Anti-HLA-DQ2.5 antibodies and their use in the treatment of celiac disease

Abstract

This invention relates to an anti-HLA-DQ2.5 antibody and its use for the treatment of celiac disease. [Solution] The present invention provides a modified anti-HLA-DQ2.5 antibody. The anti-HLA-DQ2.5 antibody of the present invention has binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide, but substantially no binding activity to a complex formed by HLA-DQ2.5 and a peptide unrelated to HLA-DQ2.5. Furthermore, the antibody of the present invention has been shown to have an inhibitory effect on T cell activation by gluten peptides.

Description

[Technical Field]

[0001] This invention relates to an anti-HLA-DQ2.5 antibody and its use for the treatment of celiac disease. [Background technology]

[0002] Celiac disease is an autoimmune disorder in which gluten intake causes damage to the small intestine in genetically susceptible individuals (Non-Patent Documents 1-5). It is estimated that about 1% of the population in Europe and North America, or 8 million people in the United States and the European Union, suffers from celiac disease; however, no significant therapeutic progress has been made since the disease was recognized in the 1940s. Human leukocyte antigens (HLAs) belonging to major histocompatibility complex (MHC) class II include HLA-DR, HLA-DP, and HLA-DQ molecules, such as the HLA-DQ2.5 isoform (hereinafter referred to as "HLA-DQ2.5" herein), which form heterodimers composed of α and β chains on the cell surface. The majority (>90%) of celiac disease patients have alleles of the HLA-DQ2.5 haplotype (Non-Patent Literature 6). This 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 T cell receptors (TCRs) on T cells. In celiac disease patients, immunogenic gluten peptides, such as gliadin peptides, are formed as a result of digesting high-gluten foods such as bread (Non-Patent Literature 2). The peptide is transported through the small intestinal epithelium to the lamina propria and deamidated by tissue transglutaminases such as transglutaminase 2 (TG2). The deamidated gliadin peptide is processed by antigen-presenting cells (APCs), which load them onto HLA-DQ2.5. The loaded peptide is presented to HLA-DQ2.5-restricted T cells, activating innate and adaptive immune responses. This causes inflammatory damage to the small intestinal mucosa as well as symptoms including various types of gastrointestinal disorders, nutritional deficiencies, and systemic symptoms (Non-Patent Literature 8, 9, and 10). Anti-HLA DQ neutralizing antibodies have been reported to inhibit gluten peptide-dependent activation of T cells derived from celiac disease patients (Non-Patent Literature 7). Currently, the only viable treatment for celiac disease is lifelong adherence to a gluten-free diet (GFD). However, in reality, completely eliminating gluten exposure is difficult even with a GFD. The tolerable amount of gluten for these patients is only about 10-50 mg / day (Non-Patent Literature 11). Secondary contamination can occur widely during GFD manufacturing, and even in patients who strictly adhere to a GFD, trace amounts of gluten can trigger symptoms of celiac disease. Given the risk of such unintentional gluten exposure, adjunctive therapies are needed for GFD. [Prior art documents] [Non-patent literature]

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

[0004] technical challenges In the above-mentioned circumstances requiring adjunctive therapy, the present invention provides an anti-HLA-DQ2.5 antigen-binding molecule. [Means for solving the problem]

[0005] Solving the problem The antigen-binding molecule of the present invention has been modified to be able to bind to two or more complexes formed by HLA-DQ2.5 and gluten peptides.

[0006] More specifically, the present invention provides the following: [1] (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 binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide. A multispecific antigen-binding molecule containing, The antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides, At least one gluten peptide in the complex to which the first antigen-binding moiety binds is different from at least one gluten peptide in the complex to which the second antigen-binding moiety binds; and The antigen-binding molecule has substantially no binding activity to either or both HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5. The antigen-binding molecule is humanized, and One or more amino acids in the heavy and / or light chains of the first antigen-binding moiety and / or second antigen-binding moiety in the multispecific antigen-binding molecule are modified. Multispecific antigen binding molecules. [1a] The multispecific antigen-binding molecule of [1], wherein the antigen-binding molecule substantially has no binding activity to Ba / F3 cells expressing HLA-DQ2.2. [1-1] A multispecific antigen-binding molecule of [1] or [1a], wherein one or more amino acids in the heavy chain and / or light chain of the first antigen-binding moiety and / or second antigen-binding moiety are substituted. [1-2] A multispecific antigen-binding molecule of [1-1] comprising at least one amino acid substitution in the variable region of the heavy chain; at least one amino acid substitution in the constant region of the heavy chain; at least one amino acid substitution in the variable region of the light chain; and at least one amino acid substitution in the constant region of the light chain. [2] A gluten peptide is one of the multispecific antigen-binding molecules from [1] to [1-2], which is an immunodominant peptide associated with celiac disease. [3] A gluten peptide is selected from the group consisting of 33mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4a gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secarin 1 peptide, secarin 2 peptide, and 26mer gliadin peptide, and one of the multispecific antigen-binding molecules from [1] to [2]. [3-1] A multispecific antigen-binding molecule from any one of [1]-[2], wherein the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, three, four, eight, nine, ten, eleven, twelve, three, four, eight, nine, six, seven, eight, nine, six, ten, eleven, twelve, three, four, seven, eight, nine, six [3-2] A gluten peptide is selected from the group consisting of 33mer gliadin peptide, α1 gliadin peptide, α2 gliadin peptide, γ1 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4a gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secarin 1 peptide, secarin 2 peptide, and 26mer gliadin peptide, and one of the multispecific antigen-binding molecules from [1] to [2]. [3-3] A multispecific antigen-binding molecule from any one of [1]-[2], wherein the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, 12, 13, four, f, six, seven, f, six, six, six, seven, eight, nine, ten, eleven, f [4] Any one of the multispecific antigen-binding molecules from [1] to [3-3], wherein the multispecific antigen-binding molecule substantially lacks binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, the unrelated peptide being at least one peptide selected from the group consisting of CLIP peptide, hepatitis B virus 1 peptide, salmonella peptide, mycobacterium bovis peptide, and tyroperoxidase peptide. [4-1] A multispecific antigen-binding molecule from any one of [1] to [3-3], which substantially lacks binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, wherein the unrelated peptide is any of the following: CLIP peptide, hepatitis B virus 1 peptide, salmonella peptide, mycobacterium bobis peptide, and tyroperoxidase peptide. [5] A multispecific antigen-binding molecule from [1] to [4-1] that has substantially no binding activity to HLA-DP, HLA-DR, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DQ7.5, and HLA-DQ8. [6] Any one of the multispecific antigen-binding molecules from [1] to [5] that (i) block the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells, and / or (ii) block the interaction between the HLA-DQ2.2 / gluten peptide complex and HLA-DQ2.2 / gluten peptide-restricted CD4+ T cells. [6-2] A multispecific antigen-binding molecule of [6] in which the gluten peptide is selected from the group consisting of α1 gliadin peptide, α1b gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ3 gliadin peptide, γ4a gliadin peptide, γ4d gliadin peptide, and BC hordein peptide. [7] One of the multispecific antigen-binding molecules from [1] to [6-2] having enhanced binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide, compared to before humanization and modification. [8] One of the multispecific antigen-binding molecules from [1] to [7] having enhanced cross-reactivity to gluten peptides compared to before humanization and modification. [8-1] A multispecific antigen-binding molecule of [8], wherein the gluten peptides are ω2 gliadin peptide, BC hordein peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ4a gliadin peptide, and γ4d gliadin peptide. [9] One of the multispecific antigen-binding molecules from [1] to [8-1], wherein one, two, three, or all of the amino acid residue sets selected from the group consisting of the following (a) to (d) sets in the heavy and light chains of the antigen-binding molecule are amino acid residues that repel each other electrostatically: (a) The amino acid residue in the heavy chain constant region (CH1) at position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) at position 131 according to Kabat numbering, (b) 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 Kabat numbering, (c) The amino acid residue in CH1 at position 175 according to EU numbering, and the amino acid residues in CL at positions 131 and 160 according to Kabat numbering, (d) 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.

[10] Furthermore, a multispecific antigen-binding molecule [9] in which two or more amino acid residues that form the interface between the heavy chain variable region and the light chain variable region are electrically repelling amino acid residues from one another.

[11] A multispecific antigen-binding molecule of

[10] in which the electrostatically repelling amino acid residues 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) The amino acid residue in the heavy chain variable region at position 39 according to Kabat numbering, and the amino acid residue in the light chain variable region at position 38 according to Kabat numbering, (b) An amino acid residue in the heavy chain variable region at position 45 according to Kabat numbering, and an amino acid residue in the light chain variable region at position 44 according to Kabat numbering.

[12] One of the multispecific antigen-binding molecules from [9] to

[11] , in which amino acid residues that repel each other electrostatically are selected from amino acid residues included in either set (X) or (Y) below: (X) Glutamic acid (E), aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[13] A multispecific antigen-binding molecule comprising any one of [9] to

[12] , further comprising an Fc domain that exhibits reduced binding affinity to the human Fcγ receptor compared to the native human IgG1 Fc domain.

[14] A multispecific antigen-binding molecule of

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

[15] A multispecific antigen-binding molecule

[13] or

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

[16] The multispecific antigen-binding molecule of

[15] in which the Fc domain contains (e1) or (e2) below, and the amino acid positions are numbered according to EU numbering: (e1) A first Fc region subunit including Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region including Cys at position 354 and Trp at position 366; (e2) A first Fc region subunit containing Glu at position 439, and a second Fc region containing Lys at position 356.

[17] Any one of the

[13] -

[16] multispecific antigen-binding molecules in which the Fc domain exhibits stronger FcRn binding affinity to human FcRn compared to the native human IgG1 Fc domain.

[18] A multispecific antigen-binding molecule of

[16] in which the first and / or second Fc domain subunit comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and the amino acid positions are numbered according to EU numbering.

[19] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xii): [1] to [8] (i) Glutamate or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) Glutamate at position 147 (EU numbering) in the heavy chain constant region; (iii) Glutamate or lysine at position 131 (Kabat numbering) in the constant region of the light chain; (iv) Glutamate or lysine at position 160 (Kabat numbering) in the constant region of the light chain; (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) Glutamate at position 439 (EU numbering) in the heavy chain constant region; (xii) Glutamate at position 440 (EU numbering) in the heavy chain constant region. [19-1] A bispecific antibody containing the following multispecific antigen-binding molecules of

[19] : The first heavy chain contains 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); The first light chain contains glutamic acid at position 131 (Kabat numbering) and glutamic acid at position 160 (Kabat numbering); The second heavy chain contains 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 containing lysine at position 131 (Kabat numbering) and lysine at position 160 (Kabat numbering). [19-2] The first heavy chain further comprises glutamic acid at position 419 (EU numbering) and proline at position 445 (EU numbering), as well as amino acid deletions at positions 446 and 447 (EU numbering); and The second heavy chain further includes lysine at position 196 (EU numbering), proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). [19-1] A multispecific antigen-binding molecule. [19-3] The first heavy chain further comprises 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 further 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 further 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 further 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). A multispecific antigen-binding molecule of [19-1] or [19-2]. [19a] A multispecific antigen-binding molecule from [1] to [19-3] that has substantially no binding activity to the gluten peptide itself.

[20] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, A multispecific antigen-binding molecule in which the first antigen-binding moiety contains one of the following (a1) to (a3): (a1) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131, and a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and 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, and 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, and 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 identity with respect to a first antibody variable region described in (a1) or (a2), and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a second antibody variable region described in (a1) or (a2).

[21] A multispecific antigen-binding molecule of

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

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

[23] A multispecific antigen-binding molecule comprising an antibody variable region in a first antigen-binding moiety and / or a second antigen-binding moiety, comprising a human antibody framework or a humanized antibody framework, one of the

[20] -[22-2].

[24] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, A multispecific antigen-binding molecule in which the first antigen-binding moiety contains one of the following (e1) to (e3): (e1) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88, and a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; (e2) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89, and a second antibody variable region containing 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 identity with respect to a first antibody variable region described in (e1) or (e2), and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a second antibody variable region described in (e1) or (e2).

[25] A multispecific antigen-binding molecule of

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

[26] A multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, The first antigen-binding site is as follows (e1)~(e3): (e1) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88, and a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; (e2) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89, and a second antibody variable region containing 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 identity with respect to a first antibody variable region described in (e1) or (e2), and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a second antibody variable region described in (e1) or (e2). It includes any one of the following, and The second antigen-binding region is as follows (f1)~(f8): (f1) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 92, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 98; (f2) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 92, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (f3) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 93, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (f4) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 94, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (f5) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 95, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (f6) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 96, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (f7) A third antibody variable region containing the amino acid sequence of SEQ ID NO: 97, and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; and (f8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a third antibody variable region described in any one of (f1) to (f7), and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a fourth antibody variable region described in any one of (f1) to (f7). A multispecific antigen-binding molecule containing one of the following. [26-2] A multispecific antigen-binding molecule comprising a first antigen-binding portion containing first and second antibody variable regions and a second antigen-binding portion containing third and fourth antibody variable regions, comprising any one of the following (1) to (15): (1) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 98; (2) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; and a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (3) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 93; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (4) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 94; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (5) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 95; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (6) First antibody variable region containing the amino acid sequence of SEQ ID NO: 88; second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; third antibody variable region containing the amino acid sequence of SEQ ID NO: 96; fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (7) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 88; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 90; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 97; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (8) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 98; (9) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 92; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (10) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 93; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (11) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 94; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (12) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 95; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (13) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 96; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 100; (14) A first antibody variable region containing the amino acid sequence of SEQ ID NO: 89; a second antibody variable region containing the amino acid sequence of SEQ ID NO: 91; a third antibody variable region containing the amino acid sequence of SEQ ID NO: 97; and a fourth antibody variable region containing the amino acid sequence of SEQ ID NO: 99; (15) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a first antibody variable region described in any one of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second antibody variable region described in any one of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a third antibody variable region described in any one of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a fourth antibody variable region described 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 containing the amino acid sequence of SEQ ID NO: 42, and a first light chain containing the amino acid sequence of SEQ ID NO: 43; (A2) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45, and a first light chain containing the amino acid sequence of SEQ ID NO: 46; and (A3) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a first heavy chain sequence described in (A1) or (A2), and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a first light chain sequence described 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 containing the amino acid sequence of SEQ ID NO: 41, and a first light chain containing the amino acid sequence of SEQ ID NO: 43; (A2) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44, and a first light chain containing the amino acid sequence of SEQ ID NO: 46; and (A3) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a first heavy chain sequence described in (A1) or (A2), and a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with respect to a first light chain sequence described in (A1) or (A2).

[28] A multispecific antigen-binding molecule of

[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 containing the amino acid sequence of SEQ ID NO: 54, and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (B2) A second heavy chain containing the amino acid sequence of SEQ ID NO: 54, and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (B3) A second heavy chain containing the amino acid sequence of SEQ ID NO: 58, and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (B4) A second heavy chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B5) A second heavy chain containing the amino acid sequence of SEQ ID NO: 63, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B6) A second heavy chain containing the amino acid sequence of SEQ ID NO: 65, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B7) A second heavy chain containing the amino acid sequence of SEQ ID NO: 67, and a second light chain containing 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 identity with a second heavy chain sequence described in any one of (B1) to (B7), and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second light chain sequence described in any one of (B1) to (B7). [28-2] A multispecific antigen-binding molecule of

[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 containing the amino acid sequence of SEQ ID NO: 53, and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (B2) A second heavy chain containing the amino acid sequence of SEQ ID NO: 53, and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (B3) A second heavy chain containing the amino acid sequence of SEQ ID NO: 57, and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (B4) A second heavy chain containing the amino acid sequence of SEQ ID NO: 59, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B5) A second heavy chain containing the amino acid sequence of SEQ ID NO: 62, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B6) A second heavy chain containing the amino acid sequence of SEQ ID NO: 64, and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (B7) A second heavy chain containing the amino acid sequence of SEQ ID NO: 66, and a second light chain containing 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 identity with a second heavy chain sequence described in any one of (B1) to (B7), and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second light chain sequence described in any one 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 containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 54 and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (2) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 54 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (3) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (4) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (5) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 63 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (6) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 54 and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (7) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 65 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (8) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 54 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (9) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (10) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 67 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (11) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 65 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (12) A first heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 67 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (13) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 63 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (14) A first heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a second light chain containing 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 identity with a first heavy chain sequence described in any one of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a first light chain sequence described in any one of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second heavy chain sequence described in any one of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second light chain sequence described in any one 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 containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (2) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (3) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 57 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (4) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (5) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 62 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (6) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a second light chain containing the amino acid sequence of SEQ ID NO: 55; (7) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 64 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (8) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (9) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 57 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (10) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 66 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (11) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 64 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (12) A first heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a first light chain containing the amino acid sequence of SEQ ID NO: 43, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 66 and a second light chain containing the amino acid sequence of SEQ ID NO: 56; (13) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 62 and a second light chain containing the amino acid sequence of SEQ ID NO: 61; (14) A first heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a first light chain containing the amino acid sequence of SEQ ID NO: 46, and a second heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a second light chain containing 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 identity with a first heavy chain sequence described in any one of (1) to (14); a second amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a first light chain sequence described in any one of (1) to (14); a third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second heavy chain sequence described in any one of (1) to (14); and a fourth amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a second light chain sequence described in any one of (1) to (14). [29a] Any one combination of (i) to (iii) below: (i) A multispecific antigen-binding molecule containing a sequence described in any one of (a1) to (a3) ​​of

[20] , and a multispecific antigen-binding molecule containing a sequence described in any one of (b1) to (b8) of

[21] ; (ii) A multispecific antigen-binding molecule containing a sequence described in any one of (e1) to (e3) of

[24] , and a multispecific antigen-binding molecule containing a sequence described in any one of (f1) to (f8) of

[25] ; and (iii) A multispecific antigen-binding molecule containing a sequence described in any one of (A1) to (A3) of

[27] or [27-2], and a multispecific antigen-binding molecule containing a sequence described in any one of (B1) to (B8) of

[28] or [28-2].

[30] A nucleic acid that encodes one of the multispecific antigen-binding molecules from [1] to

[29] . A vector containing the nucleic acids

[31]

[30] . A cell containing nucleic acids of

[32]

[30] or vectors of

[31] .

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

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

[34] The method of

[33] , further comprising the step of recovering the multispecific antigen-binding molecule from the cell culture. A pharmaceutical composition comprising one of the multispecific antigen-binding molecules from [1] to

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

[36] A composition of

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

[37] Use of any one of the multispecific antigen-binding molecules from [1] to

[29] or any combination of [29a] in the manufacture of pharmaceuticals.

[38] Use of

[37] the drug being a drug for the treatment and / or prevention of celiac disease.

[39] A method for treating an individual having celiac disease, comprising the step of administering to the individual an effective amount of one multispecific antigen-binding molecule from [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 from [1] to

[29] or a combination of [29a], and instructions for use. [Brief explanation of the drawing]

[0007] [Figure 1-1] Figure 1-1 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 micrograms (μg) / mL, while the control DQN0139bb (DQN0139bb-SG181) (WO2018 / 155692) and IC17dK were tested at 1 μg / mL). In the names of the gluten peptides, "a", "g", and "w" represent "α", "γ", and "ω", respectively. [Figure 1-2]Figure 1-2 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1270 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-3] Figures 1-3 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1352 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-4] Figures 1-4 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1527 / L0605-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-5] Figures 1-5 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1255 / L0605-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-6] Figures 1-6 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1270 / L0722-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-7]Figures 1-7 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1521 / L0605-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-8] Figures 1-8 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1270 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-9] Figures 1-9 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1352 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-10] Figure 1-10 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (all antibodies were tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-11] Figure 1-11 shows the binding results of an anti-HLA-DQ antibody (variant DQN0344H0976 / L0591 / / DQN0385H1251 / L0605-F6) to a Ba / F3 cell line expressing HLA class II (the antibody was tested at 0.05 μg / mL, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, the antibody was tested at 0.313 μg / mL, while the controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-12]Figure 1-12 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6) to Ba / F3 cell lines expressing HLA class II (antibodies were tested at 0.05 μg / mL, while 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, while controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-13] Figure 1-13 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6) to Ba / F3 cell lines expressing HLA class II (antibodies were tested at 0.05 μg / mL, while 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, while controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-14] Figure 1-14 shows the binding results of anti-HLA-DQ antibodies (variants) to HLA-DP, DR, DQ5.1, and DQ6.3 (all antibodies were tested at 0.05 μg / mL, while the control antibodies DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-15] Figure 1-15 shows the binding results of DQN0139bb to Ba / F3 cell lines expressing HLA class II (control DQN0139bb was tested at 1 μg / mL). [Figure 1-16] Figure 1-16 shows the IC17dK results for Ba / F3 cell lines expressing HLA class II (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, while the controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5-CLIP, antibodies were tested at 0.313 μg / mL; and the controls DQN0139bb and IC17dK were tested at 20 ug / mL). [Figure 3-1] Figure 3-1 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / α1 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 / α2 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 / α1b 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 / ω1 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 / ω2 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 / BC-Hordein-dependent Jurkat T cell activation. [Figure 3-7] Figure 3-7 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ1 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 / γ2 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 / γ3 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 / γ4a 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 / α1 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 / α2 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 / α1b 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 / ω1 gliadin-dependent Jurkat T cell activation. [Figure 4-5] Figures 4-5 show the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / ω2 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 / BC-Hordein-dependent Jurkat T cell activation. [Figure 4-7] Figure 4-7 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ1 gliadin-dependent Jurkat T cell activation. [Figure 4-8] Figure 4-8 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ2 gliadin-dependent Jurkat T cell activation. [Figure 4-9] Figure 4-9 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ3 gliadin-dependent Jurkat T cell activation. [Figure 4-10] Figure 4-10 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ4a gliadin-dependent Jurkat T cell activation. [Figure 5-1]Figure 5-1 shows HLA-DQ2.2 / α1α gliadin-dependent Jurkat T cell activation mediated by 33-mer gliadin. [Figure 5-2] Figure 5-2 shows HLA-DQ2.2 / α2 gliadin-dependent Jurkat T cell activation mediated by 33-mer gliadin. [Figure 5-3] Figure 5-3 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.2 / α1α gliadin-dependent Jurkat T cell activation. [Figure 5-4] Figure 5-4 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.2 / α2 gliadin-dependent Jurkat T cell activation. [Modes for carrying out the invention]

[0008] Description of the manner The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methodologies, such as the widely used methodologies described below: 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 PE Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG 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);およびCancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993)。.

[0009] In the spirit of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

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

[0011] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[0012] The term "antigen-binding moiety" or "antigen-binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to a part or all of an antigen. An antigen-binding moiety / domain may be provided, for example, by one or more antibody-variable domains (also called antibody-variable regions). Preferably, the antigen-binding moiety / domain contains 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 can bind with sufficient affinity to HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and gluten peptides, and as a result, is useful as a diagnostic and / or therapeutic agent when the antibody targets HLA-DQ2.5. In one embodiment, the degree of binding of an anti-HLA-DQ2.5 antigen-binding molecule (antibody) to an unrelated antigen is less than approximately 10% of the binding of the antibody to HLA-DQ2.5 or HLA-DQ2.5 / gluten peptide complexes, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, antibodies having "binding activity" to HLA-DQ2.5 or HLA-DQ2.5 / gluten peptide complexes are defined as having a concentration of ≤1 micromol (μM), ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0014] As used herein, the term “antigen-binding molecule” refers to any molecule containing an antigen-binding site or any molecule having antigen-binding activity, and may also refer to molecules such as peptides or proteins having a length of approximately 5 amino acids or more. Peptides and proteins are not limited to those of biological origin; for example, they may be polypeptides produced from artificially designed sequences. They may be naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, scaffold molecules containing known stable three-dimensional structures, such as α / β barrels (where a portion of the molecule becomes the antigen-binding site), are also a form of antigen-binding molecule as described herein. In some embodiments, the “antigen-binding molecule” is an antibody. In this specification, the terms “antigen-binding molecule” and “antibody” are used in their broadest sense and encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody is a multispecific antibody. In some embodiments, a multispecific antibody is a bispecific antibody.

[0015] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0016] An antibody that "binds to the same epitope as the reference antibody" is defined as an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay. Conversely, the reference antibody blocks the binding of the aforementioned antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0017] An "autoimmune disease" is a non-malignant disease or disorder that originates from and is directed toward the tissues of an individual. In this specification, an autoimmune disease explicitly excludes malignant or cancerous diseases or conditions, and in particular excludes 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, the following: inflammatory reactions such as celiac disease, psoriasis, and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome: ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma, and other conditions with T-cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (Including but not limited to lupus nephritis and cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes mellitus; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte leakage; central nervous system (CNS) Inflammatory disorders; multiple organ injury syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disorders; anti-glomerular basement membrane disorders; antiphospholipid syndromes; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorders; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0018] The term "celiac disease" refers to a hereditary autoimmune disorder caused by damage to the small intestine resulting from the ingestion of gluten in food. Symptoms of celiac disease include, but are not limited to, gastrointestinal disorders such as abdominal pain, diarrhea, and gastroesophageal reflux; central nervous system (CNS) symptoms such as vitamin deficiencies, mineral deficiencies, fatigue, and anxiety and depression; bone symptoms such as osteomalacia and osteoporosis; skin symptoms such as dermatitis; blood symptoms such as anemia and lymphopenia; and other symptoms such as infertility, hypogonadism, and growth retardation and short stature in children.

[0019] The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0020] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0021] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period of time that is effective in achieving the desired therapeutic or prophylactic outcome.

[0022] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (residue Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

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

[0024] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0025] In this specification, the term “gluten” refers collectively to a complex of storage proteins called prolamins found in wheat and other related cereals. In the lumen of 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, secarin from rye, and avenin from oat.

[0026] In celiac disease, gluten peptides are antigenic peptides recognized by T cells and are the cause of the disease. Immunodominance is a phenomenon in which the immune response is mainly triggered by a relatively small number of antigenic peptides. Such antigenic peptides are called "immunodominant peptides." In celiac disease, such immunodominant peptides include, for example, α1 gliadin (also called "α1α gliadin") and α2 gliadin (both included in the 33mer gliadin sequence), as well as ω1 gliadin, ω2 gliadin, and BC-hordein (a total of 5 peptides) (Science Translational Medicine 21 Jul 2010: Vol. 2, Issue 41, pp. 41ra51). Alternatively, immunodominant peptides include, but are not limited to, α1 gliadin, α2 gliadin, ω1 gliadin, ω2 gliadin, BC-hordein, γ1 gliadin, and γ2 gliadin (a total of 7 peptides). In this specification, such immunodominant peptides may be referred to as “immunodominant peptides associated with celiac disease.” The types and total number of such peptides are not particularly limited, as long as they are predominantly associated with celiac disease.

[0027] As used herein, the phrase "substantially no binding activity" refers to the activity of an antibody that binds to a non-target antigen at a binding level that includes non-specific binding or background binding but does not include specific binding. In other words, such an antibody "does not have specific / significant binding activity" to the non-target antigen. Specificity can be measured by any method described herein or known in the art. The level of non-specific binding or background binding may be zero, not zero but close to zero, or so low that it can be technically ignored by a person skilled in the art. For example, if a person skilled in the art cannot detect or observe any significant (or relatively strong) signal of binding between the antibody and the non-target antigen in a suitable binding assay, the antibody can be said to "substantially no binding activity" or "does not have specific / significant binding activity" to the non-target antigen. Alternatively, "substantially no binding activity" or "does not have specific / significant binding activity" can be rephrased as "does not specifically / significantly / substantially bind" (to the non-target antigen). Sometimes, the phrase "lack of binding activity" has substantially the same meaning in this technical field as the phrases "substantially no binding activity" or "lack of specific / significant binding activity."

[0028] In this specification, "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 alleles of the corresponding haplotype on 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, the HLA-DQ molecules include, but are not limited to, known subtypes (isoforms) of HLA-DQ molecules 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 the HLA-DR(DP) isoform.

[0029] The terms “host cell,” “host cell line,” and “host cell culture” refer to “cells” (including offspring of such cells) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and offspring derived from those cells, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0030] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0031] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected groups of human immunoglobulin VL or VH framework sequences. The term "Human Antibody Framework" may also be used to refer to this framework. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described 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 subgroup κI by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al.

[0032] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In one embodiment, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0033] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (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) Antigen contact 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 shown herein. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0034] An "immunoconjugate" is an antibody that has been conjugated to one or more different molecules.

[0035] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0036] In the present invention, when evaluating the binding of an anti-HLA-DQ2.5 antibody to HLA-DQ molecules such as HLA-DQ2.5, HLA-DQ2.2, and HLA-DQ7.5, CLIP peptides may be used together with the appropriate HLA-DQ molecules mentioned above.

[0037] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, 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 evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0038] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0039] An isolated nucleic acid encoding an anti-HLA-DQ2.5 antigen-binding molecule (antibody) (also simply called an "anti-HLA-DQ2.5 antigen-binding molecule (antibody) encoding nucleic acid") refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules mounted on one or more vectors, and nucleic acid molecules present at one or more locations within a host cell.

[0040] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0041] A "naked antibody" is an antibody that is not conjugated with a different part or radioactive label. Naked antibodies may be present in pharmaceutical preparations.

[0042] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed 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 a variable heavy chain domain or heavy chain variable 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 a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of an antibody may be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0043] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a sequence of bases, where these bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases containing derivatized sugars or phosphate backbone links or chemically modified residues. The nucleic acid molecules also include DNA and RNA molecules suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a target to produce antibodies in vivo (see, e.g., Stadler et al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0044] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0045] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. Here, X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0046] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained herein is effective, and which does not contain additional elements that are toxic to an unacceptable degree to the subject to which the preparation / composition is administered.

[0047] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation / composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] As used herein, the term “HLA-DQ2.5” refers to any naturally occurring HLA-DQ2.5 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both HLA-DQ2.5 that has not undergone “full-length” processing and any form of HLA-DQ2.5 resulting from processing within cells. This term also encompasses naturally occurring variants of HLA-DQ2.5, such as splice variants and allele variants. Exemplary HLA-DQ2.5 amino acid sequences are publicly available in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) accession code 4OZG and the IPD-IMGT / database.

[0049] In this specification, "TCR" means "T cell receptor," which is a membrane protein located on the surface of T cells (e.g., HLA-DQ2.5-restricted CD4+ T cells) and recognizes antigen fragments (e.g., gluten peptides) presented on MHC molecules containing HLA-DQ2.5.

[0050] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0051] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing 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. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0052] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0053] Amino acid modification The antigen-binding molecule (or antibody) of the present invention may include one or more modifications. Such modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence, and / or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions may be made to reach the final construct, provided that the final construct possesses a desired characteristic, such as antigen binding.

[0054] The antigen-binding molecule (or antibody) of the present invention may include amino acid substitutions. Conservative substitutions are shown in Table 1-1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1-1 under the heading "Exemplary Substitutions," and further described below with respect to amino acid side chain classes. The amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as antigen binding.

[0055] (Table 1-1) TIFF2026108741000001.tif165170

[0056] In this specification, expressions indicating amino acid modifications may appropriately be used, with one-letter or three-letter codes of the original and modified amino acids preceding and following a number indicating a specific position. For example, modified N100bL or Asn100bLeu, used when substituting amino acids contained in the antibody variable region, indicates the substitution of Asn at position 100b with Leu (according to Kabat numbering). That is, the number indicates the amino acid position according to Kabat numbering, the one- or three-letter amino acid code written before the number indicates the original amino acid, and the one- or three-letter amino acid code written after the number indicates the substituted amino acid. Similarly, modified P238D or Pro238Asp, used when substituting amino acids in the Fc region contained in the antibody constant region, indicates the substitution of Pro at position 238 with Asp (according to EU numbering). In other words, the numbers indicate the amino acid position according to EU numbering, the one- or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one- or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0057] Multispecific antigen binding molecules / antibodies The term "multispecific 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, an antigen-binding molecule (antibody) has at least one first antigen-binding moiety / domain capable of binding to one or more antigens (e.g., a peptide), and a second antigen-binding moiety / domain capable of binding to one or more antigens (e.g., a peptide). Some or all of the antigens to which the first antigen-binding moiety / domain binds may be different from some or all of the antigens to which the second antigen-binding moiety / domain binds. Alternatively, some of the antigens to which the first antigen-binding moiety / domain binds may be identical to some of the antigens to which the second antigen-binding moiety / domain binds.

[0058] In the context of the present invention, a “multispecific antigen-binding molecule (antibody)” can specifically bind to different types of antigens or epitopes. More specifically, a multispecific antigen-binding molecule (antibody) is specific to at least two different types of antigens or epitopes, and includes molecules / antibodies that recognize different antigens as well as molecules / antibodies that recognize different epitopes on the same antigen. For example, such a molecule usually binds to two antigens or epitopes ("bispecific antigen-binding molecule (antibody)"; used herein to mean the same as "dual-specific antigen-binding molecule (antibody)"), but may also have specificity to more antigens or epitopes (e.g., three or more types of antigens).

[0059] In this specification, terms such as “multispecificity” and “bispecificity” mean that the specificity of one antigen-binding domain / region differs from the specificity of another antigen-binding domain / region. That is, these terms mean that a single antigen-binding molecule has two or more specificities. For example, in a “bispecific” antigen-binding molecule (antibody), the first antigen-binding moiety / domain may bind to a first group of complexes formed by HLA-DQ2.5 and gluten peptides, and the second antigen-binding moiety / domain may bind to a second group of complexes formed by HLA-DQ2.5 and gluten peptides. The members (i.e., complexes) of the two groups may overlap but may not be identical. That is, some complexes may be included in both groups. Terms such as “multispecificity” and “bispecificity” can encompass this situation. The same applies to the first and second groups of complexes to which the first / second antigen-binding moiety / domains do not bind.

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

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

[0062] In the context of this disclosure, for simplicity, the term “antibody” may be used rather than “antigen-binding molecule.” However, those skilled in the art will understand that the term “antibody” may be replaced with “antigen-binding molecule” where applicable.

[0063] In one aspect, the present invention is partly based on the binding of an anti-HLA-DQ2.5 antigen-binding molecule (antibody) to HLA-DQ2.5, which presents gluten peptides to T cells. In a particular embodiment, an antibody that binds to HLA-DQ2.5 is provided.

[0064] In one aspect, the present invention provides an antigen-binding molecule or antibody having binding activity to HLA-DQ2.5, or to one or more complexes formed by HLA-DQ2.5 and gluten peptides. In a particular embodiment, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has the following functions / characteristics.

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

[0066] Anti-HLA-DQ2.5 antigen-binding molecules (antibodies) substantially lack binding activity to unintended antigens such as HLA-DQ5.1 / DQ6.3 / DQ7.3 / DQ7.5 / DQ8 / DR / DP; in other words, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) substantially do not bind to unintended antigens. For example, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) either do not have specific binding activity to HLA-DR / DP, or do not have significant binding activity to HLA-DR / DP. That is, antibodies either do not specifically bind to HLA-DR / DP, or do not bind to HLA-DR / DP significantly. Similarly, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) have substantially no binding activity to HLA-DQ molecules such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3; in other words, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) do not substantially bind to HLA-DQ molecules such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. In other words, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) do not have specific / significant binding activity to HLA-DQ molecules such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. In other words, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) do not specifically / significantly bind to HLA-DQ molecules such as HLA-DQ7.5, HLA-DQ8, HLA-DQ5.1, HLA-DQ6.3, and HLA-DQ7.3. These characteristics ("substantially absent binding activity") are desirable in order to prevent any substantial inhibitory effect on these non-targeted MHC class II molecules and to improve antibody pharmacokinetics in celiac disease patients with HLA-DQ2.5. The characteristic of having "substantially no binding activity" can be defined, for example, using the FACS results described herein. An anti-HLA-DQ2.5 antigen-binding molecule (antibody) that has "substantially no binding activity" for a particular antigen may have an MFI (mean fluorescence intensity) value of 250% or less, preferably 200% or less, and more preferably 150% or less, of the negative control under the measurement conditions described herein.

[0067] In a certain context, a bispecific antigen-binding molecule (antibody) that "substantially lacks binding activity" to a specific antigen has an MFI value of 2% or less, more preferably 1% or less, when the MFI value of IC17dK is set to 0% and the MFI value of DQN0139bb is set to 100% under the measurement conditions described herein. DQN0139bb is disclosed, for example, in WO2018 / 155692.

[0068] The anti-HLA-DQ2.5 antigen-binding molecule (antibody) has binding activity to HLA-DQ2.5 in complex with the gluten peptide described herein. In this specification, the complex formed between the HLA-DQ2.5 molecule and the gluten peptide is referred to as the "complex formed by HLA-DQ2.5 and gluten peptide," the "HLA-DQ2.5 / gluten peptide complex," or the "HLA-DQ2.5 / gluten peptide." It can also be rephrased, for example, as "gluten peptide-loaded HLA-DQ2.5," "gluten peptide-bound HLA-DQ2.5," "HLA-DQ2.5 in the form of a complex with a gluten peptide," and the "complex of HLA-DQ2.5 and gluten peptide." The above wording (for example, "a complex formed by HLA-DQ2.5 and ...[peptide]") also applies to peptides such as: 33-mer gliadin peptide, α1 gliadin peptide (also known as "α1a gliadin peptide"), α2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, BC hordein peptide, α3 gliadin peptide, α1b gliadin peptide, γ4a gliadin peptide, γ4b gliadin peptide, avenin 1 peptide, avenin 2 peptide, avenin 3 peptide, hordein 1 peptide, hordein 2 peptide, secarin 1 peptide, secarin 2 peptide, and 26-mer gliadin peptide, 14-mer Examples include 1 peptide, CLIP (hCLIP) peptide, hepatitis B virus 1 (HBV1) peptide, salmonella peptide, mycobacterium bovis (M. bovis) peptide, tyloperoxidase (TPO) peptide, etc.

[0069] On the other hand, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) substantially lack binding activity to "unrelated" peptides. In this specification, "unrelated" peptides include those that have been reported to be present on HLA-DQ2.5 but are unrelated to celiac disease or the present invention, i.e., those that are not the gluten peptides of the above-mentioned purpose. For example, unrelated peptides include, but are not limited to, CLIP (hCLIP) peptide, hepatitis B virus 1 (HBV1) peptide, salmonella peptide, mycobacterium bovis (M. bovis) peptide, and tyloperoxidase (TPO) peptide. These characteristics ("substantially absent binding activity") are desirable to prevent any substantial inhibitory effect on HLA-DQ2.5 in the form of complexes with these non-targeted MHC class II molecules and unrelated peptides, and to improve antibody PK in celiac disease patients. The property of "binding activity" can be defined, for example, using the FACS results described herein. An anti-HLA-DQ2.5 antigen-binding molecule (antibody) having "binding activity" for a specific antigen may have an MFI (mean fluorescence intensity) value of 300% or more, preferably 500% or more, and more preferably 1000% or more, of the negative control under the measurement conditions described herein.

[0070] In a certain context, a bispecific antigen-binding molecule (antibody) that has "binding activity" for a specific antigen has an MFI value of 3% or more, preferably 6% or more, preferably 10% or more, and more preferably 20% or more, when the MFI value of IC17dK is set to 0% and the MFI value of DQN0139bb is set to 100%, under the measurement conditions described herein.

[0071] When specifically referring to the specificity of binding, "binding activity" can be rephrased as "specific binding activity." The anti-HLA-DQ2.5 antigen-binding molecule (antibody) of the present invention has a dissociation constant (Kd) of 5×10 -7 M or less, preferably 4×10 -7 M or less, preferably 3×10 -7 M or less, preferably 2×10 -7 M or less, preferably 1×10 -7 M or less, preferably 9×10 -8 M or less, preferably 8×10 -8 M or less, preferably 7×10 -8 M or less, preferably 6×10 -8 M or less, preferably 5×10 -8 M or less, preferably 4×10 -8 M or less, preferably 3×10 -8 M or less, preferably 2×10 -8 M or less, preferably 1×10 -8 M or less, preferably 9×10 -9 M or less, preferably 8×10 -9 M or less, preferably 7×10 -9 M or less, preferably 6×10 -9 M or less, preferably 5×10 -9 M or less, preferably 4×10 -9 M or less, preferably 3×10 -9 M or less, preferably 2×10 -9 M or less for binding to one or more complexes formed by HLA-DQ2.5 and gluten peptides.

[0072] Suitable multispecific antigen-binding molecules of the present invention include: (1) A portion / domain containing an antibody variable region having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide; (2) A portion / domain containing an antibody variable region having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide; and (3) A portion / domain containing an Fc region having the above-described reduced Fcγ receptor binding activity, not limited to its structure. In the present invention, each of the above-mentioned domains can be directly linked by peptide bonds. For example, if F(ab')2 is used as the domain containing the antibody variable regions of (1) and (2), and these Fc regions are used as the domain containing the Fc region having reduced Fcγ receptor binding activity of (3), then the polypeptide formed by linking the antibody variable region-containing domains of (1) and (2) with the Fc region-containing domain of (3) by peptide bonds will form an antibody structure. Such antibodies can be produced by purification from the above-mentioned hybridoma culture medium, and also by purification of antibodies from the culture medium of a desired host cell that stably possesses the polynucleotide encoding the polypeptide constituting the antibody.

[0073] Examples of preferred antibody H chain variable regions of the present invention, which are contained in an 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 a CDR sequence in which its CDR1, CDR2, and CDR3 amino acid sequences are the same as those of the H chain variable regions described herein, or antibody H chain variable regions that are functionally equivalent to the above-described variable regions.

[0074] Examples of preferred antibody variable regions having T cell receptor complex binding activity according to the present invention include antibody variable regions having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide. Examples of antibody H chain variable regions contained in such antibody variable regions include antibody H chain variable regions described herein, antibody H chain variable regions having a CDR sequence in which the CDR1, CDR2, and CDR3 amino acid sequences are the same as those contained in the antibody H chain variable regions described herein, and antibody H chain variable regions functionally equivalent to the above-mentioned variable regions.

[0075] In the present invention, the phrase "functionally equivalent" means that, when used as a multispecific antigen-binding molecule, the binding affinity to the antigen is equivalent, or alternatively, the neutralizing activity to cells expressing HLA-DQ2.5 / gluten peptide (or tissues containing such cells) is equivalent. Binding affinity and neutralizing activity can be measured according to the description herein. The cells used for measuring activity may be any desired cells expressing HLA-DQ2.5 / gluten peptide (or any desired tissue containing such cells), and any suitable cell line can be used. With respect to the antibody constant region, this phrase may mean that a decrease in Fcγ receptor binding activity is equivalent.

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

[0077] The term "equivalent" does not necessarily mean the same level of activity; the activity may be enhanced. Specifically, for antigen-binding affinity, an example is when the value obtained by comparison with the binding affinity (parent KD value) of the variable region of an antibody acting as a control (KD value / 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. There is no lower limit, but an example is 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , or 10 -6 This includes. More specifically, in the present invention, the KD value / parent KD value is preferably 10 -6 ~1.5×10 -0 , more 10 -6 ~10 -1 , even more comfortable 10 -6 ~10 -2 , even more to the extent of 10 -6 ~10 -3 That is the case.

[0078] For a portion / domain containing an antibody variable region that has binding activity to HLA-DQ2.5 / gluten peptide, the KD value for HLA-DQ2.5 / gluten peptide is, for example, 2 × 10⁻⁶. -8 M or less, 1×10 -8 M or less, 9×10 -9 M or less, 8×10 -9 M or less, 7×10 -9 M or less, 6×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, or 1 × 10 -9 It may be less than M.

[0079] In the present invention, antibody variable regions that are "functionally equivalent" are not particularly limited, as long as they are variable regions of antibody H chains and / or antibody L chains that satisfy the above conditions. Examples of such antibody variable regions include regions produced by introducing substitutions, deletions, additions, and / or insertions of one or more amino acids (e.g., 1, 2, 3, 4, 5, or 10 amino acids) into the amino acid sequences of the variable regions in Tables 1 to 3 above. A method well known to those skilled in the art for introducing one or more amino acid substitutions, deletions, additions, and / or insertions into an amino acid sequence is a method for introducing mutations into a protein.For example, a person skilled in the art can prepare a variable region functionally equivalent to the antibody variable region having the above-mentioned function by appropriately introducing mutations into the amino acid sequence 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, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W., Drutsa, V., Jansen, HW, Kramer, B., Pflugfelder, M., and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer, W., and Fritz, HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad. Sci. US A. 82, 488-492).

[0080] When modifying amino acid residues, the amino acid is preferably mutated to a different amino acid that preserves the properties of its 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 hydroxyl group side chains (S, T, and Y), amino acids containing sulfur atom side chains (C and M), amino acids containing carboxylic acid and amide side chains (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 represented by a single-letter code in parentheses). Amino acid substitutions within each of these groups are called conservative substitutions. It is known that polypeptides containing modified amino acid sequences in which one or more amino acid residues in a given amino acid sequence are deleted, added, and / or substituted with other amino acids can retain their 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 region of the present invention containing such amino acid modifications has at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity with the CDR sequence, FR sequence, or amino acid sequence of the entire variable region before modification.In this specification, sequence identity is defined as the percentage ratio of residues identical to the original amino acid sequence residues in the H-chain variable region or L-chain variable region, determined after the sequences have been aligned and gaps have been appropriately introduced, if necessary, to maximize sequence identity. Amino acid sequence identity can be determined by the following method.

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

[0082] Nucleic acids that hybridize under stringent conditions to nucleic acids containing nucleotide sequences encoding the amino acid sequence of the variable region can also be isolated by gene amplification methods such as polymerase chain reaction (PCR) using primers synthesized based on information from the nucleotide sequence encoding the amino acid sequence of the variable region, instead of using the hybridization method described above.

[0083] The identity of one nucleotide or amino acid sequence to another can be determined using the BLAST algorithm by Karlin 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). To analyze a nucleotide sequence according to BLASTN, which is based on BLAST, the parameters are set to, for example, score=100 and wordlength=12. On the other hand, parameters used for analyzing an amino acid sequence using BLASTX, which is based on BLAST, include, for example, score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, the default parameters for each program are used. Specific methods for such analysis are publicly known in the art (see the National Center for Biotechnology Information (NCBI), Basic Local Alignment Search Tool (BLAST) website; http: / / www.ncbi.nlm.nih.gov).

[0084] The Fc region included in the multispecific antigen-binding molecule of the present invention is not particularly limited, as long as it is an Fc region having reduced Fcγ receptor binding activity, and examples of preferred Fc regions of the present invention include combinations of Fc region portions described herein.

[0085] Preferred examples of the multispecific antigen-binding molecules of the present invention include bispecific antibodies comprising a first antibody variable region having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide, and a second antibody variable region having binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide. Examples of such bispecific antibodies include bispecific antibodies comprising the H chain and L chain described herein, and bispecific antibodies comprising an Fc region that binds to an epitope overlapping with the epitope to which the above-mentioned antibody binds and has reduced Fcγ receptor binding activity.

[0086] Whether an antibody recognizes an epitope that overlaps with an epitope recognized by another antibody can be confirmed by competition between the two antibodies for the epitope. Antibody competition can be evaluated by competitive binding assays using methods such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer assay (FRET), and fluorescence micro-assay techniques (FMAT®). The amount of antibody bound to the antigen indirectly correlates with the binding ability of the candidate competing antibody (test antibody) that competitively binds to the overlapping epitope. In other words, as the amount or affinity of the 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, a properly labeled antibody and the antibody to be evaluated are added simultaneously to the antigen, and the resulting bound antibody is detected using labeling. The amount of antibody bound to the antigen can be easily determined by pre-labeling the antibody. This labeling is not particularly limited, and the labeling method is selected according to the assay technique used. Specifically, labeling methods include fluorescent labeling, radioactive labeling, and enzyme labeling.

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

[0088] In this specification, "antibody that binds to overlapping epitopes" means an antibody that reduces the amount of bound labeled antibody by 50% of the amount of bound labeled antibody (IC). 50 This refers to a test antibody that can reduce the level by at least 50% at concentrations that are typically 100 times higher, preferably 80 times higher, more preferably 50 times higher, even more preferably 30 times higher, and even more preferably 10 times higher than the test antibody.

[0089] A multispecific antigen-binding molecule having an antigen-binding site of an antibody that binds to an epitope that overlaps with the epitope to which the aforementioned antibody binds can exhibit excellent binding or neutralizing activity.

[0090] The multispecific antigen-binding molecules of the present invention are produced by the same method as the method for producing recombinant antibodies described herein.

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

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

[0093] Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various methods known in the art. Human antibodies are outlined 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 prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0095] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have already been described. (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 via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes 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 can also be generated by isolating selected Fv clone variable domain sequences from a human-derived phage display library. Such variable domain sequences can then be combined with a desired human constant domain. A method for selecting human antibodies from an antibody library is described below.

[0097] Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0098] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually contains one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0099] Humanized antibodies and their production methods have been reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (Describes "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (Describes "FR shuffling"); and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (Describes a "guided selection" approach for FR shuffling).

[0100] The human framework regions that can be used for humanization are not limited to these, but include: framework regions selected using the "best fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684 (1997)). (and see Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0101] In any of the above embodiments, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) is humanized. In one embodiment, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises HVR in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises HVR in any of the above embodiments and further comprises an FR1, FR2, FR3, or FR4 sequence as shown herein. In this specification, “human framework” may also be referred to as “humanized framework” with respect to the fact that the antibody is humanized.

[0102] In some embodiments, the multispecific antigen-binding molecule of the present invention is (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 binding activity to HLA-DQ2.5 in the form of a complex with a gluten peptide. Includes, The antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides. At least one gluten peptide in the complex to which the first antigen-binding moiety binds is different from at least one gluten peptide in the complex to which the second antigen-binding moiety binds; and The antigen-binding molecule has substantially no binding activity to either or both HLA-DQ2.5-positive PBMC B cells and Ba / F3 cells expressing HLA-DQ2.5 or HLA-DQ2.2. The antigen-binding molecule is humanized, and One or more amino acids in the heavy chain and / or light chain constant and / or variable regions of the first antigen-binding moiety and / or second antigen-binding moiety of the multispecific antigen-binding molecule have been modified.

[0103] In some embodiments, in a multispecific antigen-binding molecule, one or more amino acids in the heavy and / or light chains of the first antigen-binding moiety and / or the second antigen-binding moiety are substituted. In some embodiments, the multispecific antigen-binding molecule comprises at least one amino acid substitution in the variable region of the heavy chain; at least one amino acid substitution in the constant region of the heavy chain; at least one amino acid substitution in the variable region of the light chain; and at least one amino acid substitution in the constant region of the light chain. In some embodiments, gluten peptides are immunodominant peptides associated with celiac disease. In some embodiments, gluten peptides are selected from the group consisting of 33-mer gliadin peptides, α1-gliadin peptides, α2-gliadin peptides, γ1-gliadin peptides, γ2-gliadin peptides, ω1-gliadin peptides, ω2-gliadin peptides, BC-hordein peptides, α3-gliadin peptides, α1b-gliadin peptides, γ4a-gliadin peptides, γ4b-gliadin peptides, avenin-1 peptide, avenin-2 peptide, avenin-3 peptide, hordein-1 peptide, hordein-2 peptide, secarin-1 peptide, secarin-2 peptide, and 26-mer gliadin peptides. In some aspects, gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth In some embodiments, gluten peptides are selected from the group consisting of 33-mer gliadin peptides, α1-gliadin peptides, α2-gliadin peptides, γ1-gliadin peptides, ω1-gliadin peptides, ω2-gliadin peptides, BC-hordein peptides, α3-gliadin peptides, α1b-gliadin peptides, γ4a-gliadin peptides, γ4b-gliadin peptides, avenin-1 peptide, avenin-2 peptide, avenin-3 peptide, hordein-1 peptide, hordein-2 peptide, secarin-1 peptide, secarin-2 peptide, and 26-mer gliadin peptides. In some aspects, gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, t, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, three, four, f In some embodiments, multispecific antigen-binding molecules substantially lack binding activity to gluten peptides themselves or to gluten peptides themselves. In this context, the terms “themselves” and “themselves” refer to the state in which gluten peptides do not form a complex with HLA-DQ2.5. In some embodiments, the multispecific antigen-binding molecule substantially lacks binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, the unrelated peptide being at least one peptide selected from the group consisting of CLIP (hCLIP) peptide, hepatitis B virus 1 peptide, salmonella peptide, mycobacterium bobis peptide, and tyroperoxidase peptide. In some embodiments, the multispecific antigen-binding molecule substantially lacks binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, which includes all of the CLIP (hCLIP) peptide, hepatitis B virus 1 peptide, salmonella peptide, mycobacterium bobis peptide, and tyroperoxidase peptide. In some embodiments, the antigen-binding molecule has enhanced binding activity to the complex formed by HLA-DQ2.5 and the gluten peptide compared to before humanization and modification. In this context, “enhanced binding activity” means that the antigen-binding molecule binds more strongly to the complex formed by HLA-DQ2.5 and the gluten peptide than the pre-modification, i.e., pre-humanization and modification, prior antibody. In some embodiments, the antigen-binding molecule has enhanced cross-reactivity to gluten peptides compared to before humanization and modification. In some embodiments, the gluten peptides are ω2 gliadin peptide, BC hordein peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ4a gliadin peptide, and γ4d gliadin peptide. In this context, “enhanced cross-reactivity to gluten peptides” means that the antigen-binding molecule binds to or exhibits neutralizing activity to more gluten peptides than the prior antibodies before modification, i.e., before humanization and modification.

[0104] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of a heavy chain variable domain (VH) sequence disclosed herein. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference (i.e., original) sequence, but an anti-HLA-DQ2.5 antigen-binding molecule (antibody) containing such sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted with respect to the reference (i.e., original) sequence. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a VH sequence as disclosed herein, or a sequence including its post-translational modifications. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 as disclosed herein, (b) HVR-H2 as disclosed herein, and (c) HVR-H3 as disclosed herein. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0105] The amino acids contained in the amino acid sequence of the present invention may be post-translationally modified (for example, modification to pyroglutamic acid by pyroglutamylation of the N-terminal glutamine is well known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the amino acid sequence of the present invention.

[0106] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is provided, which comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of a light chain variable domain (VL) disclosed herein. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions to the reference (i.e., original) sequence, but the anti-HLA-DQ2.5 antigen-binding molecule (antibody) containing such sequence retains the ability to bind to HLA-DQ2.5. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from the reference (i.e., original) sequence. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of the HVR (i.e., within the FR). Optionally, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) comprises a VL sequence as disclosed herein, or a sequence including its post-translational modifications. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 as disclosed herein; (b) HVR-L2 as disclosed herein; and (c) HVR-L3 as disclosed herein. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

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

[0108] In a further aspect, the present invention provides antigen-binding molecules (antibodies) that bind to the same epitopes as the anti-HLA-DQ2.5 antigen-binding molecules (antibodies) provided herein. For example, in certain embodiments, molecules / antibodies that bind to the same epitopes as any of the molecules / antibodies described herein are provided. In certain embodiments, molecules / antibodies that bind to epitopes within fragments of HLA-DQ2.5 consisting of about 8 to 17 amino acids, or within complexes formed by HLA-DQ2.5 and gluten peptides are provided. In this context, the gluten peptide may be any of the gluten peptides described herein.

[0109] In a further aspect, the present 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 a particular embodiment, 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 a further aspect of the present invention, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) according to any of the above embodiments is a monoclonal antigen-binding molecule (antibody) comprising a chimeric, humanized, or human antigen-binding molecule (antibody). In a preferred embodiment, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) of the present invention is a humanized antigen-binding molecule (antibody). In one embodiment, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) is an antibody fragment, for example, Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, a complete IgG1 antibody, or another antibody class or isotype as defined herein.

[0111] In further contexts, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) in any of the above embodiments may incorporate any of the following characteristics, either alone or in combination.

[0112] Antibody affinity In certain embodiments, the antibodies provided herein are ≤1 micromol (μM), ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0113] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125 I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 micrograms (μg) / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 microliters (μL) / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0114] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μL / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μL / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1When it exceeds, the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) at 25 °C of 20 nM of anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO (trademark) spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0115] antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, as well as other fragments described hereinafter. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab’)2 fragments that include salvage receptor binding epitope residues and have an increased half-life in vivo, see U.S. Patent No. 5,869,046.

[0116] A diabody is an antibody fragment with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0117] A single-domain antibody is an antibody fragment that includes all or a portion of the heavy-chain variable domain of an antibody, or all or a portion of the light-chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0118] Antibody fragments can be made by a variety of methods including, but not limited to, proteolytic digestion of a full-length antibody as described herein, production by recombinant host cells (e.g., E. coli or phage).

[0119] Fc region mutant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to thereby generate Fc region variants. Fc region variants may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0120] Antibodies with increased half-life and increased binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring 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 U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding affinity of the Fc region to FcRn. Such Fc variants include those involving substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (for example, substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0121] Fc area In this specification, the terms “Fc region” or “Fc domain” are used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native Fc region and mutant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0122] Fc receptor The term “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. In one embodiment, FcR is a naturally occurring human FcR. In another embodiment, FcR is one that binds to an IgG antibody (γ receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, which also include allelic variants and alternative splice forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs have been reviewed, for example, 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 included in the term “FcR” as used herein.

[0123] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly 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 human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered with polypeptides containing mutant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0125] Fcγ receptor Fcγ receptors are receptors that can bind to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies, and include all members of the family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI(CD64), e.g., isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), e.g., isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), e.g., isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all unidentified human Fcγ receptors, Fcγ receptor isoforms, and their allotypes. However, Fcγ receptors are not limited to these examples. Without limiting themselves, Fcγ receptors include those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors may originate from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as all unidentified mouse Fcγ receptors, Fcγ receptor isoforms, and their allotypes. Such preferred Fcγ receptors include, for example, human FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16), and / or FcγRIIIB(CD16).Whether or not the Fcγ receptor has binding activity to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be evaluated using methods other than the FACS and ELISA formats described above, such as the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0126] On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to the antibody Fc domain to form an Fc / Fc ligand complex. The molecule may originate from any organism. Binding of an Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fcγ receptors, Fcα receptors, Fcβ receptors, FcRn, C1q, and C3, mannan-binding lectins, mannose receptors, Staphylococcus protein A, Staphylococcus protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to the Fcγ receptor. Fc ligands also include unidentified molecules that bind to Fc.

[0127] Fcγ receptor binding activity The reduced binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB can be evaluated using the FACS and ELISA formats described above, as well as the BIACORE method based on ALPHA screen (amplified luminescence proximity homogeneous assay) and surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0128] The ALPHA screen is performed using the ALPHA technology, based on the principle described below, which employs two types of beads: donor beads and acceptor beads. A luminescence signal is detected only when a molecule linked to a donor bead biologically interacts with a molecule linked to an acceptor bead, and when these two beads are located in close proximity. When excited by a laser beam, the photosensitizer in the donor bead converts the oxygen surrounding the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, a chemiluminescent reaction is induced within the acceptor bead. This reaction ultimately results in luminescence. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and the chemiluminescent 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)-tagged Fcγ receptor is immobilized on acceptor beads. In the absence of antigen-binding molecules and antibodies containing competing mutant Fc domains, the Fcγ receptor interacts with antigen-binding molecules or antibodies containing wild-type Fc domains, resulting in a signal in the 520–620 nm range. Antigen-binding molecules or antibodies with untagged mutant Fc domains compete with those containing wild-type Fc domains for interaction with the Fcγ receptor. Relative binding affinity can be measured by quantifying the decrease in fluorescence as a result of competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies, using sulfo-NHS-biotin are known. Appropriate methods for attaching a GST tag to the Fcγ receptor include fusing a polypeptide encoding the Fcγ receptor with GST in frame, expressing the fused gene using cells into which a vector carrying the fused gene has been introduced, and then purifying the result using a glutathione column. The induced signal can preferably be analyzed by fitting it to a one-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0130] One of the substances used to observe their interactions is immobilized as a ligand on a thin layer of gold on the sensor tip. When light is shone on the back surface of the sensor tip so that total internal reflection occurs at the interface between the gold layer and the glass, the intensity of the reflected light partially decreases at a specific site (SPR signal). The other substance used to observe their interactions is injected as an analyte onto the surface of the sensor tip. The mass of the immobilized ligand molecule increases when the analyte binds to the ligand. This changes the refractive index of the solvent on the surface of the sensor tip. The change in refractive index causes a position shift of the SPR signal (conversely, dissociation returns the signal to its original position). In the Biacore system, the amount of the above shift (i.e., the change in mass on the sensor tip surface) is plotted on the vertical axis, and thus the change in mass over time is displayed as experimental data (sensorgram). Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and affinity (KD) is determined from the ratio between these two constants. Inhibition assays are preferred for use in the BIACORE method. Examples of such inhibitory assays are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0131] Fc region with reduced Fcγ receptor binding activity In this specification, "reduced Fcγ receptor binding activity" means, for example, that the competitive activity of the test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the competitive activity of the control antigen-binding molecule or antibody, based on the analysis method described above.

[0132] An antigen-binding molecule or antibody comprising the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be suitably used as a control antigen-binding molecule or antibody. The Fc domain structure is shown in RefSeq accession number AAC82527.1, RefSeq accession number AAB59393.1, RefSeq accession number CAA27268.1, and RefSeq accession number AAB59394.1. Further, when an antigen-binding molecule or antibody comprising an Fc domain variant of an antibody of a specific isotype is used as a test substance, the effect of the mutation of the variant on the Fcγ receptor binding activity is evaluated using, as a control, an antigen-binding molecule or antibody comprising the Fc domain of the same isotype. As described above, an antigen-binding molecule or antibody comprising an Fc domain variant determined to have reduced Fcγ receptor binding activity is preferably prepared.

[0133] Such known variants include, for example, a variant having a deletion of amino acids 231A - 238S (EU numbering) (WO2009 / 011941), and variants 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] Specifically, preferred antigen-binding molecules or antibodies include those containing an Fc domain having a mutation (such as a substitution) at at least one amino acid position selected from the following amino acids that form the Fc domain of a particular isotype of antibody: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The isotype of the antibody from which the Fc domain originates is not particularly limited, and a suitable Fc domain derived from monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0135] In the present invention, SG181 may be used as an Fcγ receptor silencing Fc that weakens Fc binding to the Fcγ receptor. 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 the antigen-binding molecule or antibody of the present invention for reduced Fcγ receptor binding.

[0136] Other preferred antigen-binding molecules or antibodies include, for example, those containing an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is substituted with the amino acid at the corresponding position in the EU numbering of the corresponding IgG2 or IgG4.

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

[0138] H chain / L chain association and regulation of other properties Another aspect of the present invention relates to an antigen-binding molecule in which the association of heavy chains and light chains is regulated, a method for producing an antigen-binding molecule in which the association of heavy chains and light chains is regulated, and a method for regulating the association of heavy chains and light chains 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 light chain is regulated, the heavy chain and light chain constituting the antigen-binding molecule are a combination of the heavy chain and light chain of the choice, and the amino acid residues at given locations in the constant region (CH1) of the heavy chain and the constant region (CL) of the light chain are amino acid residues that repel each other electrically (have the same charge).

[0140] In the present invention, by replacing the amino acid residues at given locations in CH1 and CL of an undesirable combination of heavy and light chains with amino acid residues that electrically repel each other (i.e., have the same charge), the formation of an undesirable combination of heavy and light chains can be prevented by utilizing this charge repulsion, and as a result, a desirable combination of heavy and light chains can be formed.

[0141] In this invention, the phrases "regulating association" and "association being regulated" refer to regulation to achieve desired association conditions, and more specifically, regulation to prevent the formation of undesirable associations between heavy and light chains.

[0142] In the present invention, the term "interface" usually refers to an association surface resulting from association (interaction), and the amino acid residues forming the interface are typically one or more amino acid residues included in the polypeptide region involved in the association, and more preferably amino acid residues that approach each other during the association and participate in the interaction. More specifically, this interaction includes, for example, cases in which amino acid residues approach each other during the association to form hydrogen bonds, electrostatic interactions, or salt bridges.

[0143] In the present invention, the phrase "amino acid residues forming an interface" more specifically refers to amino acid residues included in the polypeptide region constituting the interface. For example, the polypeptide region constituting the interface refers to the polypeptide region responsible for selective intermolecular binding in, for example, an antigen-binding molecule (e.g., an antibody), a ligand, a receptor, or a substrate. More specifically, in an antigen-binding molecule, such examples include the heavy chain constant region, the heavy chain variable region, the light chain constant region, and the light chain variable region.

[0144] In a preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding molecule has electrically repelling (same charge) amino acid residues at a given location in CH1 and CL of an undesirable combination of heavy and light chains before association regulation.

[0145] By modifying the amino acid residues in the aforementioned antigen-binding molecule to create amino acid residues that electrically repel each other (have the same charge), it is thought that the association of these amino acid residues is inhibited by the repulsive force of their charges.

[0146] Therefore, in the aforementioned antigen-binding molecule, the amino acid residues to be modified are preferably amino acid residues that approach each other upon association in the polypeptide region that forms the interface.

[0147] The amino acid residues that come into close proximity during association can be determined, for example, by analyzing the three-dimensional structure of the polypeptide and by examining the amino acid sequence of the polypeptide region that forms the interface during polypeptide association. The amino acid residues of the interface that come into close proximity to each other are preferred targets for "modification" in the antigen-binding molecule of the present invention.

[0148] It is known that some amino acids are charged. Generally, lysine (K), arginine (R), and histidine (H) are known to be positively charged amino acids. Aspartic acid (D), glutamic acid (E), etc., are known to be 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), etc., are known to be uncharged or nonpolar amino acids.

[0149] Therefore, in this invention, amino acids that repel each other electrically (have the same charge) mean the following: (1) Amino acids in which one amino acid is a positively charged amino acid and the other amino acid is also a positively charged amino acid, and (2) An amino acid in which one amino acid is a load-electric amino acid and the other amino acid is also a load-electric amino acid.

[0150] Examples of amino acid modifications include the modification of uncharged or nonpolar amino acids to positively charged amino acids, the modification of uncharged or nonpolar amino acids to charged amino acids, the modification of positively charged amino acids to charged amino acids, and the modification of charged amino acids to positively charged amino acids. Furthermore, the modification of uncharged or nonpolar amino acids to different uncharged or nonpolar amino acids, the modification of positively charged amino acids to different positively charged amino acids, and the modification of charged amino acids to different charged amino acids are also included in the amino acid modifications of the present invention.

[0151] Modifying amino acids in this invention includes making one modification to each of the heavy chain and the light chain, or making multiple modifications to each of the heavy chain and the light chain. In addition, the number of modifications made to the heavy chain and the light chain may be the same or different.

[0152] The modification of amino acids in this invention includes making multiple modifications to positively charged amino acids on either the heavy chain or the light chain, and making multiple modifications to uncharged amino acids on the other chain. Furthermore, the multiple modifications to positively charged amino acids and the multiple modifications to uncharged amino acids may be performed on the same heavy chain or light chain. In these modifications, modifications to uncharged amino acids or nonpolar amino acids and modifications of uncharged amino acids or nonpolar amino acids may also be suitably combined.

[0153] In modifications of the present invention, for example, an amino acid on one side of the chain may be used in an unmodified state, in which case both the heavy and light chains do not need to be modified, and only one side of the chain 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 antibody light chain constant regions include IgK(κ), IgL1, IgL2, IgL3, IgL6, and IgL7(λ) type constant regions. The light chain constant region of the antigen-binding molecule of the present invention is not particularly limited; if multiple types of light chains are used, the light chains may be of different types, for example, κ and λ. Several allotype sequences obtained by genetic polymorphism are described as human IgK(κ) constant regions and human IgL7(λ) constant regions in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, and any of these may be used in the present invention.

[0155] The antibody constant region, particularly the heavy chain constant region, may be modified as necessary to improve the function or stability of the antigen-binding molecule. Examples of modifications to improve the function of the antigen-binding molecule include modifications to strengthen or weaken the binding between the antigen-binding molecule and the Fcγ receptor ("FcγR"), modifications to strengthen or weaken the binding between the antigen-binding molecule and FcRn, and modifications to strengthen or weaken the cytotoxic activity of the antigen-binding molecule (e.g., ADCC activity and CDC activity). In addition, modifications to improve the heterogeneity of the antigen-binding molecule, as well as modifications to improve non-immunogenicity and / or pharmacokinetics, may also be included.

[0156] Furthermore, heterogeneity in the heavy chain C-terminal sequence of IgG antibodies has been reported, including amidation of the C-terminal carboxyl group due to the deletion of the C-terminal amino acid lysine residue, or the deletion of two C-terminal amino acids, glycine and lysine (Anal. Biochem. 2007 Jan 1:360(1):75-83). Therefore, in the present invention, it is preferable to use IgG lacking C-terminal lysine or C-terminal lysine and glycine in order to reduce the heterogeneity of the heavy chain C-terminus. Chimeric antibodies and humanized antibodies, which use human-derived sequences, are expected to be useful when administered to humans for therapeutic purposes, as their antigenicity in the human body is weakened.

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

[0158] A preferred example of the multispecific antibody of the present invention is a bispecific antibody. Therefore, a preferred example of the antigen-binding molecule of the present invention is a bispecific antibody 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 antibody" of a preferred embodiment of the antigen-binding molecule of the present invention, the "first heavy chain" refers to one of the two heavy chains (H chains) that form 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 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) that form 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 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 originate from the same antibody that binds to a specific antigen (or epitope), and the second L chain and the second H chain also originate from the same antibody that binds to a specific antigen (or epitope). In this specification, an L-chain-H-chain pair formed by a first H-chain and an L-chain is referred to as the first pair, and an L-chain-H-chain pair formed by a second H-chain and an L-chain is referred to as the second pair. The antigen (or epitope) used to produce the antibody derived from the second pair is preferably different from the antigen used to produce the antibody derived from the first pair. More specifically, the antigens recognized by the first pair and the second pair may be the same, but preferably the pairs bind to different antigens (or epitopes). In this case, the H-chain and L-chain of the first pair and the second pair preferably have different amino acid sequences from each other. When the first pair and the second pair bind to different epitopes, the first pair and the second pair may recognize completely different antigens, or they may recognize different sites (different epitopes) on the same antigen. Furthermore, one of them may recognize an antigen such as a protein, peptide, gene, or sugar, while the other may recognize a cytotoxic substance such as a radioactive substance, chemotherapeutic agent, or cell-derived toxin. However, if it is desired to produce antibodies having pairs formed by a specific combination of H chains and L chains, those specific H chains and L chains may be arbitrarily determined to be the first pair and the second pair.

[0160] A more detailed explanation is provided below for the case of IgG-type bispecific antibodies having two types of heavy chain constant regions CH1 (CH1-A and CH1-B) and two types of light chain constant regions (CL-A and CL-B); however, the present invention may also be applied to other antibodies.

[0161] If we want to obtain a bispecific antibody that recognizes one epitope by a first CH1-A and a first CL-A, and binds to another epitope by a second CH1-B and a second CL-B, then theoretically, if we express each of the four types of chains to produce that antibody, we could potentially produce 10 types of antibody molecules.

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

[0163] Examples include modifying the amino acid residues forming the interface between CH1-A and CL-B to be positively charged amino acid residues, and modifying the amino acid residues forming the interface between CH1-B and CL-A to be negatively charged amino acid residues. As a result of these modifications, unintended association between CH1-A and CL-B is inhibited because both amino acid residues forming the interface are positively charged, and association between CH1-B and CL-A is also inhibited because both amino acid residues forming the interface are negatively charged. Thus, unintended associations between CH1-A and CL-B and associations between CH1-B and CL-A are inhibited because the amino acid residues forming the interface have the same charge. As a result, antibodies with the intended associations between CH1-A and CL-A and between CH1-B and CL-B can be efficiently obtained. Furthermore, the intended association between CH1-A and CL-A is facilitated because the amino acid residues forming the interface have different types of charges; and the intended association between CH1-B and CL-B is also facilitated because the amino acid residues forming the interface have different types of charges. As a result, antibodies with the intended association can be efficiently obtained.

[0164] Another example involves modifying the amino acid residues forming the interface between CL-A and CH1-B to positively charged amino acids, when these residues are otherwise uncharged or nonpolar amino acids. As a result of this modification, unintended association between CH1-A and CL-B is inhibited because both interface-forming amino acid residues are positively charged. On the other hand, since the interface-forming amino acid residues are not electrically repelling from each other, intended associations between CH1-A and CL-A and between CH1-B and CL-B are considered to occur more easily than when the amino acids are electrically repelling from each other. Consequently, antibodies having the intended associations between CH1-A and CL-A and between CH1-B and CL-B can be efficiently obtained. However, in this example, if the amino acid residues forming the interface between CL-A and CH1-B are not otherwise uncharged or nonpolar amino acids, they may be modified to become otherwise uncharged or nonpolar amino acids.

[0165] Furthermore, in another example, if 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 modified to be a positively charged amino acid residue, while the other is modified to be an electrically 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 the modification made to CH1-A. As a result of this modification, the intended 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, but the intended association between CH1-B and CL-B is not inhibited because the amino acid residues forming the interface are amino acids that do not electrically repel each other. As a result, antibodies having the intended association between CH1-A and CL-A, and the intended association between CH1-B and CL-B, can be efficiently obtained. On the other hand, in this example, if the amino acid residues forming the interface between CL-B and CH1-B are not uncharged amino acids or nonpolar amino acids in CH1-B, they may be modified to become uncharged amino acids or nonpolar amino acids.

[0166] In addition, the use of association control according to the present invention makes it possible to suppress association between CH1 (CH1-A and CH1-B) or between CL (CL-A and CL-B).

[0167] Those skilled in the art will be able to suitably determine the type of amino acid residues that approach the CH1-CL interface during association in a desired polypeptide where the association regulation according to the present invention is desirable.

[0168] Furthermore, those skilled in the art can also suitably obtain sequences that can be used as CH1 or CL in antibodies in organisms such as humans, monkeys, mice, and rabbits by using public databases, etc. More specifically, amino acid sequence information of CH1 or CL can be obtained by the means described in the following examples.

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

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

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

[0172] In the aforementioned antigen-binding molecule, the "amino acid residues that repel each other electrically" or "amino acid residues that have the same charge" are preferably selected from amino acid residues contained in either of the following sets (X) or (Y): (X) Glutamic acid (E) or aspartic acid (D); or (Y) Lysine (K), arginine (R), or histidine (H).

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

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

[0175] The present invention provides an antigen-binding molecule in which one, 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 and light chains of the antigen-binding molecule are amino acid residues that repel each other electrically: (a1) An amino acid residue contained in CH1 at position 175 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and an amino acid residue contained in CL at position 160 according to EU numbering, which is glutamic acid (E) or aspartic acid (D); (a2) an amino acid residue contained in CH1 at position 175 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and an amino acid residue contained in CL at position 160 according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (b1) An amino acid residue contained in CH1 at position 175 according to EU numbering, which is glutamic acid (E) or aspartic acid (D), and an amino acid residue contained in CL at position 131 according to EU numbering, which is glutamic acid (E) or aspartic acid (D); (b2) an amino acid residue contained in CH1 at position 175 according to EU numbering, which is lysine (K), histidine (H), or arginine (R), and an amino acid residue contained in CL at position 131 according to EU numbering, which is lysine (K), histidine (H), or arginine (R); (c1) The amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, each being glutamic acid (E) or aspartic acid (D), and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, each being glutamic acid (E) or aspartic acid (D); (c2) The amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, each being lysine (K), histidine (H), or arginine (R), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, each being lysine (K), histidine (H), or arginine (R).

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

[0177] In addition to the above, a method for inhibiting unintended association of CH1 / CL by introducing charge repulsion on the interface between CH1 and CL (WO 2013 / 065708) can be further applied to the antigen-binding molecule of the present invention. More specifically, the present invention provides an antigen-binding molecule having CH1 and CL, wherein one, two or more sets of amino acid residues selected from the group consisting of the amino acid residue sets shown in (a) to (d) below are electrically repelled from each other: (a) an amino acid residue located at position 147 in the heavy chain constant region (CH1) according to EU numbering, and an amino acid residue located at position 160 in the light chain constant region (CL) according to EU numbering; (b) The amino acid residue contained in CH1 at position 147 according to EU numbering, and the amino acid residue contained in CL at position 131 according to EU numbering; (c) The amino acid residue contained in CH1 at position 175 according to EU numbering, and the amino acid residue contained in CL at position 160 according to EU numbering; (d) The amino acid residue contained in CH1 at position 213 according to EU numbering, and the amino acid residue contained in CL at position 123 according to EU numbering.

[0178] The antigen-binding molecule of the present invention can be further modified to have an electric charge, such as a method for introducing electric repulsion at the interface of the second constant region (CH2) or the third constant region (CH3) of the heavy chain to suppress undesirable association between heavy chains, a method for introducing electric repulsion at the interface between the heavy chain variable region and the light chain variable region to suppress unintended association between heavy chains and light chains, or a method for modifying the amino acid residues that form a hydrophobic core at the interface between the heavy chain variable region and the light chain variable region to have an electric charge, in order to suppress unintended association between heavy chains and light chains (see WO 2006 / 106905).

[0179] In techniques that suppress unintended association between heavy chains by introducing electrical repulsion at the CH2 or CH3 interface, examples of amino acid residues that come into contact at the interface of other constant regions of the heavy chain include positions 356 (EU numbering) and 439 (EU numbering), 357 (EU numbering) and 370 (EU numbering), and positions 399 (EU numbering) and 409 (EU numbering) in the CH3 region. For the numbering of antibody constant regions, refer to the publication by Kabat et al. (Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, NIH); for the numbering of heavy chain constant regions, EU numbering is indicated.

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

[0181] Furthermore, the antibody may have a second heavy chain CH3 region distinct from the first heavy chain CH3 region described above, wherein the amino acid residue set is selected from the amino acid residue sets shown in (1) to (3) above, and one to three amino acid residue sets corresponding to the amino acid residue sets shown in (1) to (3) above that repel each other electrically in the first heavy chain CH3 region do not repel each other electrically with the corresponding amino acid residues in the first heavy chain CH3 region.

[0182] The amino acid residues described in (1) to (3) above approach each other when they associate. Those skilled in the art can find sites corresponding to the amino acid residues described in (1) to (3) above for a desired heavy chain CH3 region or heavy chain constant region by homology modeling using commercially available software, and can suitably modify the amino acid residues at those sites.

[0183] In the aforementioned antigen-binding molecules, "electrically repelling," "having the same charge," or "possessing the same charge" means, for example, that any two or more amino acid residues include amino acid residues contained in either group (X) or (Y) as described herein.

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

[0185] In the present invention, the amino acid residues subjected to "modification" are not limited to the amino acid residues of the antigen-binding molecule variable region or antibody constant region described above. Those skilled in the art can identify amino acid residues that form interfaces in polypeptide variants or heteromer polymers and modify the amino acid residues at those sites to regulate their association, for example, by homology modeling using commercially available software. Homology modeling is a method 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, first, a protein that has been determined to have a three-dimensional structure with high homology to the protein is searched for. Next, this three-dimensional structure is used as a template to construct the structure of the protein with the unknown structure, and the structure is further optimized by molecular dynamics or the like to predict the three-dimensional structure of the unknown protein.

[0186] In a method for introducing electrical repulsion at the interface between the heavy chain variable region and the light chain variable region to suppress undesirable association between the heavy chain and the light chain, examples of amino acid residues that come into contact at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include glutamine (Q) at position 39 according to Kabat numbering in the VH (FR2 region) and glutamine (Q) at position 38 according to Kabat numbering in the opposing (contacting) VL (FR2 region). Further preferred examples are leucine (L) at position 45 according to Kabat numbering in the VH (FR2) and proline (P) at position 44 according to Kabat numbering in the opposing VL (FR2). The publication by Kabat et al. (Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, NIH) was referenced for the numbering of these sites.

[0187] Since these amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. 2003; 16: 113-120), the association of the variable region of antigen-binding molecules can be regulated for 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 a multispecific antigen-binding molecule, two or more amino acid residues that form the interface between the heavy chain variable region and the light chain variable region are amino acid residues that repel each other electrostatically.

[0189] A specific example is an antigen-binding molecule in which two or more amino acid residues forming the interface between VH and VL are electrically repelling amino acid residues from each other. More specifically, examples include antigen-binding molecules having 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) an amino acid residue contained in VH at position 39 according to Kabat numbering, and an amino acid residue contained in VL at position 38 according to Kabat numbering; or (b) The amino acid residue contained in VH at position 45 according to Kabat numbering, and the amino acid residue contained in VL at position 44 according to Kabat numbering.

[0190] In some embodiments, in a 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) The amino acid residue in the heavy chain variable region at position 39 according to Kabat numbering, and the amino acid residue in the light chain variable region at position 38 according to Kabat numbering, (b) An amino acid residue in the heavy chain variable region at position 45 according to Kabat numbering, and an amino acid residue in the light chain variable region at position 44 according to Kabat numbering.

[0191] Each of the amino acid residues described in (a) or (b) above approaches each other when they associate. A person skilled in the art can find the sites corresponding to the amino acid residues described in (a) or (b) above in a desired VH or VL by homology modeling using commercially available software, and can suitably modify the amino acid residues at those sites.

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

[0193] In a method for modifying the amino acid residues that form a hydrophobic core at the VH-VL interface to create charged polar amino acids, thereby suppressing unintended association between heavy and light chains, preferred examples of amino acid residues that can form a hydrophobic core at the VH-VL interface include leucine (L) at position 45 in VH(FR2) according to Kabat numbering, and proline (P) at position 44 in the opposing VL(FR2) according to Kabat numbering. For the numbering of these sites, we used Kabat et al. (Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, NIH) as a reference.

[0194] Generally, the term "hydrophobic core" refers to the region formed within an associated polypeptide by the assembly of hydrophobic amino acid side chains. Examples of hydrophobic amino acids include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Furthermore, amino acid residues other than hydrophobic amino acids (e.g., tyrosine) can also be involved in the formation of a hydrophobic core. This hydrophobic core, along with a hydrophilic surface where hydrophilic amino acid side chains are exposed to the outside, becomes a driving force for promoting the association of water-soluble polypeptides. When hydrophobic amino acids from two different domains are present on the molecular surface and exposed to water molecules, entropy and free energy will increase. Therefore, the two domains associate with each other to decrease their free energy and become stable, and the hydrophobic amino acids at the interface will be embedded into the interior of the molecule to form a hydrophobic core.

[0195] It is thought that when polypeptide association occurs, modifying the hydrophobic amino acids that form the hydrophobic core to polar amino acids with a charge inhibits the formation of the hydrophobic core; and consequently, peptide association is inhibited.

[0196] Those skilled in the art will be able to recognize the presence or absence of a hydrophobic core, its formation site (region), etc., by analyzing the amino acid sequence of a desired antigen-binding molecule. That is, the antigen-binding molecule of the present invention is characterized in that an amino acid residue capable of forming a hydrophobic core at its interface has been modified to be a charged amino acid residue. More specifically, examples include antigen-binding molecules in which the amino acid residue shown in either (1) or (2) below is a charged amino acid residue. 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 at position 45 according to Kabat numbering, contained in VH; and (2) The amino acid residue at position 44 according to Kabat numbering, which is contained in VL.

[0197] Preferred examples of charged amino acid residues in the aforementioned antigen-binding molecules 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] Generally, the amino acid residues described in (1) and (2) above in humans and mice are as follows: (1) Leucine (L), and (2) Proline (P). Therefore, in a preferred embodiment of the present invention, these amino acid residues are subjected to modification (e.g., substitution with charged amino acids). Furthermore, the types of amino acid residues described in (1) and (2) above are not necessarily limited to the aforementioned amino acid residues, but may be other amino acids equivalent to these amino acid residues.

[0199] Other known methods can be applied to the antigen-binding molecule of the present invention. For example, to promote the association of a first VH (VH1) with a first VL (VL1) and / or a second VH (VH2) with a second VL (VL2), an amino acid side chain present in one variable region of the H chain can be replaced with a larger side chain (knob), and an amino acid side chain present in the opposite variable region of the other H chain can be replaced with a smaller side chain (hole), so that the knob may be located in a hole, thereby promoting the association of VH1 with VL1 and / or VH2 with VL2; and consequently, further suppressing the association of VH1 with VL2 and / or VH2 with VL1.

[0200] For example, in the case of human IgG1, modifications Y349C and T366W are made to make the amino acid side chain in the CH3 region of one H chain larger (knob), while modifications D356C, T336S, L368A, and Y407V are made to make the amino acid side chain in the CH3 region of the other H chain smaller.

[0201] In some embodiments, in a multispecific antigen-binding molecule, the Fc domain is composed of a first Fc domain subunit and a second Fc domain subunit that are capable of stable association. In some embodiments, in a multispecific antigen-binding molecule, the Fc domain comprises either (e1) or (e2): (e1) A first Fc region subunit including Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region including Cys at position 354 and Trp at position 366; (e2) A first Fc region subunit containing Glu at position 439, and a second Fc region containing Lys at position 356. (Amino acid positions are numbered according to EU numbering.)

[0202] For example, the knob-into-hole technique is described, for instance, in US 5731168; US 7695936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the projection is located within the cavity to promote heterodimer formation and prevent homodimer formation. The projection is constructed by replacing a smaller amino acid side chain derived from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine).

[0203] Furthermore, other known methods can be applied to the antigen-binding molecule of the present invention. By complementary association of CH3 using a chain exchange domain CH3, a portion of the CH3 of one H chain of the antigen-binding molecule is changed to a sequence derived from IgA corresponding to that portion, and a sequence derived from IgA corresponding to that portion is introduced into the complementary portion of the CH3 of the other H chain, thereby efficiently preparing the target antigen-binding molecule (Protein Engineering Design & Selection, 23: 195-202, 2010).

[0204] Furthermore, other known methods can be applied to the antigen-binding molecule of the present invention. For example, when producing bispecific antibodies, target bispecific antibodies can be prepared by providing differences in isoelectric points by making different amino acid modifications to each of the variable regions of two types of H chains, and by utilizing these differences in isoelectric points for purification by ion-exchange chromatography (WO 2007 / 114325).

[0205] A method of modifying the amino acid residue at position 435, according to EU numbering, which is a site related to the binding between IgG and protein A, to an amino acid with a different binding strength to protein A, such as Arg, may also be used for the antigen-binding molecule of the present invention in combination with the method described above. By using this method, the interaction between the H chain and protein A can be altered, and only heterodimeric antigen-binding molecules can be efficiently purified using a protein A column. This method can also be used independently without being combined with the method described above.

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

[0207] Multiple, for example, two or more of the aforementioned known methods 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 in which the aforementioned known methods have been modified.

[0208] In addition, the present invention provides a method for producing an antigen-binding molecule in which the association between the heavy chain and the light chain is regulated. A preferred embodiment of the production method of the present invention is a method for producing an antigen-binding molecule in which the association between the heavy chain and the light chain is regulated, comprising the following steps: (1) A step of modifying the nucleic acid encoding CH1 and CL such that one set of amino acid residues 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 repel each other electrostatically: (a) The amino acid residue in the heavy chain constant region (CH1) at position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) at position 131 according to Kabat numbering, (b) 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 Kabat numbering, (c) The amino acid residue in CH1 at position 175 according to EU numbering, and the amino acid residues in CL at positions 131 and 160 according to Kabat numbering, (d) 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, (2) the step of introducing a modified nucleic acid into a host cell, and the step of culturing the host cell so that the nucleic acid is expressed; and (3) A step of recovering antigen-binding molecules from a cell culture of host cells.

[0209] In addition, the present invention relates to a production method comprising modifying a nucleic acid such that, in step (1) described above, the amino acid residues that electrically repel each other are selected from among the amino acid residues contained in either group (X) or (Y) described above.

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

[0211] The aforementioned amino acid residues that repel each other electrically are preferably selected from amino acid residues contained in either of the sets (X) and (Y) described 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 heavy and light chains of an antigen-binding molecule is a method for regulating the association of the heavy and light chains of an antigen-binding molecule, comprising the step of modifying a nucleic acid such that one set of amino acid residues 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 repel each other electrostatically: (a) an amino acid residue in the heavy chain constant region (CH1) at position 175 according to EU numbering, and an amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering, (b) 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, (c) The amino acid residue in CH1 at position 175 according to EU numbering, and the amino acid residues in CL at positions 131 and 160 according to EU numbering, (d) Amino acid residues in CH1 that are at positions 147 and 175 according to EU numbering, and amino acid residues in CL that are at positions 131 and 160 according to EU numbering.

[0213] In addition, the present invention relates to a method for regulating association, comprising modifying a nucleic acid such that in step (1) described above, the amino acid residues that electrostatically repel each other are selected from amino acid residues contained in either group (X) or (Y) described above.

[0214] Furthermore, the present invention relates to a method for regulating association, comprising modifying a nucleic acid in step (1) above such that two or more amino acid residues forming the VH-VL interface are electrostatically repelling amino acid residues from one another. Here, the electrostatically repelling amino acid residues are preferably any one set of amino acid residues selected from the group consisting of, for example, the sets 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, (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.

[0215] According to the method for regulating the association of the present invention, desired bispecific antibodies can be obtained preferentially and efficiently, as previously described. That is, desired heteromeric polymers in the form of bispecific antibodies can be efficiently formed from monomer mixtures.

[0216] In the above-described method of the present invention, the phrase "modify a nucleic acid" means modifying a nucleic acid so that it corresponds to the amino acid residues to be derived by the "modification" of the present invention. More specifically, this means modifying a nucleic acid encoding an original (pre-modification) amino acid residue to a nucleic acid encoding the amino acid residue to be derived by the modification. Typically, this means performing a genetic manipulation or mutagenesis that will result in at least one nucleotide insertion, deletion, or substitution of the original nucleic acid so that a codon encoding the target amino acid residue is formed. More specifically, the codon encoding the original amino acid residue is replaced with a codon encoding the amino acid residue to be derived by the modification. Such nucleic acid modification can be suitably performed by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis. In addition, the present invention provides nucleic acids encoding the antigen-binding molecule of the present invention. Furthermore, vectors containing nucleic acids are also included in the present invention.

[0217] In some embodiments, the Fc domain of a multispecific antigen-binding molecule consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the multispecific antigen-binding molecule described herein comprises one or fewer Fc domains.

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

[0219] In some embodiments, the Disclosure provides a multispecific antigen-binding molecule further comprising an Fc domain that exhibits reduced binding affinity to the human Fcγ receptor compared to the native human IgG1 Fc domain, the Fc domain further exhibiting stronger FcRn binding affinity to human FcRn compared to the native human IgG1 Fc domain.

[0220] In some embodiments, the Disclosure provides a multispecific antigen-binding molecule further comprising an Fc domain that exhibits reduced binding affinity to the human Fcγ receptor compared to the native human IgG1 Fc domain, wherein the first and / or second Fc domain subunits contained in the Fc domain comprise Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, with the amino acid positions numbered according to EU numbering.

[0221] In some embodiments, in multispecific antigen-binding molecules, the Fc domain exhibits even stronger FcRn binding affinity to human FcRn compared to the native human IgG1 Fc domain. In some embodiments, in a multispecific antigen-binding molecule, the first and / or second Fc domain subunits include Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, with the amino acid positions numbered according to EU numbering.

[0222] IgG-type bispecific antibodies are secreted by introducing the genes for the light and heavy chains (L and H chains) of the two desired types of IgG into cells—a total of four genes—and co-expressing them. However, the number of possible combinations of H and L chains of IgG that can be produced by these methods is theoretically limited to 10. Therefore, purifying IgG containing the desired H and L chain combination from 10 types of IgG is difficult. Furthermore, theoretically, the amount of IgG secreted with the desired combination is expected to be significantly reduced, thus requiring large-scale culture and further increasing production costs.

[0223] Therefore, methods for promoting the association of H chains with desired combinations and between L chains and H chains can be applied to the multispecific antigen-binding molecules of the present invention. For example, a technique for suppressing undesirable H chain association by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody H chain 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 aforementioned amino acid residues in the antibody variable region or the antibody constant region. Those skilled in the art can identify amino acid residues that form interfaces in mutant polypeptides or heteromultimers by homology modeling using commercially available software, for example; and then subject the amino acid residues at these positions to modification in order to regulate association.

[0225] Other known methods can also be used for the association of the multispecific antibodies of the present invention. By substituting an amino acid side chain present in one of the Fc regions of the antibody's H chain with a larger side chain (knob), and substituting an amino acid side chain present in the corresponding Fc region of the other H chain with a smaller side chain (hole), thereby enabling the placement of the knob into the hole, Fc region-containing polypeptides containing different amino acids can be efficiently associated with each other. The knob-into-hole method is discussed elsewhere in this specification.

[0226] In addition, other known methods can also be used for the formation of the multispecific antibodies of the present invention. Complementary association of CH3 molecules using a chain exchange domain CH3, produced by modifying one portion of the CH3 molecules of one H chain of an antibody with a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the other H chain CH3 molecule, can efficiently induce the association of polypeptides having different sequences (Protein Engineering Design & Selection, 23; 195-202, 2010). This known method can also be used to efficiently form the desired multispecific antibodies.

[0227] In addition, techniques for antibody production using the association of antibody CH1 and CL and VH and VL, as described in WO 2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb; 32(2):191-8; techniques for producing bispecific antibodies using a combination of separately prepared monoclonal antibodies (Fab arm exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for regulating the association between antibody heavy chain CH3, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; Christoph et al. (Nature Biotechnology Vol. 31, p 753-758) Techniques for producing bispecific antibodies using two bacterial cell lines that individually express one of the antibody chains, each containing a single H chain and a single L chain, as described in (2013), may be used for the formation of multispecific antibodies.

[0228] Alternatively, even if the desired multispecific antibody cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating and purifying the desired multispecific antibody from the produced antibody. For example, a method has been reported to enable the purification of two types of homomer forms and the desired heteromer antibody by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of H chains to create a difference in isoelectric points (WO2007114325). To date, a method has been reported for purifying heterodimer antibodies using protein A, which includes a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A (WO98050431 and WO95033844). Furthermore, heterodimer antibodies can be efficiently purified by using an H chain that includes substitutions of amino acids such as Tyr and His at the IgG-protein A binding sites at EU numbering positions 435 and 436, which result in different protein A affinitys, or by using H chains with different protein A affinitys obtained according to the method of Reference Example 5 to alter the interaction between each H chain and protein A, and then by using a protein A column.

[0229] Furthermore, an Fc region in which the heterogeneity of the C-terminus of the Fc region has been improved can be appropriately used as the Fc region of the present invention. More specifically, the present invention provides an Fc region produced by deleting glycine at position 446 and lysine at position 447, as specified by EU numbering, from the amino acid sequences of two polypeptides constituting an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0230] Multiple, for example, two or more of these techniques can be used in combination. Furthermore, these techniques can be appropriately and separately applied to the two H chains to be associated. Moreover, these methods can be used in combination with the aforementioned Fc region having reduced binding activity to the Fcγ receptor. Furthermore, the antigen-binding molecule of the present invention may be a molecule produced separately based on the antigen-binding molecule subjected to the above modifications, having the same amino acid sequence.

[0231] In some embodiments, the multispecific antigen-binding molecule contains one or more of the following amino acid residues (i) to (xii): (i) Glutamate or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) Glutamate at position 147 (EU numbering) in the heavy chain constant region; (iii) Glutamate or lysine at position 131 (Kabat numbering) in the constant region of the light chain; (iv) Glutamate or lysine at position 160 (Kabat numbering) in the constant region of the light chain; (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) Glutamate at position 439 (EU numbering) in the heavy chain constant region; (xii) Glutamate at position 440 (EU numbering) in the heavy chain constant region. In some embodiments, the multispecific antigen-binding molecule is a bispecific antibody comprising: The first heavy chain contains 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); The first light chain contains glutamic acid at position 131 (Kabat numbering) and glutamic acid at position 160 (Kabat numbering); The second heavy chain contains 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 containing lysine acid at position 131 (Kabat numbering) and lysine at position 160 (Kabat numbering). In some embodiments, in multispecific antigen-binding molecules: The first heavy chain further includes glutamic acid at position 419 (EU numbering) and proline at position 445 (EU numbering), as well as amino acid deletions at positions 446 and 447 (EU numbering); and The second heavy chain further includes 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, in multispecific antigen-binding molecules: The first heavy chain further 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 further 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 further 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 further 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 The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies exhibiting one or more desired activities. For example, various methods are known in the art for generating phage display libraries and for screening such libraries for antibodies possessing desired binding characteristics. Such methods have been reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further, for example, 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. This is described in 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).

[0233] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically present antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunosource without requiring the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self-antigens without immunization. Finally, naive libraries can also be synthesized synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning the pre-reorganization V-gene segment from stem cells and using PCR primers containing random sequences that encode the hypervariable CDR3 region and achieve in vitro rearrangement. Patent documents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0234] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0235] Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0236] If the antibody contains an Fc region, the carbohydrate to which it is attached may be modified. Native antibodies produced by mammalian cells typically contain branched, bifurcated oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, the modification of oligosaccharides in the antibody of the present invention may be carried out to produce antibody variants with specific improved properties.

[0237] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. 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 lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0238] Further antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0239] In a preferred embodiment, the antibodies described above may have a first H chain CH3 region and a second H chain CH3 region that are crosslinked 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, and size exclusion chromatography. The actual conditions used to purify a particular protein are thought to depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, 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 binds can be used. For example, for affinity chromatography purification of the multispecific antigen-binding molecules of the present invention, a matrix having protein A or protein G may be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the multispecific antigen-binding molecules. The purity of the multispecific antigen-binding molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0241] Cysteine-modified antibody variant In certain embodiments, it would be desirable to produce cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0242] antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either 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 propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0243] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

[0244] Recombination method and configuration For example, as described in U.S. Patent No. 4,816,567, antibodies can be produced using recombinant methods or configurations. In one embodiment, an isolated nucleic acid encoding an anti-HLA-DQ2.5 antigen-binding molecule (antibody) as described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method is provided for producing an anti-HLA-DQ2.5 antigen-binding molecule (antibody), comprising culturing host cells containing the nucleic acid encoding the antibody as described above under conditions suitable for the expression of the anti-HLA-DQ2.5 antibody, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0245] For the recombinant production of anti-HLA-DQ2.5 antigen-binding molecules (antibodies), nucleic acids encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind 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, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 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, for the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from the bacterial cell paste into a soluble fraction and further purified.

[0247] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0248] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0249] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0250] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific 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] Measurement method (assay) 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 activity by various assay methods known in the art.

[0252] Combined measurement methods and other measurement methods In one aspect, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0253] In another context, competitive assays may be used to identify antibodies that compete with any of the antibodies described above for binding to HLA-DQ2.5 (or the HLA-DQ2.5 / gluten peptide complex). In certain embodiments, such competing antibodies bind to the same epitopes (e.g., linear or conformational epitopes) to which the antibodies described above bind. Detailed exemplary methods for mapping the epitopes to which antibodies bind 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 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 the first antibody to bind to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex), any excess unbound antibody is removed, and the amount of label bound to the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is measured. If the amount of label bound to the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to HLA-DQ2.5 (or 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 (e.g., HLA-DQ2.5 or HLA-DQ2.5 / gluten peptide complex). The antigen can be prepared as a recombinant protein using any method, such as as described herein. Antibody-containing samples, such as blood and spleen, are collected from the immunized animals. For B cell selection, for example, a biotinylated antigen is prepared, antigen-binding B cells are conjugated to the biotinylated antigen, and the cells are subjected to cell sorting and culture for selection. The specific binding of the cells to the antigen can be evaluated by any suitable method, such as ELISA. This method can also be used to evaluate the lack of cross-reactivity to unintended antigens. To isolate the selected antibody or determine its sequence, for example, RNA is purified from the cells, and the DNA encoding the antibody region is prepared by reverse transcription of RNA and PCR amplification. Furthermore, for further analysis, the cloned antibody gene can be expressed in suitable cells, and the antibody can be purified from the culture supernatant.

[0256] To test whether an anti-HLA-DQ2.5 antigen-binding molecule (antibody) binds to a target antigen (e.g., a complex formed by HLA-DQ2.5 and a gluten peptide, such as those described herein), any method for evaluating binding can be used. For example, when using a FACS-based cell sorting method, cells expressing the antigen are incubated with the test antibody, and then an appropriate secondary antibody against the test antibody (i.e., the primary antibody) 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 described in “Antibody Affinity” herein can be used. For example, Kd measurement by BIACORE surface plasmon resonance assay can be used to evaluate binding between the test antibody and the target antigen described herein.

[0257] In a particular embodiment, the method of the present invention further comprises the steps of: testing whether an antibody has neutralizing activity against the binding of HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) to the 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 having said neutralizing activity. In a particular embodiment, the method of the present invention further comprises the steps of: testing whether an antibody has neutralizing activity against the binding of HLA-DQ2.2 (or HLA-DQ2.2 / gluten peptide complex) to the 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 having said neutralizing activity. These steps can be carried out in the presence of gluten peptides, such as those described herein, i.e., using HLA-DQ2.5 or HLA-DQ2.2 bound to the peptide. Neutralizing activity can be evaluated, for example, as described herein. Briefly, for immobilization on a plate, beads, e.g., yellow particles coated with streptavidin, are appropriately prepared, and soluble HLA-DQ bound to the peptide is added to the beads. The plate is washed and blocked, and an antibody is added and incubated. When evaluating the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR, for example, D2 TCR tetramer-PE can be added and incubated. The binding between these two can be evaluated based on the color development / fluorescent labeling of the TCR bound to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex).

[0258] In some embodiments, multispecific antigen-binding molecules block the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. In some embodiments, multispecific antigen-binding molecules block the interaction between the HLA-DQ2.2 / gluten peptide complex and HLA-DQ2.2 / gluten peptide-restricted CD4+ T cells. In this context, the gluten peptide is the peptide in the complex to which any of the above antigen-binding molecules / domains bind. In some embodiments, the gluten peptide is selected from the group consisting of α1 gliadin peptide, α1b gliadin peptide, α2 gliadin peptide, ω1 gliadin peptide, ω2 gliadin peptide, γ1 gliadin peptide, γ2 gliadin peptide, γ3 gliadin peptide, γ4a gliadin peptide, γ4d gliadin peptide, and BC-hordein peptide.

[0259] In some embodiments, the multispecific antigen-binding molecule has substantially 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 present invention has enhanced 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 enhancement may be determined by comparing it with the binding activity to a complex formed by HLA-DQ2.5 and an unrelated peptide, or to cells that do not have the complex of interest, such as HLA-DQ2.5-positive PBMC B cells and / or Ba / F3 cells expressing HLA-DQ2.5 or HLA-DQ2.2.

[0261] The bispecific antibody of the present invention comprises the heavy and light chains of a first arm / half-antibody and the heavy and light chains of a second arm / half-antibody. The terms “arm” or “half-antibody” refer to a portion of an antibody that contains one heavy chain and one 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, the bispecific antibody of the present invention derives the first arm / half-antibody from DQN0344xx (DQN0344Hx / DQN0344Lx) described herein, and the second arm / half-antibody from DQN0385ee (DQN0385He / DQN0385Le) described herein. The sequence ID numbers (SEQ ID numbers) of VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and the full-length heavy (H) chain and light (L) chain of the bispecific antibody of the present invention are shown in Tables 2-3 to 2-6 (below). In some embodiments, the antibody of the present invention comprises HCDR1 containing the sequence of SEQ ID NO: 129 or 164; HCDR2 containing the sequence of SEQ ID NO: 130 or 165; and HCDR3 containing the sequence of SEQ ID NO: 131 or 166. In some embodiments, the antibody of the present invention comprises LCDR1 containing the sequence of SEQ ID NO: 132 or 167; LCDR2 containing the sequence of SEQ ID NO: 133 or 168; and LCDR3 containing the sequence of SEQ ID NO: 134 or 169. In some embodiments, the antibody of the present invention includes a heavy chain variable region comprising the sequence of SEQ ID NO: 88 or 89. In some embodiments, the antibody of the present invention comprises a heavy chain constant region containing the sequence of SEQ ID NO: 105 or 162. In some embodiments, the antibody of the present invention includes a light chain variable region comprising the sequence of SEQ ID NO: 90 or 91. In some embodiments, the antibody of the present invention comprises a light chain constant region containing the sequence of SEQ ID NO: 106. In some embodiments, the antibody of the present invention comprises a (full-length) heavy chain containing the sequence of SEQ ID NO: 41, 42, 44, or 45. In some embodiments, the antibody of the present invention comprises a (full-length) light chain containing the sequence of SEQ ID NO: 43 or 46. In some embodiments, the antibodies of the present invention include HCDR1 containing the sequence of SEQ ID NO: 135, 144, 147, 153, 156, or 159; HCDR2 containing the sequence of SEQ ID NO: 136, 145, 148, 154, 157, or 160; and HCDR3 containing the sequence of SEQ ID NO: 137, 146, 149, 155, 158, or 161. In some embodiments, the antibody of the present invention comprises LCDR1 containing the sequence of SEQ ID NO: 138, 141, or 150; LCDR2 containing the sequence of SEQ ID NO: 139, 142, or 151; and LCDR3 containing the sequence of SEQ ID NO: 140, 143, or 152. In some embodiments, the antibody of the present invention includes a heavy chain variable region comprising the sequence of SEQ ID NOs: 92, 93, 94, 95, 96, or 97. In some embodiments, the antibody of the present invention comprises a heavy chain constant region containing the sequence of SEQ ID NO: 104 or 163. In some embodiments, the antibody of the present invention includes a light chain variable region comprising the sequence of SEQ ID NO: 98, 99, or 100. In some embodiments, the antibody of the present invention comprises a light chain constant region containing the sequence of SEQ ID NO: 107. In some embodiments, the antibody of the present invention comprises a (full-length) heavy chain containing the sequence of SEQ ID NOs: 53, 54, 57, 58, 59, 60, 62, 63, 64, 65, 66, or 67. In some embodiments, the antibody of the present invention comprises a (full-length) light chain containing the sequence of SEQ ID NO: 55, 56, or 61.

[0263] Table 1-2 shows the specific sequences of the full-length H chain and L chain of the arm / half-antibody (included in the bispecific antibody of the present invention).

[0264] (Table 1-2) Full-length H chain and L chain of bispecific antibodies The H (or L) chain contains HCDR1, HCDR2, and HCDR3 (or LCDR1, LCDR2, and LCDR3) from the N-terminus to the C-terminus, and these are underlined in this table. TIFF2026108741000002.tif226151TIFF2026108741000003.tif232151TIFF20261087410 00004.tif230151TIFF2026108741000005.tif223151TIFF2026108741000006.tif108151

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

Claims

1. A multispecific antigen-binding molecule that binds to a complex formed by HLA-DQ2.5 and a gluten peptide, comprising a first antigen-binding portion containing first and second antibody variable regions and a second antigen-binding portion containing third and fourth antibody variable regions, and comprising any one of the following (1) to (14): (1) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, and CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; (2) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, and CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, and CDR 3 of SEQ ID NO: 140; (3) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, and CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO: 143; (4) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, and CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO: 143; (5) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, and CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; (6) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, and CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; (7) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131; a second antibody variable region including CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, and CDR 3 of SEQ ID NO: 134; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, and CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO: 143; and (8) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, and CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, and CDR 3 of SEQ ID NO: 140; (9) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, and CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO: 143; (10) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, and CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO: 143; (11) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, and CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; (12) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, and CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; (13) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, and CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, and CDR 3 of SEQ ID NO: 152; and (14) A first antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, and CDR 3 of SEQ ID NO: 166; a second antibody variable region including CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, and CDR 3 of SEQ ID NO: 169; a third antibody variable region including complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, and CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region including CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, and CDR 3 of SEQ ID NO:

143.

2. A multispecific antigen-binding molecule according to claim 1, which is a bispecific antibody.

3. A nucleic acid encoding a multispecific antigen-binding molecule according to claim 1 or 2.

4. A vector comprising the nucleic acid described in claim 3.

5. A cell comprising the nucleic acid according to claim 3 or the vector according to claim 4.

6. A method for producing a multispecific antigen-binding molecule, comprising the step of culturing the cells described in claim 5 so as to produce the multispecific antigen-binding molecule.

7. The method according to claim 6, further comprising the step of recovering the multispecific antigen-binding molecule from the cell culture.

8. A pharmaceutical composition comprising a multispecific antigen-binding molecule according to claim 1 or 2, and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, which is a pharmaceutical composition for use in the treatment and / or prevention of celiac disease.

10. Use of a multispecific antigen-binding molecule according to claim 1 or 2 in the manufacture of a pharmaceutical product.

11. The use according to claim 10, wherein the pharmaceutical is a pharmaceutical for the treatment and / or prevention of celiac disease.

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