Fc region (U.S. Patent No. 7,371,826). 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 on other examples of Fc region variants.

[0121] Fc area The term "Fc region" or "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes both native sequence Fc regions and variable Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, a C-terminal lysine (Lys447) or glycine-lysine (residues 446 to 447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the European Union numbering system, also called the European Union 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.

[0122] Fc receptor The term "Fc receptor" or "FcR" refers to a receptor that binds the Fc region of an antibody. In some embodiments, an FcR is a naturally occurring human FcR. In some embodiments, an FcR binds an IgG antibody (gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subtypes, including allelic variants and alternative forms of those receptors. Fc gamma RII receptors include Fc gamma RIIA (an "activating receptor") and Fc gamma RIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc gamma RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The Fc gamma inhibitory receptor RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For example, FcRs are described 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). Other FcRs, including those identified in the future, are referred to herein as "FcRs".

[0123] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and for the regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding 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.)).

[0124] The in vivo binding to human FcRn and the plasma half-life of high-affinity binding polypeptides to human FcRn can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in mammals administered variable Fc region polypeptides. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcRs. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0125] Fc gamma receptor Fc gamma receptor refers to a receptor that is capable of binding to the Fc domain of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies and includes all members of the family of proteins that are primarily encoded by a single Fc gamma receptor gene. In humans, the family includes Fc gamma RI (CD64) including the isoforms Fc gamma RIa, Fc gamma RIb, and Fc gamma RIc; Fc gamma RII (CD32) including the isoforms Fc gamma RIIa (including allotypes H131 and R131), Fc gamma RIIb (including Fc gamma RIIb-1 and Fc gamma RIIb-2), and Fc gamma RIIc; and Fc gamma RIII (CD16) including the isoforms Fc gamma RIIIa (including allotypes V158 and F158) and Fc gamma RIIIb (including allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2); and all unidentified human Fc gamma receptors, Fc gamma receptor isoforms and allotypes thereof. However, Fc gamma receptor is not limited to these examples. Without being limited thereto, Fc gamma receptor includes receptors derived from human, mouse, rat, rabbit and monkey. Fc gamma receptor may be derived from any living organism.Mouse Fc gamma receptor includes, without limitation, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16) and Fc gamma RIII-2 (CD16-2) as well as all unidentified mouse Fc gamma receptors, Fc gamma receptor isoforms and allotypes thereof. Such preferred Fc gamma receptors include, for example, human Fc gamma RI (CD64), Fc gamma RIIA (CD32), Fc gamma RIIB (CD32), Fc gamma RIIIA (CD16) and / or Fc gamma RIIIB (CD16). Whether a gamma Fc receptor has binding activity to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be assessed by the ALPHA screen (enhanced fluorescent proximity homogeneity assay), the surface plasmon resonance (SPR)-based BIACORE method, and other methods (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to the FACS and ELISA formats described above.

[0126] Meanwhile, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to the Fc domain of an antibody and forms an Fc / Fc ligand complex. This molecule may be derived from any organism. Binding of an Fc ligand to Fc preferably induces one or more effector functions. These Fc ligands include Fc receptors, Fc gamma receptor, Fc alpha receptor, Fc beta receptor, FcRn, C1q and C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, staphylococcal protein G, and viral Fc gamma receptors. Fc ligands also include Fc receptor homologues (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136) which are a family of Fc receptors homologous to the Fc gamma receptor. Fc ligands also include unknown molecules that bind to Fc.

[0127] Fc gamma receptor binding activity Impairment of Fc domain binding activity to any of the Fc gamma receptors, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and / or Fc gamma RIIIB is assessed using the FACS and ELISA formats described above, as well as the ALPHA screen (fluorescently amplified proximity homogeneity assay) and the surface plasmon resonance (SPR)-based BIACORE method.

[0128] ALPHA display is performed by ALPHA technology based on the principle described below using two types of beads: donor and acceptor beads. A fluorescent signal is detected only when molecules attached to the donor beads interact biologically with molecules attached to the acceptor beads and when the two beads are placed in close proximity to each other. Excited by a laser beam, the photosensitizer in a donor bead converts the oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor beads and reaches the acceptor beads located in close proximity, a fluorescent chemical reaction is induced within the acceptor beads. This reaction ultimately results in the emission of light. If the molecules attached to the donor beads do not interact with the molecules attached to the acceptor beads, the singlet oxygen produced by the donor beads does not reach the acceptor beads and the chemical fluorescent reaction does not occur.

[0129] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on donor beads and a glutathione S-transferase (GST)-labeled Fc gamma receptor is immobilized on acceptor beads. In the absence of an antigen-binding molecule or antibody containing a competing mutant Fc domain, the Fc gamma receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, resulting in a signal between 520 and 620 nm. An antigen-binding molecule or antibody containing an unlabeled mutant Fc domain competes with an antigen-binding molecule or antibody containing a wild-type Fc domain for interaction with the Fc gamma receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from competition. Methods for biotinylating antigen-binding molecules or antibodies, such as those known to be biotinylated using sulfo-NHS-biotin or the like.Suitable methods for adding a GST tag to an Fc gamma receptor include methods that involve combining polypeptides encoding the Fc gamma receptor and GST in-frame, expressing the fusion gene using cells transfected with the gene vector, and then purifying using a glutathione column. The induced signal can be analyzed, for example, by fitting a one-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0130] One of the materials to observe their interaction is as a ligand on a thin gold layer of an immobilized sensor chip. When light is shone on the back surface of the sensor chip so that total reflection occurs at the interface between the thin gold layer and the glass, the intensity of the reflected light is partially reduced at a specific location (the SPR signal). Another material to observe their interaction is as an analyte injected onto the surface of the sensor chip. The mass of the immobilized ligand molecule increases as the analyte binds to the ligand. This causes a change in the refractive index of the solvent on the surface of the sensor chip. The change in the refractive index causes a shift in the position of the SPR signal (inversely, it returns the signal resolution to the original position). In the Biacore system, the displacement value described above (i.e., the change in mass on the surface of the sensor chip) is plotted on the vertical axis and thus the change in mass over time is shown as measured data (sensorgram). Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and the affinity (KD) is determined from the ratio between these two constants.The inhibition assay is preferably used in the BIACORE method. Examples of this inhibition assay are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0131] Fc region with reduced binding activity to the Fc gamma receptor Here, "reduced Fc gamma receptor binding activity" means that, for example, based on the analytical method described above, the competitive activity of a molecule or antibody binding to the test antigen is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and 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 of the competitive activity of a molecule or antibody binding to the control antigen.

[0132] Antigen-binding molecules or antibodies comprising the Fc domain of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be suitably used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in RefSeq Accession No. AAC82527.1, RefSeq Accession No. AAB59393.1, RefSeq Accession No. CAA27268.1 and RefSeq Accession No. AAB59394.1. In addition, when an antigen-binding molecule or antibody comprising an Fc domain mutant of an antibody of a particular isotype was used as a test material, the effect of the mutation on the Fc gamma receptor binding activity was evaluated using an antigen-binding molecule or antibody comprising an Fc domain mutant of the same isotype as a control. As described above, antigen-binding molecules or antibodies comprising an Fc domain mutant whose Fc gamma receptor binding activity is reduced are suitably prepared.

[0133] These known mutants include, for example, mutants with deletions of amino acids 231A-238S (EU numbering) (WO 2009 / 011941) as well as the 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).

[0134] In particular, preferred antigen-binding molecules or antibodies include those comprising an Fc domain with a mutation (e.g., substitution) of at least one amino acid selected from the following amino acid positions: 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), in the amino acids constituting the Fc domain of an antibody of a are isotype specific. The isotype of the antibody from which the Fc domain is derived is not limited, and a suitable Fc domain derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used. Fc domains derived from IgG1 antibodies are preferably used.

[0135] In the present invention, SG181 may be used as a gamma Fc receptor, a silenced Fc that reduces Fc binding to gamma Fc receptors. In some embodiments, SG181.S3n (SEQ ID NO: 101) and SG181.S3p (SEQ ID NO: 102) may be used as heavy chain constant region sequences. These heavy chain constant region sequences may be included in antigen-binding molecules or antibodies of the present invention to reduce binding to gamma Fc receptors.

[0136] Preferred antigen-binding molecules or antibodies are, for example, those consisting of an Fc domain in which each amino acid at positions 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids constituting the Fc domain of an IgG1 antibody is replaced by an amino acid at the corresponding position in EU numbering in the corresponding IgG2 or IgG4.

[0137] In some embodiments, the multispecific antigen-binding molecule of the invention further comprises an Fc domain that exhibits reduced binding affinity for the human Fc gamma receptor compared to a human IgG1 Fc domain. In some embodiments, in the multispecific antigen-binding molecule, the Fc domain comprises Arg at position 235 and Arg at position 236, where the amino acid positions are numbered according to the European Union numbering.

[0138] Setting the H-chain / L-chain relationship and other features Another embodiment of the present invention relates to an antigen-binding molecule in which the heavy chain and light chain association are regulated, a method for making an antigen-binding molecule in which the heavy chain and light chain association are regulated, and a method for regulating the heavy chain and light chain association in an antigen-binding molecule.

[0139] The antigen-binding molecule of the present invention relates to an antigen-binding molecule in which the association of the heavy chain and the light chain is regulated, wherein the heavy chain and the light chain constituting the antigen-binding molecule are a combination of the desired heavy chain and light chain, and wherein amino acid residues at specific locations in the heavy chain constant region (CH1) and the light chain constant region (CL) mutually electrically repel amino acid residues (having the same charge).

[0140] In the present invention, by converting amino acid residues at specific locations in CH1 and CL from the undesirable combination of heavy chain and light chain to amino acid residues that are electrically mutually repelling (i.e., have the same charge), the formation of undesirable combinations can be prevented by utilizing this charge repulsion of the heavy chain and light chain, thereby forming the desired combination of heavy chain and light chain.

[0141] In the present invention, the terms "association adjustment" and "association is adjusted" refer to adjustment to achieve desired association conditions, and specifically refer to adjustment in such a way that undesirable associations are not created between the heavy chain and the light chain.

[0142] In the present invention, the term "interface" generally refers to the level of association resulting from the association (interaction), and the amino acid residues that form the interface are usually one or more amino acid residues present in the polypeptide regions participating in the association, and are preferably amino acid residues that come into close proximity to each other during the association and participate in the interaction. In particular, this interaction includes, for example, cases where the amino acid residues come into close proximity during the association to form hydrogen bonds, electrostatic interactions, or salt bridges with each other.

[0143] In the present invention, the term "amino acid residues forming a linker" specifically refers to amino acid residues present in the polypeptide region that form the linker. For example, polypeptide regions that form the linker refer to polypeptide regions responsible for selective binding between molecules such as antigen-binding molecules (e.g., antibodies), ligands, receptors, or substrates. Specifically, in antigen-binding molecules, such examples include heavy chain constant regions, heavy chain variable regions, light chain constant regions, and light chain variable regions.

[0144] In a preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding molecule has amino acid residues at specific positions in CH1 and CL of the undesired combination of heavy chain and light chain prior to association that electrically repel each other (i.e., have the same charge).

[0145] It is thought that by changing the amino acid residues in the aforementioned antigen-binding molecule to amino acid residues that are electrically repulsive (have the same charge), the association of these amino acid residues is inhibited by the repulsive force of electrical charges.

[0146] Therefore, in the aforementioned antigen-binding molecule, the modified amino acid residues are preferably amino acid residues that, upon association, come close to each other in the polypeptide regions that form the interface.

[0147] The amino acid residues that come into close proximity during the association can be determined by analyzing the three-dimensional structure of a polypeptide and examining the amino acid sequences of the polypeptide regions that form a linker during the polypeptide association. The amino acid residues in the linker that come into mutual proximity are preferred targets for "modification" in the antigen-binding molecule of the present invention.

[0148] Some amino acids have an electrical charge. In general, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids). Aspartic acid (D), glutamic acid (E), and the like are known as negatively charged amino acids (negatively charged amino acids). In addition, alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), and the like are known as uncharged or nonpolar amino acids.

[0149] Therefore, amino acids that are electrically mutually repelling (have the same charge) in the present invention refer to the following: (1) Amino acids in which one of the amino acids is a positively charged amino acid and the other amino acid is also a positively charged amino acid, and (2) Amino acids in which one of the amino acids is a negatively charged amino acid and the other amino acid is also a negatively charged amino acid.

[0150] Examples of amino acid modifications include modification of an uncharged or nonpolar amino acid to a positively charged amino acid, modification of an uncharged or nonpolar amino acid to a negatively charged amino acid, modification of a positively charged amino acid to a negatively charged amino acid, and modification of a negatively charged amino acid to a positively charged amino acid. In addition, modification of an uncharged or nonpolar amino acid to a different uncharged or nonpolar amino acid, modification of a positively charged amino acid to a different positively charged amino acid, and modification of a negatively charged amino acid to a different negatively charged amino acid are also included in the amino acid modifications of the present invention.

[0151] Amino acid modifications in the present invention include making one modification in each of the heavy and light chains or making multiple modifications in each of the heavy and light chains. Furthermore, the number of modifications added to the heavy and light chains may be the same or different.

[0152] Amino acid modifications in the present invention include multiple modifications to positively charged amino acids in the heavy chain or light chain and multiple modifications to negatively charged amino acids in the other chain. In addition, multiple modifications to positively charged amino acids as well as multiple modifications to negatively charged amino acids may be made in the same heavy chain or light chain. In these modifications, modifications to uncharged amino acids or nonpolar amino acids as well as modifications to uncharged amino acids or nonpolar amino acids may also be appropriately combined.

[0153] In modifications of this invention, for example, the amino acids in one of the chains can be used without modification, and in such cases, the heavy chain and light chain do not need to be modified, and only one of the chains may be modified.

[0154] The light chain constant region of the antigen-binding molecule of the present invention is preferably a human light chain constant region. Examples of the antibody light chain constant region include IgK (kappa), IgL1, IgL2, IgL3, IgL6 and IgL7 (lambda) type constant regions. The light chain constant region of the antigen-binding molecule of the present invention is not particularly limited. When using multiple types of light chains, the light chains may be different types of light chains, for example, kappa and lambda. Several allotype sequences obtained by genetic polymorphism are described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242 as human IgK (kappa) constant region and human IgL7 (lambda) constant region, and any of these may be used in the present invention.

[0155] Antibody constant regions, in particular, heavy chain constant regions, may be modified as necessary to improve the function or stability of an antigen-binding molecule. Examples of modifications to improve the function of an antigen-binding molecule include modifications that enhance or weaken the binding between an antigen-binding molecule and an Fc gamma receptor ("Fc gamma R"), modifications that enhance or weaken the binding between an antigen-binding molecule and FcRn, modifications that enhance or weaken the cytotoxic activity (such as ADCC activity and CDC activity) of an antigen-binding molecule, and the like. In addition, modifications that improve the heterogeneity of the antigen-binding molecule and modifications that improve non-immunogenicity and / or pharmacokinetics may also be included.

[0156] In addition, as a result of C-terminal sequence heterogeneity of the IgG antibody heavy chain, amidation of the C-terminal carboxyl group by deletion of the C-terminal amino acid, lysine residue, or by deletion of two C-terminal amino acids, glycine and lysine, has been reported (Anal. Biochem. 2007 Jan 1:360(1):75-83). Therefore, in the present invention, to reduce the heterogeneity of the C-terminus of the heavy chain, an IgG in which the C-terminal lysine or the C-terminal lysine and glycine are deleted is preferably used. Because their antigenicity is attenuated in the human body, chimeric and humanized antibodies using human-derived sequences are expected to be useful when administered to humans for therapeutic purposes or the like.

[0157] A preferred example of the antigen-binding molecule of the present invention is a heteromeric multimer having two or more CH1 types and two or more CL types. This heteromeric multimer preferably binds to two or more epitopes, and an example thereof is a multispecific antibody.

[0158] A preferred example of a multispecific antibody of the present invention is a bispecific antibody; therefore, an example of a preferred embodiment of the antigen-binding molecule of the present invention is a bispecific antibody that is composed of two types of heavy chains (a first heavy chain and a second heavy chain) and two types of light chains (a first light chain and a second light chain).

[0159] To more precisely describe the "bispecific antibodies" of the preferred embodiments of the antigen-binding molecules of the present invention, the above "first heavy chain" refers to one of the two heavy chains (H chains) constituting the antibody and the "second H chain" refers to the other H chain that is different from the first H chain; that is, of the two H chains, one of them can be arbitrarily defined as the first H chain and the other as the second H chain. Similarly, the "first light chain" refers to one of the two light chains (L chains) constituting the bispecific antibody and the "second L chain" refers to the other L chain that is different from the first L chain. Of the two L chains, one of them can be arbitrarily defined as the first L chain and the other as the second L chain. Typically, the first L chain and the first H chain are derived from the same antibody that binds to a specific antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody that binds to a specific antigen (or epitope).Here, the LH chain pair formed by the first H chain and the L chain is referred to as the first pair, and the LH chain pair formed by the second H chain and the second L chain is referred to as the second pair. The antigen (or epitope) used to produce the antibody from which the second pair is derived is preferably different from the antigen used to produce the antibody from which the first pair is derived. In particular, the antigens recognized by the first pair and the second pair may be the same, but these pairs preferably bind to different antigens (or epitopes). In this case, the H chains and L chains of the first pair and the second pair preferably have amino acid sequences that are different from each other. When the first pair and the second pair bind to different epitopes, the first pair and the second pair may recognize a completely different antigen, or they may recognize different locations (different epitopes) on the same antigen.In addition, one of them may recognize an antigen such as a protein, peptide, gene, or sugar, and the other may recognize cytotoxic substances such as radioactive substances, chemotherapeutic agents, or cell-derived toxins. However, when one wishes to produce antibodies that have pairs consisting of specific combinations of H chains and L chains, their H chains and L chains may be arbitrarily designated as the first pair and the second pair.

[0160] A more detailed description is provided below of a bispecific IgG antibody having two types of CH1 heavy chain constant regions (CH1-A and CH1-B) and two types of light chain constant regions (CL-A and CL-B); however, the present invention can be similarly applied to other antibodies.

[0161] When one wants to obtain a bispecific antibody that recognizes one epitope with the first CH1-A and the first CL-A and binds to another epitope by the second CH1-B and the second CL-B, theoretically there is a possibility that 10 types of antibody molecules will be produced when each of the four types of chains is expressed to produce that antibody.

[0162] In this case, the desired antibody molecules can be preferentially obtained if, for example, the association is adjusted such that the association between CH1-A and CL-B and / or between CH1-B and CL-A is inhibited.

[0163] An example is the modification of the amino acid residues forming a linker between CH1-A and CL-B to positively charged amino acid residues and the modification of the amino acid residues forming a linker between CH1-B and CL-A to negatively charged amino acid residues. As a result of these modifications, the undesired association between CH1-A and CL-B is inhibited because the amino acid residues forming the linker are both positively charged, and the association between CH1-B and CL-A is inhibited because the amino acid residues forming the linker are both negatively charged; therefore, the undesired association between CH1-A and CL-B and the association between CH1-B and CL-A are inhibited because the amino acid residues forming the links have the same charge. As a result, antibodies with the desired affinity between CH1-A and CL-A and the desired affinity between CH1-B and CL-B can be efficiently obtained.In addition, the desired association between CH1-A and CL-A is promoted because the amino acid residues that form the linker have different types of charges from each other; and the desired association between CH1-B and CL-B is also promoted because the amino acid residues that form the linker have different types of charges from each other. As a result, antibodies with the desired association can be efficiently obtained.

[0164] Another example is the modification of the amino acid residues forming the linker between CH1-A and CL-B to positively charged amino acid residues, when the amino acid residues forming the linker between CL-A and CH1-B are uncharged or nonpolar amino acids. As a result of this modification, the undesired association between CH1-A and CL-B is inhibited because the amino acid residues forming the linker are both positively charged. On the other hand, since the amino acid residues forming the links are amino acids that do not mutually electrically repel each other, the desired association between CH1-A and CL-A and the desired association between CH1-B and CL-B occur more easily than when the amino acids are electrically repel each other. As a result, antibodies having the desired association between CH1-A and CL-A and the desired association between CH1-B and CL-B can be efficiently obtained.Meanwhile, in this example, if the amino acid residues that form the interface between CL-A and CH1-B are not mutually uncharged or nonpolar amino acids, they may be modified to become uncharged or nonpolar amino acids.

[0165] Furthermore, in another example, when the amino acid residues forming the interface between CL-B and CH1-B are uncharged or nonpolar amino acids in CH1-B, one of the amino acid residues forming the interface between CH1-A and CL-A is changed to a positively charged amino acid residue while the other is changed to a negatively charged amino acid residue; and the amino acid residues forming the interface between CL-B and CH1-B in CL-B are modified to have the same charge as when modified in CH1-A. As a result of this modification, while the desired association between CH1-A and CL-A is promoted because the amino acid residues forming the interface are a combination of positive and negative charges, the desired association between CH1-B and CL-B is not inhibited because the amino acid residues forming the interface are amino acids that are mutually electrically repelling. As a result, an antibody can be effectively obtained that has the desired association between CH1-A and CL-A and the desired association between CH1-B and CL-B.Meanwhile, in this example, when the amino acid residues forming the interface between CL-B and CH1-B are not uncharged or nonpolar amino acids in CH1-B, they may be modified to become uncharged or nonpolar amino acids.

[0166] Furthermore, the use of the communication setting of the present invention makes it possible to suppress communication between CH1s (CH1-A and CH1-B), or communication between CLs (CL-A and CL-B).

[0167] Those skilled in the art can appropriately determine the types of amino acid residues that are brought into close proximity during association at the CH1 and CL interface in a polypeptide of interest for which regulation of association by the present invention is desirable.

[0168] In addition, those skilled in the art can also appropriately obtain sequences that can be used as CH1 or CL of an antibody in an organism such as a human, monkey, mouse, rabbit, and the like by using a public database, etc. More specifically, amino acid sequence information of CH1 or CL can be obtained by the methods described in the examples described below.

[0169] For example, with respect to the bispecific antibodies described in the examples below, specific examples of amino acid residues that come into close proximity (that face or are in contact) during association at the CH1 and CL interface include the combinations shown below: - Glutamine (Q) at position 175 according to EU numbering in CH1 and glutamine (Q) facing (contact) or glutamic acid (E) at position 160 according to Kabat numbering in CL; - Glutamine (Q) at position 175 according to EU numbering in CH1 and threonine (T) facing (contact) or serine (S) at position 131 according to Kabat numbering in CL; - Glutamine (Q) at position 175 according to EU numbering in CH1 and serine (S) or threonine (T) facing (contact) at position 131 and glutamine (Q) or glutamic acid (E) at position 160 according to Kabat numbering in CL; and - Lysine (K) at position 147 and glutamine (Q) at position 175 according to EU numbering in CH1 and serine (S) or threonine (T) facing (contact) at position 131 and glutamine (Q) or glutamic acid (E) at position 160 according to Kabat numbering in CL.

[0170] The numbers described in the EU numbering in the present invention are shown in accordance with the EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242). In the present invention, the terms "an amino acid residue at position X according to the EU numbering" and "an amino acid at position X according to the EU numbering" (where X is an arbitrary number) can also be read as "an amino acid residue corresponding to position X according to the EU numbering" and "an amino acid corresponding to position X according to the EU numbering". As shown in the examples described below, the molecules binding to the desired antigen can be preferably obtained by modifying these amino acid residues and carrying out the methods of the present invention.

[0171] In one embodiment, the present invention provides an antigen-binding molecule in which the association of the heavy chain and the light chain is regulated in which one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below in the heavy chain and the light chain of the antigen-binding molecule are amino acid residues that are mutually electrically repel each other: (a) The amino acid residue in CH1 at position 175 according to the European Union numbering and the amino acid residue in CL at position 160 according to the Kabat numbering; (b) the amino acid residue in CH1 at position 175 according to the European Union numbering and the amino acid residue in CL at position 131 according to the Kabat numbering; (c) amino acid residues in CH1 at positions 147 and 175 according to the European Union numbering and amino acid residues in CL at positions 131 and 160 according to the Kabat numbering; and (d) The amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residue in CL at positions 131 and 160 according to Kabat numbering.

[0172] In the above-mentioned antigen-binding molecule, the "amino acid residues that mutually electrically repel each other" or "amino acid residues with the same charge" are preferably selected from amino acid residues present in, for example, one of the sets (X) or (Y) below: (X) Glutamic acid (E) or aspartic acid (D); or (Y) Lysine (K), Arginine (R), or Histidine (H).

[0173] In the above antigen-binding molecule, specific examples of sets of amino acid residues that electrically repel each other include the following sets of amino acid residues: (a) the amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residue in CL at position 160 according to EU numbering; (b) the amino acid residue in CH1 at position 175 according to the European Union numbering and the amino acid residue in CL at position 131 according to the Kabat numbering; (c) amino acid residues in CH1 at positions 147 and 175 according to EU numbering and amino acid residues in CL at positions 131 and 160 according to Kabat numbering; (d) The amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residue in CL at positions 131 and 160 according to Kabat numbering.

[0174] In some embodiments, in the multispecific antigen-binding molecule, one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (d) below in the heavy chain and light chain of the antigen-binding molecule are amino acid residues that electrostatically repel each other: (a) an amino acid residue in a heavy chain constant region (CH1) located at position 175 according to EU numbering and an amino acid residue in a light chain constant region (CL) located at position 131 according to Kabat numbering; (b) an amino acid residue in CH1 located at position 175 according to EU numbering and an amino acid residue in CL located at position 160 according to Kabat numbering; (c) the amino acid residue in CH1 located at position 175 according to the European Union numbering and the amino acid residue in CL located at positions 131 and 160 according to the Kabat numbering; (d) Amino acid residues in CH1 located at positions 147 and 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to Kabat numbering.

[0175] The present invention provides an antigen-binding molecule wherein one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a1) to (c2) below in the heavy chain and the light chain of the antigen-binding molecule are amino acid residues that are mutually electrically repelled: (a1) the amino acid residue in CH1 at position 175 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D) and the amino acid residue in CL at position 160 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D); (a2) the amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H) or arginine (R) and the amino acid residue in CL at position 160 according to EU numbering is lysine (K), histidine (H) or arginine (R); (b1) the amino acid residue in CH1 at position 175 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D) and the amino acid residue in CL at position 131 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D); (b2) the amino acid residue in CH1 at position 175 according to the European Union numbering system is lysine (K), histidine (H) or arginine (R) and the amino acid residue in CL at position 131 according to the European Union numbering system is lysine (K), histidine (H) or arginine (R); (c1) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering, each of which is glutamic acid (E) or aspartic acid (D), and the amino acid residues in CL at positions 131 and 160 according to EU numbering, each of which is glutamic acid (E) or aspartic acid (D); (c2) The amino acid residues in CH1 at positions 147 and 175 according to EU numbering, each of which is lysine (K), histidine (H) or arginine (R), and the amino acid residues in CL at positions 131 and 160 according to EU numbering, each of which is lysine (K), histidine (H) or arginine (R).

[0176] In the above antigen-binding molecule, specific examples of amino acid residues that electrically repel each other include the following amino acid residues: (a1) the amino acid residue in CH1 at position 175 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D) and the amino acid residue in CL at position 160 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D); (a2) the amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H) or arginine (R) and the amino acid residue in CL at position 160 according to EU numbering is lysine (K), histidine (H) or arginine (R); (b1) the amino acid residue in CH1 at position 175 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D) and the amino acid residue in CL at position 131 according to the European Union numbering system is glutamic acid (E) or aspartic acid (D); (b2) the amino acid residue in CH1 at position 175 according to the European Union numbering system is lysine (K), histidine (H) or arginine (R) and the amino acid residue in CL at position 131 according to the European Union numbering system is lysine (K), histidine (H) or arginine (R); (c1) the amino acid residues in CH1 at positions 147 and 175 according to EU numbering, each of which is glutamic acid (E) or aspartic acid (D), and the amino acid residues in CL at positions 131 and 160 according to EU numbering, each of which is glutamic acid (E) or aspartic acid (D); (c2) amino acid residues in CH1 at positions 147 and 175 according to EU numbering, each of which is lysine (K), histidine (H) or arginine (R), and amino acid residues in CL at positions 131 and 160 according to EU numbering, each of which is lysine (K), histidine (H) or arginine (R); (d1) the amino acid residue in CH1 at position 175 according to EU numbering is glutamic acid (E) or aspartic acid (D) and the amino acid residues in CL at positions 131 and 160 according to EU numbering are either glutamic acid (E) or aspartic acid (D); (d2) The amino acid residue in CH1 at position 175 according to EU numbering is lysine (K), histidine (H) or arginine (R) and the amino acid residue in CL at positions 131 and 160 according to EU numbering is lysine (K), histidine (H) or arginine (R), respectively.

[0177] In addition to the above, the technique of inhibiting undesired associated CH1 / CL by creating an electrical charge repulsion at the interface between CH1 and CL (WO 2013 / 065708) can be further applied to the antigen-binding molecule of the present invention. In particular, the present invention provides an antigen-binding molecule with CH1 and CL, wherein one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (d) below are mutually electrically repelled: (a) the amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering and the amino acid residue in the light chain constant region (CL) at position 160 according to EU numbering; (b) the amino acid residue in CH1 at position 147 according to EU numbering and the amino acid residue in CL at position 131 according to EU numbering; (c) the amino acid residue in CH1 at position 175 according to EU numbering and the amino acid residue in CL at position 160 according to EU numbering; (d) The amino acid residue in CH1 at position 213 according to EU numbering and the amino acid residue in CL at position 123 according to EU numbering.

[0178] A technique for creating an electrical repulsion at the interface of the second heavy chain constant region (CH2) or the third heavy chain constant region (CH3) to suppress the undesirable association between the heavy chains, a technique for creating an electrical repulsion at the interface of the heavy chain variable region and the light chain variable region to suppress the undesirable association between the heavy chain and the light chain, or a technique for modifying the amino acid residues forming a hydrophobic core present at the interface of the heavy chain variable region and the light chain variable region to polar amino acids with an electrical charge to suppress the undesirable association between the heavy chain and the light chain can be further applied to the antigen-binding molecules of the present invention (see WO 2006 / 106905).

[0179] In a technique that suppresses undesired association between heavy chains by creating electrical repulsion at the CH2 or CH3 interface, examples of amino acid residues that are in contact at the interface with other heavy chain constant regions include the regions corresponding to positions 356 (EU numbering) and 439 (EU numbering), positions 357 (EU numbering) and 370 (EU numbering), and positions 399 (EU numbering) and 409 (EU numbering) in the CH3 region. For numbering of antibody constant regions, see Kabat, EA, et al. (1991, Sequences of Proteins of Immunological Interest, NIH); and for numbering of heavy chain constant regions, EU numbering is shown.

[0180] Specifically, for example, in an antigen-binding molecule containing two types of heavy chain CH3 regions, one to three sets of amino acid residues in the first heavy chain CH3 region selected from the following amino acid residue sets (1) to (3) may be electrically made to repel each other: (1) Amino acid residues in the CH3 region of the heavy chain at positions 356 and 439 according to the European Union numbering; (2) amino acid residues in the CH3 region of the heavy chain at positions 357 and 370 according to EU numbering; and (3) Amino acid residues in the CH3 region of the heavy chain at positions 399 and 409 according to the European Union numbering.

[0181] Furthermore, the antibody can be an antibody with a set of amino acid residues in the second CH3 region of the heavy chain distinct from the first CH3 region of the heavy chain mentioned above, wherein the set of amino acid residues is selected from the set of amino acid residues shown in (1) to (3) above, and wherein one to three sets of amino acid residues corresponding to the sets of amino acid residues shown in (1) to (3) above that are mutually electrically repulsive in the first CH3 region of the heavy chain are not mutually electrically repulsive from the corresponding amino acid residues in the first CH3 region of the heavy chain.

[0182] The amino acid residues described in (1) to (3) above are brought closer together during association. Those skilled in the art can use homology modeling and the like using commercially available software to find the corresponding positions of the amino acid residues described in (1) to (3) for the CH3 region of the heavy chain or the heavy chain constant region of interest and modify the amino acid residues at those positions appropriately.

[0183] In the above antigen-binding molecule, "electrophoretic repulsion", "having the same charge", or "carrying the same charge" means that, for example, two or more amino acid residues have amino acid residues that are in one of the groups (X) and (Y) listed herein.

[0184] In a preferred embodiment of the above antigen-binding molecule, the first CH3 region of the heavy chain and the second CH3 region of the heavy chain may be linked by disulfide bonds.

[0185] In the present invention, an amino acid residue that is subject to "modification" is not limited to the amino acid residue of the variable region of the antigen-binding molecule or the constant region of the antibody mentioned above. Those skilled in the art can find amino acid residues that form an interface in a polypeptide or heteromeric multimer by homology modeling and the like using commercially available software, and modify the amino acid residues at those locations to regulate the association. Homology modeling is a technique for predicting the three-dimensional structure of a protein using commercially available software. When constructing the structure of a protein with an unknown three-dimensional structure, one first searches for a protein that is known to have a three-dimensional structure that is highly similar to the protein. In the next step, using this 3D structure as a template, the structure of the protein with the unknown structure is constructed, and the structure is then optimized using molecular dynamics methods and the like to predict the 3D structure of the unknown protein.

[0186] In the technique of creating an electrical repulsion at the interface of the heavy chain variable region and the light chain variable region to suppress the undesirable association of the heavy chain and the light chain, examples of amino acid residues that are in contact at the interface of the heavy chain variable region (VH) and the light chain variable region (VL) include glutamine (Q) at position 39 according to Kabat numbering in VH (FR2 region) and glutamine (Q) facing (contact) at position 38 according to Kabat numbering in VL (FR2 region). In addition, a preferred example is leucine (L) at position 45 according to Kabat numbering in VH (FR2) and proline (P) facing at position 44 facing in VL (FR2). Kabat, EA, et al. (1991, Sequence of Proteins of Immunological Interest, NIH) is referred to for the numbering of these sites.

[0187] Since these amino acid residues are highly conserved in humans and mice (J. Mol. Recognit. 2003; 16: 113-120), the association of the variable regions of antigen-binding molecules can be adjusted for the VH-VL association of antigen-binding molecules other than those shown in the examples by modifying the amino acid residues corresponding to the aforementioned amino acid residues.

[0188] In some embodiments, in the multispecific antigen-binding molecule, two or more amino acid residues that form an interface between a heavy chain variable region and a light chain variable region are amino acid residues that electrostatically repel each other.

[0188] A specific example is an antigen-binding molecule in which two or more amino acid residues forming the VH and VL interface are amino acid residues that are electrically repelled from each other. In particular, examples include an antigen-binding molecule with one or two sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) or (b) below: (a) the amino acid residue in VH at position 39 according to Kabat numbering and the amino acid residue in VL at position 38 according to Kabat numbering; or (b) The amino acid residue in VH at position 45 according to Kabat numbering and the amino acid residue in VL at position 44 according to Kabat numbering.

[0190] In some embodiments, in the multispecific antigen-binding molecule, the amino acid residues that electrostatically repel each other are one or two sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) and (b): (a) an amino acid residue in the variable region of the heavy chain located at position 39 according to Kabat numbering and an amino acid residue in the variable region of the light chain located at position 38 according to Kabat numbering; (b) An amino acid residue in the variable region of the heavy chain located at position 45 according to Kabat numbering and an amino acid residue in the variable region of the light chain located at position 44 according to Kabat numbering.

[0191] Each of the amino acid residues described in (a) or (b) above is brought close to each other after association. Those skilled in the art can find locations that correspond to the amino acid residues described in (a) or (b) in the desired VH or VL by homology modeling and the like, using commercially available software, and appropriately modify the amino acid residues at those locations.

[0192] In some embodiments, in the multispecific antigen-binding molecule, the amino acid residues that electrostatically repel each other are selected from amino acid residues present in either set (X) or (Y) below: (X) Glutamic acid (E), Aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[0193] In the technique of modifying amino acid residues forming a hydrophobic core present at the VH and VL interface to polar amino acids with an electrical charge to suppress undesired heavy chain and light chain association, preferred examples of amino acid residues capable of forming a hydrophobic core at the VH and VL interface include leucine (L) at position 45 according to Kabat numbering in VH (FR2) and proline (P) facing position 44 according to Kabat numbering in VL (FR2). Kabat et al. (1991, Sequences of Proteins of Immunological Interest, NIH) was used as a reference for the numbering of these positions.

[0194] In general, the term "hydrophobic core" refers to the region formed by a set of hydrophobic amino acid side chains within the associated polypeptides. Examples of hydrophobic amino acids include: alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In addition, amino acid residues other than hydrophobic amino acids (e.g., tyrosine) may be involved in the formation of a hydrophobic core. This hydrophobic core, together with a hydrophilic surface where the hydrophilic amino acid side chains are exposed to the outside, becomes the driving force for promoting the association of water-soluble polypeptides. When hydrophobic amino acids from two different domains are present on a molecular surface and exposed to water molecules, the entropy increases and the free energy increases. Accordingly, the two domains associate with each other to reduce the free energy and become stable, and the hydrophobic amino acids at the interface are buried inside the molecule to form a hydrophobic core.

[0195] It is thought that when polypeptide linkage occurs, the formation of a hydrophobic core is inhibited by modifying the hydrophobic amino acids that form the hydrophobic core to electrically charged polar amino acids; and as a result, polypeptide linkage is thought to be inhibited.

[0196] Those skilled in the art can determine the presence or absence of a hydrophobic core, the formation site (region), and the like by analyzing the amino acid sequence of a target antigen-binding molecule; that is, the antigen-binding molecule of the present invention is an antigen-binding molecule characterized in that amino acid residues capable of forming a hydrophobic core at an interface are modified to electrically charged amino acid residues. Specifically, examples include an antigen-binding molecule in which the amino acid residues shown in (1) or (2) below are electrically charged amino acid residues. The side chains of the amino acid residues shown in (1) and (2) below are adjacent to each other and can form a hydrophobic core: (1) the amino acid residue in VH at position 45 according to Kabat numbering; and (2) The amino acid residue in VL at position 44 according to Kabat numbering.

[0197] Preferred examples of electrically charged amino acid residues in the above antigen-binding molecule include glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine (H). More preferred examples include glutamic acid (E) and lysine (K).

[0198] In general, the amino acid residues described in (1) and (2) above in humans and mice, respectively, are: (1) Leucine (L) and (2) Proline (P). Therefore, in a preferred embodiment of the present invention, these amino acid residues are subject to modification (such as substitution with charged amino acids). Furthermore, the types of amino acid residues mentioned above (1) and (2) are not necessarily limited to the above-mentioned amino acid residues, but may also be other amino acids equivalent to these amino acid residues.

[0199] Other known techniques can be applied to the antigen-binding molecules of the present invention. For example, in order to promote the association of the first VH (VH1) and the first VL (VL1) and / or the second VH (VH2) and the second VL (VL2), an amino acid side chain in the variable region of one of the H chains can be replaced with a larger side chain (protrusion) and an amino acid side chain in the opposite variable region of the other H chain can be replaced with a smaller side chain (concavity), so that the handle may be arranged in the hole and the association of VH1 and VL1 and / or VH2 and VL2 is promoted; and consequently, the association of VH1 and VL2 and / or VH2 and VL1 can be further suppressed.

[0200] For example, in the case of human IgG1, the Y349C and T366W changes are made to convert an amino acid side chain in the CH3 region of one H chain into a larger side chain (bulge), and the D356C, T336S, L368A, and Y407V changes are made to convert an amino acid side chain in the CH3 region of the other H chain into a smaller side chain.

[0201] In some embodiments, in the multispecific antigen-binding molecule, an Fc domain is composed of a first Fc region subunit and a second Fc region subunit that are capable of stable association. In some embodiments, in the multispecific antigen-binding molecule, the Fc domain comprises (e1) or (e2) of the following: (e1) the first subunit of the Fc region includes Cys at position 349, Ser at position 366, Ala at position 368 and Val at position 407 and the second Fc region includes Cys at position 354 and Trp at position 366; (e2) The first subunit of the Fc region contains Glu at position 439 and the second Fc region contains Lys at position 356, In which amino acid positions are numbered according to the European Union numbering system.

[0202] For example, the ridge-to-groove technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, this method involves creating a ridge ("handle") at the interface of a first polypeptide and a corresponding groove ("hole") at the interface of a second polypeptide, such that the ridge can be placed in the groove to promote heterodimer formation and prevent homodimer formation. The ridges are made by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). Compensatory indentations of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (such as alanine or threonine).

[0203] Other known techniques can also be applied to the antigen-binding molecules of the present invention. A target antigen-binding molecule can be efficiently prepared by complementary CH3 linkage using an engineered domain with CH3 strand exchange in which a portion of CH3 from one H chain of one antigen-binding molecule is changed to the IgA-derived sequence corresponding to that portion and a complementary portion of CH3 from another H chain with the IgA-derived sequence corresponding to that portion is inserted (Protein Engineering Design & Selection, 23: 195-202, 2010).

[0204] Other known techniques can also be used for the antigen-binding molecules of the present invention. When producing bispecific antibodies, a target bispecific antibody can be prepared by creating a difference in isoelectric point by making different amino acid modifications in each of the variable regions of the two types of H chains and using that difference in isoelectric point for purification by ion exchange chromatography (WO 2007 / 114325).

[0205] The technique of modifying the amino acid residue at position 435 according to the European Union numbering, which is the binding site between IgG and protein A, to an amino acid with a different binding strength than protein A, such as Arg, may also be used on the antigen-binding molecule of the present invention in combination with the above techniques. By using this technique, the interaction between the H chain and protein A can be changed and only the heterodimeric molecules bound to the antigen can be effectively purified using the protein A column. This technique can also be used independently without combination with the above techniques.

[0206] Modifications of the invention can be applied to antigen-binding molecules such as, for example, an antigen-binding molecule having a structure in which, to promote the association of a first VH (VH1) and a first VL (VL1) and / or a second VH (VH2) and a second VL (VL2), VH1 is linked to an Fc region via a first CH1 and VL1 to a first CL, and VH2 is linked to another Fc region via a second CL and VL2 to a second CH1 (WO 09 / 80254).

[0207] For example, two or more of the above known techniques can be used in combination for the antigen binding molecule of the present invention. Furthermore, the antigen binding molecule of the present invention may be prepared based on an antibody by making modifications to the above-mentioned known techniques.

[0208] Furthermore, the present invention provides a method for producing an antigen-binding molecule in which the association between a heavy chain and a light chain is regulated. A preferred embodiment of the method of production of the present invention is a method for producing an antigen-binding molecule in which the association between a heavy chain and a light chain is regulated, comprising: (1) Modifying nucleic acids encoding CH1 and CL such that one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below are amino acid residues that electrostatically repel each other: (a) an amino acid residue in a heavy chain constant region (CH1) located at position 175 according to EU numbering and an amino acid residue in a light chain constant region (CL) located at position 131 according to Kabat numbering; (b) an amino acid residue in CH1 located at position 175 according to EU numbering and an amino acid residue in CL located at position 160 according to Kabat numbering; (c) amino acid residues in CH1 located at positions 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to Kabat numbering; (d) Amino acid residues in CH1 located at positions 147 and 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to Kabat numbering. (2) introducing the modified nucleic acids into a host cell and culturing the host cell such that the nucleic acids are expressed; and (3) Collecting an antigen-binding molecule from the host cell culture.

[0209] Furthermore, the present invention relates to a production method which comprises, in the above step (1), modifying nucleic acids such that amino acid residues that electrically repel each other are selected from amino acid residues present in each of the aforementioned groups (X) and (Y).

[0210] Furthermore, the present invention relates to a production method which comprises in the above step (1) modifying nucleic acids such that two or more amino acid residues forming the VH and VL interface are amino acid residues that electrically repel each other. Preferably, the amino acid residues that electrically repel each other are a set of amino acid residues selected from the group consisting of, for example, a set of amino acid residues shown in (a) and (b) below: (a) the amino acid residue in VH at position 39 according to Kabat numbering and the amino acid residue in VL at position 38 according to Kabat numbering. or (b) The amino acid residue in VH at position 45 according to Kabat numbering and the amino acid residue in VL at position 44 according to Kabat numbering.

[0211] The aforementioned amino acid residues that electrically repel each other are preferably selected from amino acid residues present in both sets of (X) and (Y) mentioned above.

[0212] In addition, the present invention provides a method for regulating the association of the heavy and light chains of an antigen-binding molecule. A preferred embodiment of the method for regulating the association of the present invention is a method for regulating the association of the heavy and light chains of an antigen-binding molecule comprising modifying nucleic acids such that one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (c) below are amino acid residues that electrostatically repel each other: (a) an amino acid residue in a heavy chain constant region (CH1) located at position 175 according to EU numbering and an amino acid residue in a light chain constant region (CL) located at position 131 according to EU numbering, (b) an amino acid residue in CH1 located at position 175 according to EU numbering and an amino acid residue in CL located at position 160 according to EU numbering, (c) amino acid residues in CH1 located at positions 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to EU numbering, (d) Amino acid residues in CH1 located at positions 147 and 175 according to EU numbering and amino acid residues in CL located at positions 131 and 160 according to EU numbering.

[0213] Furthermore, the present invention relates to a method for regulating communication, which in the above-mentioned step (1) comprises modifying the nucleic acids such that the amino acid residues that electrostatically repel each other are selected from amino acid residues present in each of the above-mentioned groups (X) or (Y).

[0214] Furthermore, the present invention relates to a method for regulating the association, which comprises in the above step (1) modifying nucleic acids such that two or more amino acid residues forming a VH-VL interface are amino acid residues that electrostatically repel each other. Here, the amino acid residues that electrostatically repel each other are preferably any set of amino acid residues selected from the group consisting of, for example, a set of amino acid residues shown in (a) and (b) below: (a) an amino acid residue in VH located at position 39 according to Kabat numbering and an amino acid residue in VL located at position 38 according to Kabat numbering, (b) An amino acid residue in VH located at position 45 according to Kabat numbering and an amino acid residue in VL located at position 44 according to Kabat numbering.

[0215] According to the linkage adjustment method of the present invention, a desired bispecific antibody can be preferentially and efficiently obtained as previously described; that is, a desired heteromeric multimer in the form of a bispecific antibody can be efficiently formed from the monomer mixture.

[0216] The term "modifying nucleic acids" in the above-mentioned methods of the present invention refers to modifying nucleic acids such that they correspond to the amino acid residues introduced by the "modifications" of the present invention. More specifically, this term refers to modifying nucleic acids encoding the original (pre-modified) amino acid residues into nucleic acids encoding the amino acid residues to be introduced by the modification. Typically, this means performing gene manipulations or mutation operations that result in the insertion, deletion or substitution of at least one nucleotide into the original nucleic acid, such that codons encoding the desired amino acid residues are formed. Specifically, codons encoding the original amino acid residues are replaced by codons encoding the amino acid residues to be introduced by the modification. This modification of the nucleic acid can be conveniently carried out by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis.Furthermore, the present invention provides nucleic acids encoding an antigen-binding molecule of the present invention. Furthermore, vectors carrying the nucleic acids are also included in the present invention.

[0217] In some embodiments, the Fc domain of the multispecific antigen-binding molecule is composed of a pair of polypeptide chains that include the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which includes the constant heavy chain domains CH2 and CH3 of IgG. The two Fc domain subunits are capable of stably associating with each other. In one embodiment, the multispecific antigen-binding molecule described herein does not include more than one Fc domain.

[0218] In one embodiment described herein, the Fc domain of the multispecific antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In another specific embodiment, the Fc domain is a human IgG1 Fc region.

[0219] In some embodiments, the present disclosure provides a multispecific antigen-binding molecule that further comprises an Fc domain that exhibits reduced binding affinity for the human Fc gamma receptor compared to the human IgG1 Fc domain, wherein the Fc domain exhibits a stronger FcRn binding affinity for human FcRn compared to a natural human IgG1 Fc domain.

[0220] In some embodiments, the present disclosure provides a multispecific antigen-binding molecule that further comprises an Fc domain that exhibits reduced binding affinity for the human Fc gamma receptor compared to the human IgG1 Fc domain, wherein the first and / or second subunit of the Fc region formed in the Fc domain comprises Leu at position 428, Ala at position 434, Arg at position 438 and Glu at position 440, In which amino acid positions are numbered according to the European Union numbering system.

[0221] In some embodiments, in the multispecific antigen-binding molecule, the Fc domain exhibits a stronger binding affinity for human FcRn than a human IgG1 Fc domain. In some embodiments, in the multispecific antigen-binding molecule, the first and / or second subunit of the Fc region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, In which amino acid positions are numbered according to the European Union numbering system.

[0222] Bispecific IgG antibodies are secreted into cells by introducing the genes of the L chains and H chains constituting the two desired IgG types, i.e., a total of four genes, and expressing them together. However, the number of combinations of IgG H and L chains that can be produced by these methods is theoretically ten. Accordingly, it is difficult to purify an IgG containing the desired combination of H and L chains from ten IgG types. In addition, the theoretical secretion rate of IgG containing the desired combination is significantly reduced, and therefore large-scale cultivation will be necessary, further increasing production costs.

[0223] Therefore, techniques for enhancing the association between H chains and between L and H chains having desired conformations can be applied to the multispecific antigen-binding molecules of the present invention. For example, techniques for suppressing undesired H chain association by creating electrostatic repulsion at the interface of the second constant region or the third constant region of the antibody H chain (CH2 or CH3) can be applied to multispecific antibody association (WO2006 / 106905).

[0224] In the present invention, the amino acid residues subject to modification are not limited to the above-mentioned amino acid residues in the antibody variable regions or antibody constant regions. Those skilled in the art can identify amino acid residues that form a link in the mutant polypeptides or heteromultimers using commercially available software, and the amino acid residues at these positions can be subjected to modification to regulate the link.

[0225] Other known techniques can also be used to link the multispecific antibodies of the present invention. Polypeptides containing an Fc region comprising different amino acids can be effectively linked by replacing an amino acid side chain present in one of the Fc regions of the antibody H chain with a larger side chain (protrusion) and replacing an amino acid side chain present in the corresponding Fc region of the other H chain with a smaller side chain (recess) to place the protrusion within the recess. The protrusion(s)-in-recess(s) method is discussed elsewhere herein.

[0226] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. The linkage of polypeptides with different sequences can be efficiently prepared by complementary CH3 linkage using a domain engineered with CH3 strand exchange in which a portion of the CH3 from one H chain of an antigen-binding molecule is changed to the IgA-derived sequence corresponding to that portion and a complementary portion of the CH3 from another H chain with the IgA-derived sequence corresponding to that portion is inserted (Protein Engineering Design & Selection, 23: 195-202, 2010). This known technique can also be used to efficiently form multispecific antibodies.

[0227] In addition, technologies for producing antibodies using antibody CH1 and CL linkage and VH and VL linkage are described in WO 2011 / 028952, WO2014 / 018572 and Nat Biotechnol. 2014 Feb; 32(2):191-8; technologies for producing bispecific antibodies using separate monoclonal antibodies prepared in combination (Fab arm swapping) are described in WO2008 / 119353 and WO2011 / 131746; technologies for regulating the linkage between CH3 heavy chain antibodies are described in WO2012 / 058768 and WO2013 / 063702; Technology for producing bispecific antibodies consisting of two types of light chains and one type of heavy chain described in WO2012 / 023053; technologies for producing bispecific antibodies using two bacterial cell strains separately expressing one of the chains of an antibody comprising a single H chain and a single L chain described by Christof et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)); and the like may be used to form multispecific antibodies.

[0228] Alternatively, even when a desired multispecific antibody cannot be efficiently formed, a multispecific antibody of the present invention can be obtained by isolating and purifying the desired multispecific antibody from the produced antibodies. For example, a method for enabling purification of two types of homomeric forms and a desired heteromeric antibody by ion exchange chromatography by creating a difference in isoelectric points by introducing amino acid substitutions in the variable regions of two types of H chains has been reported (WO2007114325). To date, as a method for purifying heteromeric antibodies, methods using protein A to purify a heterodimeric antibody comprising a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A have been reported (WO98050431 and WO95033844).In addition, a heterodimeric antibody can be efficiently purified by using H chains alone, which include replacing the amino acid residues at EU numbering positions 435 and 436, which are the IgG-protein A binding sites, with Tyr, His, or amino acids that confer different affinity to protein A, or by using H chains with different protein A affinity obtained according to the method of Example 5, to alter the interaction of each of the H chains with protein A, and then from a protein A column.

[0229] In addition, an Fc region whose C-terminal heterogeneity of the Fc region is improved can be suitably used as an Fc region of the present invention. In particular, the present invention provides Fc regions produced by deleting glycine at position 446 and lysine at position 447, as specified in the European Union numbering, from the amino acid sequence of two polypeptides constituting an Fc region derived from IgG1, IgG2, IgG3 or IgG4.

[0230] Many of these technologies can be used in combination, such as two or more technologies. In addition, these technologies can be suitably applied separately to the two H chains. In addition, these techniques can be used in combination with the aforementioned Fc region having reduced Fc gamma receptor binding activity. In addition, an antigen-binding molecule of the present invention may be a molecule that is separately produced so that it has the same amino acid sequence based on the antigen-binding molecule subjected to the modifications described above.

[0231] In some embodiments, the multispecific antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xii): (i) glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) leucine at position 428 (EU numbering) in the heavy chain constant region; (ix) Alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region. In some embodiments, the multispecific antigen-binding molecule is a bispecific antibody, comprising: A first heavy chain comprising lysine at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering), glutamic acid at position 439 (EU numbering), and glutamic acid at position 440 (EU numbering); A first light chain containing glutamic acid at position 131 (Kabat numbering) and glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising glutamic acid at position 147 (EU numbering), glutamic acid at position 175 (EU numbering), arginine at position 235 (EU numbering), arginine at position 236 (EU numbering), lysine at position 356 (EU numbering), leucine at position 428 (EU numbering), alanine at position 434 (EU numbering), arginine at position 438 (EU numbering) and glutamic acid at position 440 (EU numbering); and A second light chain contains lysine at position 131 (Kabat numbering) and lysine at position 160 (Kabat numbering). In some embodiments, the multispecific antigen-binding molecule: The first heavy chain additionally contains glutamic acid at position 419 (EU numbering) and proline at position 445 (EU numbering) and amino acid deletions at positions 446 and 447 (EU numbering); and The second heavy chain additionally contains lysine at position 196 (EU numbering), proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). In some embodiments, the multispecific antigen-binding molecule: The first heavy chain additionally contains glycine at position 16 (Kabat numbering), alanine at position 32 (Kabat numbering), valine at position 35a (Kabat numbering), alanine at position 50 (Kabat numbering), lysine at position 61 (Kabat numbering), glutamic acid at position 64 (Kabat numbering), threonine at position 73 (Kabat numbering), glutamic acid at position 95 (Kabat numbering), and valine at position 102 (Kabat numbering); The first light chain additionally contains glutamic acid at position 28 (Kabat numbering), tyrosine at position 55 (Kabat numbering), glutamic acid or tyrosine at position 56 (Kabat numbering), glutamic acid at position 92 (Kabat numbering), valine at position 94 (Kabat numbering), and alanine at position 95a (Kabat numbering); The second heavy chain additionally comprises glutamic acid at position 28 (Kabat numbering), alanine or glutamic acid at position 30 (Kabat numbering), glutamic acid at position 31 (Kabat numbering), tryptophan at position 32 (Kabat numbering), phenylalanine at position 34 (Kabat numbering) and methionine at position 35 (Kabat numbering), serine at position 35a (Kabat numbering), serine at position 50 (Kabat numbering), glutamic acid or glycine at position 61 (Kabat numbering), glutamic acid at position 64 (Kabat numbering) and glutamic acid at position 65 (Kabat numbering); and The second light chain additionally contains threonine at position 25 (Kabat numbering), lysine at position 54 (Kabat numbering), glutamic acid at position 56 (Kabat numbering), leucine at position 67 (Kabat numbering), glutamine at position 79 (Kabat numbering), and lysine at position 94 (Kabat numbering).

[0232] Library-derived antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and are 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, in Methods in Molecular Biology 248:161-175 (Lo, ed., 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) is further described.

[0233] In specific phage display methods, VH and VL gene sets are cloned separately by polymerase chain reaction (PCR) and randomly assembled into phage libraries, 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 fragments (scFv) or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies without the need to construct hybridomas for the immunogen. Alternatively, simple pools (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self as well as self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).Finally, simple libraries can be constructed synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and performing in vitro rearrangement 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.

[0234] Antibodies or antibody fragments isolated from human antibody libraries are considered herein to be human antibodies or human antibody fragments.

[0235] Types of glycosylation In certain embodiments, the antibody provided herein is modified to increase or decrease the extent of glycosylation of the antibody. Adding or removing glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or deleted.

[0236] When an antibody is composed of an Fc region, the carbohydrate attached to it may vary. Natural antibodies produced by mammalian cells typically comprise a branched binary oligosaccharide that is generally N-linked to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may comprise a variety of carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the binary oligosaccharide structure. In some embodiments, modifications may be made to the oligosaccharide in an antibody of the invention to create antibody variants with improved properties.

[0237] In one embodiment, antibody variants are provided with a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297, relative to the sum of all glycostructure structures attached to Asn 297 (e.g., complex, hybrid, and high mannose structures) measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region. However, Asn297 may also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in antibodies. These types of fucosylation may improve 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 publications relating to "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 Lec13 CHO cells deficient in protein fucosylation (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., especially in Example 11) and knockout cell lines, such as alpha-6,1-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006) and WO2003 / 085107).

[0238] Antibody variants are further provided with bisected oligosaccharides, for example, in which a bibranched oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. These antibody variants may improve fucosylation and / or ADCC function. Examples of antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. These antibody variants may improve CDC function. Antibody species are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0239] In a preferred embodiment, the antibodies mentioned above may have their first CH3 region of the H chain and their second CH3 region of the H chain linked by disulfide bonds.

[0240] The multispecific antigen-binding molecules prepared as described herein may be purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the multispecific antigen-binding molecule is bound may be used. For example, for affinity chromatography purification of the multispecific antigen-binding molecules of the invention, a matrix with protein A or protein G may be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a multispecific antigen-binding molecule.The purity of the multispecific antigen-binding molecule can be determined by any of a variety of known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, and the like.

[0241] Cysteine-engineered antibody variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs" in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By replacing those residues with cysteine, active thiol groups are placed at accessible sites on the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or drug-linker moieties, to create an immunoconjugate, as described below. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) heavy chain; and S400 (EU numbering) heavy chain Fc region. Cysteine-engineered antibodies may be produced as described, for example, in U.S. Patent No. 7,521,541.

[0242] Antibody derivativesIn certain embodiments, an antibody provided herein may be further modified to contain additional non-protein moieties that are known in the art and readily available. Suitable moieties for derivatizing an antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-6,3,1-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propylaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched.The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0243] In another embodiment, conjugates of an antibody and a non-protein moiety are provided that may be selectively heated upon 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 may be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein moiety to a temperature at which cells in the vicinity of the antibody-non-protein moiety are killed.

[0244] Recombinant methods and compounds Antibodies may be produced 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 a molecule (antibody) that binds to the anti-HLA-DQ2.5 antigen is provided. The nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (such as the light and / or heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., 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 eukaryotic, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making a molecule (antibody) that binds to an anti-HLA-DQ2.5 antigen is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as set forth above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0245] To recombinantly produce a molecule (antibody) that binds to the anti-HLA-DQ2.5 antigen, nucleic acid encoding an antibody is isolated, for example, as described above, and inserted into one or more vectors for further cloning and / or expression in a host cell. This nucleic acid can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

[0246] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc factor function are not required. For expression of 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, describing expression of antibody fragments in Escherichia coli). After expression, the antibody may be isolated from the bacterial cell wall in a soluble fraction and can be further purified.

[0247] In addition to prokaryotes, eukaryotic microbes 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," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0248] Suitable host cells for glycosylated antibody expression have also been derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Several baculovirus strains have been identified that can be used with insect cells, particularly for transduction of Spodoptera frogiperda cells.

[0249] 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 PLANTIBODIESTM technology for producing antibodies in transgenic plants).

[0250] Vertebrate cells may 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 include the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293 cells, described, for example, in Graham et al., J. Gen Virol. 36:59 (1977); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney (CV1) cells; African green monkey kidney (VERO-76) cells; human cervical cancer cells (HELA); canine kidney (MDCK) cells; Buffalo mouse liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.Other 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 some 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).

[0251] Measurements The anti-HLA-DQ2.5 antigen-binding molecules (antibodies) provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0252] Connectivity and other measurements In one aspect, an antibody of the invention is tested for its antigen binding activity, for example, by known methods such as ELISA, Western blot, etc.

[0253] In another aspect, a competition assay may be used to identify an antibody that competes with, for example, any of the antibodies listed above for binding to HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex). In certain embodiments, this competing antibody binds to the same epitope (e.g., a linear or structural epitope) as is bound by the antibodies listed above. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0254] In an exemplary competitive assay, immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is incubated in a solution containing a first labeled antibody that binds to HLA-DQ2.5 (or 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 HLA-DQ2.5 / gluten peptide complex). The second antibody may be present in the hybridoma supernatant. As a control, immobilized HLA-DQ2.5 (or 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 that allow binding of the first antibody to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex), excess unbound antibody is removed and the amount of label associated with the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is measured. If the amount of label associated with the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is greater than the amount of label associated with the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex),).5 / gluten peptide) is significantly reduced in the test sample compared to the control sample, thus indicating that the second antibody competes with the first 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).

[0255] Animals such as rabbits, mice, rats, and other animals suitable for immunization are immunized with an antigen (such as HLA-DQ2.5 or an HLA-DQ2.5 / gluten peptide complex). The antigen may be prepared as a recombinant protein using any of the methods mentioned herein, for example. Samples containing antibody, such as blood and spleen, are collected from the immunized animals. For example, for B cell selection, a biotinylated antigen is prepared and B cells that bind to the antigen are bound by the biotinylated antigen, and the cells are subjected to sorting and cell culture for selection. Specific binding of the cells to the antigen may be assessed by any suitable method, such as an ELISA. This method may also be used to assess the absence of cross-reactivity to non-target antigens. To isolate or sequence the selected antibody, for example, RNAs are purified from cells and DNAs encoding antibody regions are prepared by reverse transcription of the RNAs and PCR amplification.Additionally, the cloned antibody genes may be expressed in appropriate cells and the antibody may be purified from the culture supernatant for further analysis.

[0256] To test whether an anti-HLA-DQ2.5 antigen-binding molecule (antibody) binds to an antigen of interest (e.g., a complex formed by HLA-DQ2.5 and a gluten peptide such as those described herein), any method for assessing binding can be used. For example, when a FACS-based cell sorting method is used, cells expressing the antigen are incubated with the test antibody, and then an appropriate secondary antibody raised against the test antibody (i.e., primary) is added and incubated. Binding between the antigen and the test antibody is detected by FACS analysis, for example, using a chromogenic / fluorescent label attached to the secondary antibody (e.g., as described herein). Alternatively, any of the measurement methods listed under "Antibody Affinity" in this specification can be used.For example, Kd measurement by a BIACORE surface plasmon resonance assay can be used to assess the binding between the tested antibody and the antigen of interest mentioned herein.

[0257] In certain embodiments, the method of the present invention further comprises: testing whether the antibody has neutralizing activity against the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR (or the interaction between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and HLA-DQ2.5-restricted CD4+ T cells); and selecting an antibody that has neutralizing activity. In certain embodiments, the method of the present invention further comprises: testing whether the antibody has neutralizing activity against the binding between HLA-DQ2.2 (or HLA-DQ2.2 / gluten peptide complex) and TCR (or the interaction between HLA-DQ2.2 (or HLA-DQ2.2 / gluten peptide complex) and HLA-DQ2.2-restricted CD4+ T cells); and selecting an antibody that has neutralizing activity. These steps can be performed in the presence of a gluten peptide such as those described herein, for example, using HLA-DQ2.5 or HLA-DQ2.2 bound by the peptide. For example, neutralizing activity can be assessed as described herein.Briefly, beads such as yellow particles coated with streptavidin are prepared appropriately and soluble HLA-DQ bound to a peptide is added to the beads for immobilization on a plate. The plate is washed and blocked, and antibody is added and incubated. When the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR is assessed, for example, D2 TCR tetramer-PE may be added and incubated. The binding between the two may be assessed based on the chromogenic / fluorescent label of the TCR bound by HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex).

[0258] In some embodiments, the multispecific antigen-binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and a CD4+ T cell restricted by HLA-DQ2.5 / gluten peptide. In some embodiments, the multispecific antigen-binding molecule blocks the interaction between the HLA-DQ2.2 / gluten peptide complex and a CD4+ T cell restricted by HLA-DQ2.2 / gluten peptide. In this context, the gluten peptide is the peptide in the complex that is restricted by any of the antigen-binding molecules / domains described above. In some embodiments, the gluten peptide is selected from the group consisting of: alpha 1 gliadin peptide, alpha 1b gliadin peptide, alpha 2 gliadin peptide, omega 1 gliadin peptide, omega 2 gliadin peptide, gamma 1 gliadin peptide, gamma 2 gliadin peptide, gamma 3 gliadin peptide, gamma 4a gliadin peptide, gamma 4d gliadin peptide, and hordein BC peptide.

[0259] In some embodiments, the multispecific antigen-binding molecule has essentially no binding activity to HLA-DP, HLA-DR, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DQ7.5, and HLA-DQ8.

[0260] In some embodiments, the antigen-binding molecule of the invention has improved binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide. In this context, the gluten peptide may be any of the gluten peptides described above. The degree of improvement may be determined in comparison to binding activity to a complex formed by HLA-DQ2.5 and an unrelated peptide, or to a cell without the complex of interest, for example, an HLA-DQ2.5 positive PBMC B cell and / or a Ba / F3 cell expressing HLA-DQ2.5 or HLA-DQ2.2.

[0261] The bispecific antibody of the invention comprises a heavy chain and a light chain of a first arm / half-antibody and a heavy chain and a light chain of a second arm / half-antibody. The term "arm" or "half-antibody" refers to a portion of an antibody comprising a heavy chain and a light chain. In some embodiments, the bispecific antibody comprises the VH (heavy chain variable region) and VL (light chain variable region) of the first arm / half-antibody and the VH and VL of the second arm / half-antibody. In some embodiments, the bispecific antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the first arm / half-antibody and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the second arm / half-antibody.

[0262] In some embodiments, for the bispecific antibodies of the invention, the first antibody arm / half is derived from DQN0344xx (DQN0344Hx / DQN0344Lx) described herein and the second antibody arm / half is derived from DQN0385ee (DQN0385He / DQN0385Le). The sequence identification numbers (SINs) of the VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 and the full-length heavy (H) and light (L) chains of the bispecific antibodies of the invention are shown in Tables 2-3 to 2-6 (below). In some embodiments, the antibody of the invention comprises: HCDR1 comprising the sequence of SEQ ID NO: 129 or 164; HCDR2 comprising the sequence of SEQ ID NO: 130 or 165; and HCDR3 comprising the sequence of SEQ ID NO: 131 or 166. In some embodiments, the antibody of the invention comprises: LCDR1 comprising the sequence of SEQ ID NO: 132 or 167; LCDR2 comprising the sequence of SEQ ID NO: 133 or 168; and LCDR3 comprising the sequence of SEQ ID NO: 134 or 169. In some embodiments, the antibody of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 88 or 89. In some embodiments, the antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 105 or 162. In some embodiments, the antibody of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 90 or 91. In some embodiments, the antibody of the invention comprises a light chain constant region comprising the sequence of SEQ ID NO: 106. In some embodiments, the antibody of the invention comprises: a heavy chain (full length) comprising the sequence of SEQ ID NO: 41, 42, 44 or 45. In some embodiments, the antibody of the invention comprises: a light chain (full length) comprising the sequence of SEQ ID NO: 43 or 46. In some embodiments, the antibody of the invention comprises: HCDR1 comprising the sequence of SEQ ID NO: 135, 144, 147, 153, 156, or 159; HCDR2 comprising the sequence of SEQ ID NO: 136, 145, 148, 154, 157, or 160; and HCDR3 comprising the sequence of SEQ ID NO: 137, 146, 149, 155, 158, or 161. In some embodiments, the antibody of the invention comprises: LCDR1 comprising the sequence of SEQ ID NO: 141, or 150; LCDR2 comprising the sequence of SEQ ID NO: 142, or 151; and LCDR3 comprising the sequence of SEQ ID NO: 140, 143, or 152. In some embodiments, the antibody of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 92, 93, 94, 95, 96, or 97. In some embodiments, the antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 104 or 163. In some embodiments, the antibody of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 98, 99, or 100. In some embodiments, the antibody of the invention comprises a light chain constant region comprising the sequence of SEQ ID NO: 107. In some embodiments, the antibody of the invention comprises: a heavy chain (full length) comprising the sequence of SEQ ID NO: 53, 54, 57, 58, 59, 60, 62, 63, 64, 65, 66, or 67. In some embodiments, the antibody of the invention comprises: a light chain (full length) comprising the sequence of SEQ ID NO: 55, 56 or 61.

[0263] The specific sequences of the full-length H and L chains for the arms / half-antibodies (present in the bispecific antibodies of the invention) are shown in Table 1-2.

[0264] [Table 1-2] Full-length H and L chains for arms / half-antibodies The H (or L) chains, from the N-terminus to the C-terminus, include HCDR1, HCDR2, and HCDR3 (or LCDR1, LCDR2, and LCDR3), which are underlined in this table.

[0265] In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding moiety comprises any of the following (a1) to (a3): (a1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (a2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (a3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (a1) or (a2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (a1) or (a2). In some embodiments, in the multispecific antigen-binding molecule, the second antigen-binding moiety comprises any of the following (b1) to (b8): (b1) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (b2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b3) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b4) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b5) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b6) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b7) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (b8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (b1) to (b7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (b1) to (b7). In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding moiety comprises any of the following (c1) to (c3): (c1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (c2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166 and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (c3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (c1) or (c2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (c1) or (c2), wherein the second antigen-binding moiety comprises any of the following (d1) to (d8): (d1) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (d2) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d3) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d4) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d5) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d6) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d7) a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161 and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (d8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (d1) to (d7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (d1) to (d7). In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion comprising the first and second variable regions of an antibody and a second antigen-binding portion comprising the third and fourth variable regions of an antibody, wherein the multispecific antigen-binding molecule comprises any of (1) to (15) below: (1) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (2) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (3) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (4) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (5) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (6) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (7) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (8) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (9) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (10) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (11) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (12) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (13) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (14) a first antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (15) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in any of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in any of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (1) to (14). In some embodiments, in the multispecific antigen-binding molecule, the antibody variable region present in the first and / or second antigen-binding portion comprises human antibody frameworks or humanized antibody frameworks. In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion; wherein the first antigen-binding moiety comprises any of the following (e1) to (e3): (e1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; (e2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 91; and (e3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the first variable region of the antibody expressed in (e1) or (e2) and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the second variable region of the antibody expressed in (e1) or (e2). In some embodiments, in the multispecific antigen-binding molecule, the second antigen-binding moiety comprises any of the following (f1) to (f8): (f1) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 98; (f2) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 92 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f3) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 93 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f4) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 94 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f5) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f6) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 96 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; (f7) a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 97 and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 99; (f8) a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the third variable region of the antibody expressed in any of (f1) to (f7) and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence similarity to the fourth variable region of the antibody expressed in any of (f1) to (f7). In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion; wherein the first antigen-binding moiety comprises any of the following (e1) to (e3): (e1) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88 and an antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; (e2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 89 and an antibody vari...

Claims

[ادعاها] [ادعای 1] یک مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن که به یک کمپلکس تشکیل شده توسط HLA-DQ2.5 و یک پپتید گلوتن متصل می‌شود و که شامل یک قسمت اول متصل‌شونده به آنتی‌ژن شامل نواحی متغیر آنتی‌بادی اول و دوم و یک قسمت دوم متصل‌شونده به آنتی‌ژن شامل نواحی متغیر آنتی‌بادی سوم و چهارم است که در آن مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن شامل هر یک از (1) تا (14) زیر است: (1) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 95؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100؛ (2) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 92؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 98؛ (3) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 92؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99؛ (4) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 93؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99؛ (5) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 94؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100؛ (6) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 96؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100؛ (7) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 88؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 90؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 97؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99؛ (8) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 92؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 98؛ (9) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ و یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 92؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99؛ (10) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 93؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99؛ (11) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 94؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100؛ (12) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 95؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100؛ (13) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 96؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 100 و (14) یک ناحیه متغیر اول آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 89؛ یک ناحیه متغیر دوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 91؛ یک ناحیه متغیر سوم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 97؛ و یک ناحیه متغیر چهارم آنتی‌بادی شامل توالی اسیدآمینه از شماره شناسایی توالی: 99.[ادعای 2] یک مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن که به یک کمپلکس تشکیل شده توسط HLA-DQ2.5 و یک پپتید گلوتن متصل می‌‌شود، شامل یک ترکیب از چهار زنجیره پلی پپتیدی انتخاب شده از گروه متشکل از (1) تا (14) زیر: (1) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 63 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61؛ (2) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 54 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 55؛ (3) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 54 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (4) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 58 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (5) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 60 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61؛ (6) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 54 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 55؛ (7) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 65 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61؛ (8) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 54 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (9) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 58 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (10) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 67 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (11) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 65 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61؛ (12) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 42 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 43 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 67 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 56؛ (13) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 63 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61 و (14) یک زنجیره سنگین اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 45 و یک زنجیره سبک اول شامل توالی اسیدآمینه از شماره شناسایی توالی: 46 و یک زنجیره سنگین دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 60 و یک زنجیره سبک دوم شامل توالی اسیدآمینه از شماره شناسایی توالی: 61.[ادعا 3] مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن ادعای 1 یا 2، که یک آنتی‌ بادی دو اختصاصی است.[ادعای 4] یک اسید نوکلئیک که مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن هر یک از ادعاهای 1 تا 3 را کد می‌کند.[ادعای 5] یک وکتور شامل اسید نوکلئیک ادعای 4.[ادعای 6] یک سلول شامل اسید نوکلئیک ادعای 4 یا وکتور ادعای 5.[ادعای 7] یک روش برای تولید یک مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن شامل کشت سلول ادعای 6 به‌طوری‌که مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن تولید شود.[ادعای 8] روش ادعای 7 که علاوه بر این شامل بازیابی مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن از کشت سلول است.[ادعای 9] یک ترکیب دارویی شامل مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن هر یک از ادعاهای 1 تا 3 و یک حامل قابل‌قبول دارویی.[ادعای 10] ترکیب ادعای 9 که یک ترکیب دارویی برای استفاده در درمان و / یا پیشگیری از بیماری سلیاک است.[ادعای 11] استفاده از مولکول چند اختصاصی متصل‌شونده به آنتی‌ژن هر یک از ادعاهای 1 تا 3 در ساخت یک دارو.[ادعای 12] استفاده از ادعای 11 که در آن دارو، یک دارو برای درمان و / یا پیشگیری از بیماری سلیاک است. ***

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