Anti-HLA-DQ2.5 antibody preparation

Abstract

The present invention provides an anti-HLA-DQ2.5 antigen-binding molecule and a formulation (particularly an injectable formulation) containing the same. [Solution] The anti-HLA-DQ2.5 antibody of the present invention has binding activity to complexes formed between HLA-DQ2.5 and gluten peptides, but has substantially no binding activity to complexes formed between HLA-DQ2.5 and unrelated peptides. Furthermore, the antibody of the present invention has been shown to have an inhibitory effect on T cell activation by gluten peptides. The present invention also relates to formulations, particularly injectable formulations, containing the anti-HLA-DQ2.5 antibody.

Description

[Technical Field]

[0001] The present invention relates to an anti-HLA-DQ2.5 antibody and a formulation containing the same. [Background technology]

[0002] Celiac disease (also known as coeliac disease) is an autoimmune disorder in which gluten ingestion causes damage to the small intestine in genetically susceptible individuals (Non-Patent Documents 1-5). Celiac disease is thought to affect approximately 1% of the Western population, or 8 million people in the United States and the European Union; however, no significant therapeutic progress has been achieved since the disease was recognized in the 1940s. Human leukocyte antigens (HLA), 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"), which form heterodimers composed of α and β chains on the cell surface. The majority (>90%) of celiac disease patients possess alleles of the HLA-DQ2.5 haplotype (Non-Patent Document 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 the digestion of gluten-rich foods, such as bread (Non-Patent Document 2). These peptides are transported through the small intestinal epithelium to the lamina propria and are deamidated by tissue transglutaminases such as transglutaminase 2 (TG2). The deamidated gliadin peptides are processed by antigen-presenting cells (APCs), which then load them onto HLA-DQ2.5. The loaded peptides are presented to HLA-DQ2.5-restricted T cells, activating innate and adaptive immune responses. This leads to inflammatory damage of the small intestinal mucosa and symptoms including various types of gastrointestinal disorders, nutritional deficiencies, and systemic symptoms (Non-Patent Documents 8, 9, and 10). Anti-HLA DQ neutralizing antibodies have been reported to inhibit gluten peptide-dependent activation of T cells from celiac disease patients (Non-Patent Document 7). The currently available treatment for celiac disease is lifelong adherence to a gluten-free diet (GFD). However, in reality, it is difficult to completely eliminate gluten exposure even with a GFD. The tolerable gluten intake for these patients is only approximately 10–50 mg / day (Non-Patent Document 11). Cross-contamination can occur extensively during GFD production, and even in patients who adhere well to a GFD, trace amounts of gluten can trigger celiac disease symptoms. Given the risk of unintentional gluten exposure, adjunctive therapies to a GFD are needed.

[0003] In recent years, various antibody preparations have been developed and put into practical use, but most antibody preparations are used as intravenous injection preparations. Meanwhile, due to needs in medical practice, there is a growing demand for the development of antibody-containing preparations as self-injectable subcutaneous injection preparations. In particular, there is a high demand for the development of solution preparations enclosed in prefilled syringes due to their convenience.

[0004] When designing antibody-containing formulations for subcutaneous injection, the amount of antibody administered per dose is large (approximately 80 to 200 mg), but subcutaneous injections generally have limitations on the amount of injection solution, making it essential to increase the concentration of the antibody in the administration solution.

[0005] In recent years, prefilled syringes have come to be used in medical settings as prefilled syringe preparations for self-injection, each of which includes a cylindrical syringe body filled with a drug, an injection needle attached to the tip of the syringe body, a syringe cap that covers the detachably attached injection needle, and a plunger that is inserted into the syringe body and can slide in the axial direction of the syringe body.

[0006] When using a prefilled syringe, the syringe cap is removed, the needle is inserted into the administration site, and the drug solution is dispensed and administered by moving the plunger forward with the plunger rod. Generally, to ensure the sliding properties of the plunger, a lubricant such as silicone oil is applied to the inner wall and plunger of the prefilled syringe.

[0007] Particle formation in aqueous solutions is a problem for antibody-containing formulations. These particles are larger than multimers, such as dimers and trimers, but are generally difficult to see with the naked eye. These include subvisible particles (SVPs), which are small particles with diameters of 1.5 μm to less than 50 μm, and visible particles (VPs, larger than 100 μm) that can be visually detected at standard illuminance (approximately 2,000-3,000 lx). While the visual detection rate of visible particles in pharmaceutical formulations varies significantly between practitioners, it has been reported that at the standard illuminance (approximately 2,000-3,000 lx) specified in the Pharmacopoeia, the detection sensitivity for 100 μm particles is approximately 40%, for 150 μm particles is approximately 70%, and for 200 μm particles is nearly 100% (Non-Patent Document 12). Furthermore, by increasing the illuminance when observing the pharmaceutical preparation or by extending the observation time, it is actually possible to visually detect particles with even smaller diameters, down to a minimum of about 40 μm. In this specification, such particles with a diameter of 40 μm or more to 100 μm are particularly referred to as particles that can be visually detected only under high illuminance. Furthermore, particles with a diameter of 40 μm or more are particles that can be visually detected under high illuminance, and are referred to as visually detectable particles.

[0008] Antibodies generally have the property of adsorbing to and aggregating at interfaces such as air-liquid and solid-liquid interfaces. The presence of these interfaces may contribute to the formation of the visually detectable particles. It has been reported that applying mechanical stress to a syringe filled with an antibody solution results in a significant increase in microparticles due to the presence of interfaces (Non-Patent Document 13). The antibody solution filled in the syringe forms an air-liquid interface due to the presence of air bubbles, and forms a solid-liquid interface upon contact with the plunger and syringe barrel. Furthermore, when the plunger and barrel of a prefilled syringe are coated with silicone, the antibody solution comes into contact with the silicone on the solid surface and forms a new solid-liquid interface. It has also been reported that proteins adsorbed and aggregated at the solid-liquid interface peel off into the liquid due to the movement of air in the prefilled syringe, appearing as visible particles (Non-Patent Document 14).

[0009] One way to reduce the stress experienced at various interfaces is to reduce the amount of air bubbles in the prefilled syringe. By reducing the amount of air bubbles, the amount of air moving through the prefilled syringe can be reduced, which is thought to result in suppressing adsorption to the gas-liquid interface and solid-liquid interface, as well as detachment of aggregates. [Prior art documents] [Non-patent literature]

[0010] [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 [Non-Patent Document 12] James A. Melchore, AAPS PharmSciTech; 2011; 12(1): 215-221. [Non-Patent Document 13] Torisu et al., J. Pharm. Sci. 106 (2017) 2966-2978. [Non-Patent Document 14] Gerhardt et al., J. Pharm. Sci. 103 (2014) 1601-1612. Summary of the Invention [Problem to be solved by the invention]

[0011] technical challenges In the above-mentioned situations requiring adjuvant therapy, the present invention provides anti-HLA-DQ2.5 antigen-binding molecules and preparations containing them (particularly injectable preparations). [Means for solving the problem]

[0012] Resolving the issue The antigen-binding molecules of the present invention are modified and are capable of binding to two or more complexes formed by HLA-DQ2.5 and gluten peptides.

[0013] More specifically, the present invention provides the following: [1] (i) a first antigen-binding portion having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide; and (ii) a second antigen-binding moiety having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide; A multispecific antigen-binding molecule comprising: the antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides, At least one of the gluten peptides in the complex to which the first antigen-binding moiety binds is different from at least one of the gluten peptides 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 of 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 chain and / or light chain of the first antigen-binding moiety and / or the 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 has substantially no binding activity to Ba / F3 cells expressing HLA-DQ2.2. [1-1] The 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 portion and / or the second antigen-binding portion of the multispecific antigen-binding molecule have been substituted. [1-2] The multispecific antigen-binding molecule of [1-1], comprising at least one amino acid substitution in the heavy chain variable region; at least one amino acid substitution in the heavy chain constant region; at least one amino acid substitution in the light chain variable region; and at least one amino acid substitution in the light chain constant region. [2] The multispecific antigen-binding molecule of any one of [1] to [1-2], wherein the gluten peptide is an immunodominant peptide associated with celiac disease. [3] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide is selected from the group consisting of 33-mer gliadin peptide, 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, secalin 1 peptide, secalin 2 peptide, and 26-mer gliadin peptide. [3-1] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or all of a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26-mer gliadin peptide. [3-2] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptide is selected from the group consisting of a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26-mer gliadin peptide. [3-3] The multispecific antigen-binding molecule of any one of [1] to [2], wherein the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or all of a 33-mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26-mer gliadin peptide. [4] The multispecific antigen-binding molecule of any one of [1] to [3-3], which has substantially no binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, wherein the unrelated peptide is at least one peptide selected from the group consisting of a CLIP peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, and a thyroperoxidase peptide. [4-1] A multispecific antigen-binding molecule according to any one of [1] to [3-3], which has substantially no binding activity to HLA-DQ2.5 in the form of a complex with an unrelated peptide, wherein the unrelated peptides are all of a CLIP peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, and a thyroperoxidase peptide. [5] Any one of the multispecific antigen-binding molecules of [1] to [4-1], which 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 of [1] to [5] that blocks (i) the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells, and / or (ii) the interaction between the HLA-DQ2.2 / gluten peptide complex and HLA-DQ2.2 / gluten peptide-restricted CD4+ T cells. [6-2] The multispecific antigen-binding molecule of [6], wherein 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] The multispecific antigen-binding molecule of any one of [1] to [6-2], wherein the antigen-binding molecule has enhanced binding activity to a complex formed by HLA-DQ2.5 and a gluten peptide, compared to before the humanization and modification. [8] The multispecific antigen-binding molecule of any one of [1] to [7], wherein the antigen-binding molecule has enhanced cross-reactivity to gluten peptides compared to before the humanization and modification. [8-1] The 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] The multispecific antigen-binding molecule of any one of [1] to [8-1], wherein one, two, three, or all of the amino acid residue sets selected from the group consisting of the amino acid residue sets shown in the following (a) to (d) in the heavy chain and light chain of the antigen-binding molecule are amino acid residues that electrostatically repel each other: (a) the amino acid residue in the heavy chain constant region (CH1) that is position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) that is position 131 according to Kabat numbering; (b) the amino acid residue in CH1 that is position 175 according to EU numbering and the amino acid residue in CL that is position 160 according to Kabat numbering; (c) the amino acid residue in CH1 that is position 175 according to EU numbering, and the amino acid residues in CL that are positions 131 and 160 according to Kabat numbering; (d) Amino acid residues in CH1 that are positions 147 and 175 according to EU numbering, and amino acid residues in CL that are positions 131 and 160 according to Kabat numbering.

[10] Furthermore, the multispecific antigen-binding molecule of [9], wherein two or more amino acid residues forming the interface between the heavy chain variable region and the light chain variable region are amino acid residues that electrostatically repel each other.

[11] The multispecific antigen-binding molecule of

[10] , wherein the mutually electrostatically repulsive 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 that is position 39 according to the Kabat numbering system, and the amino acid residue in the light chain variable region that is position 38 according to the Kabat numbering system; (b) The amino acid residue in the heavy chain variable region that is position 45 according to Kabat numbering, and the amino acid residue in the light chain variable region that is position 44 according to Kabat numbering.

[12] Any one of the multispecific antigen-binding molecules of [9] to

[11] , wherein the mutually electrostatically repulsive amino acid residues 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] Any one of the multispecific antigen-binding molecules of [9] to

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

[14] The multispecific antigen-binding molecule of

[13] , wherein 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 according to

[13] or

[14] , wherein the Fc domain is composed of a first Fc region subunit and a second Fc region subunit capable of stable association.

[16] The multispecific antigen-binding molecule of

[15] , wherein the Fc domain comprises the following (e1) or (e2), and the amino acid positions are numbered according to EU numbering: (e1) a first Fc region subunit comprising Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region comprising 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] The multispecific antigen-binding molecule of any one of

[13] to

[16] , wherein the Fc domain further exhibits stronger FcRn-binding affinity to human FcRn compared to a native human IgG1 Fc domain.

[18] The multispecific antigen-binding molecule of

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

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

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

[21] The multispecific antigen-binding molecule of

[20] , wherein the second antigen-binding portion comprises any one of the following (b1) to (b8): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b4) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b5) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b6) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b7) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (b8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 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, wherein the second antigen-binding portion comprises any one of the following (b1) to (b8): (b1) a first antibody variable region comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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, 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, 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 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 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 portion and a second antigen-binding portion, The first antigen-binding portion is selected from the following (c1) to (c3): (c1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (c2) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (c3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a 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 to a second antibody variable region described in (c1) or (c2). including one of the following: The second antigen-binding portion is selected from the following (d1) to (d8): (d1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (d2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d4) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d5) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d6) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d7) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (d8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 to a fourth antibody variable region described in any one of (d1) to (d7). A multispecific antigen-binding molecule comprising any one of: [22-2] A multispecific antigen-binding molecule comprising a first antigen-binding portion comprising a first and a second antibody variable region and a second antigen-binding portion comprising a third and a fourth antibody variable region, the multispecific antigen-binding molecule comprising any one of the following (1) to (15): (1) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 135, a CDR 2 of SEQ ID NO: 136, and a CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 138, a CDR 2 of SEQ ID NO: 139, and a CDR 3 of SEQ ID NO: 140; (2) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 135, a CDR 2 of SEQ ID NO: 136, and a CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a CDR 3 of SEQ ID NO: 143; (3) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 144, a CDR 2 of SEQ ID NO: 145, and a CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a CDR 3 of SEQ ID NO: 143; (4) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 147, a CDR 2 of SEQ ID NO: 148, and a CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (5) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 153, a CDR 2 of SEQ ID NO: 154, and a CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (6) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 156, a CDR 2 of SEQ ID NO: 157, and a CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (7) A first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 159, a CDR 2 of SEQ ID NO: 160, and a CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a CDR 3 of SEQ ID NO: 143; and (8) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 135, a CDR 2 of SEQ ID NO: 136, and a CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 138, a CDR 2 of SEQ ID NO: 139, and a CDR 3 of SEQ ID NO: 140; (9) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 135, a CDR 2 of SEQ ID NO: 136, and a CDR 3 of SEQ ID NO: 137; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a CDR 3 of SEQ ID NO: 143; (10) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 144, a CDR 2 of SEQ ID NO: 145, and a CDR 3 of SEQ ID NO: 146; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a CDR 3 of SEQ ID NO: 143; (11) A first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 147, a CDR 2 of SEQ ID NO: 148, and a CDR 3 of SEQ ID NO: 149; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (12) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 153, a CDR 2 of SEQ ID NO: 154, and a CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (13) A first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 156, a CDR 2 of SEQ ID NO: 157, and a CDR 3 of SEQ ID NO: 158; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (14) A first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 164, a CDR 2 of SEQ ID NO: 165, and a CDR 3 of SEQ ID NO: 166; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 167, a CDR 2 of SEQ ID NO: 168, and a CDR 3 of SEQ ID NO: 169; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 159, a CDR 2 of SEQ ID NO: 160, and a CDR 3 of SEQ ID NO: 161; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 141, a CDR 2 of SEQ ID NO: 142, and a 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 to 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 to 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 to 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 to a fourth antibody variable region described in any one of (1) to (14).

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

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

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

[25] The multispecific antigen-binding molecule of

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

[28] The multispecific antigen-binding molecule of

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

[27] or [27-2], further comprising a combination of two polypeptide chains selected from the group consisting of the following (B1) to (B8): (B1) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53, and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (B2) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53, and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B3) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57, and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (B4) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B5) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B6) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (B7) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66, and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; and (B8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the 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 to the 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 the following (1) to (15): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (4) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (9) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (10) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (11) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (12) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (13) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (14) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 45 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; and (15) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to 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 to 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 to 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 to 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 the following (1) to (15): (1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (2) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (3) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (4) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (5) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (6) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 55; (7) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (8) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (9) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (10) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (11) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (12) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a second light chain comprising the amino acid sequence of SEQ ID NO: 56; (13) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; (14) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a first light chain comprising the amino acid sequence of SEQ ID NO: 46, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61; and (15) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to 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 to 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 to 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 to a second light chain sequence described in any one of (1) to (14). [29a] Any combination of (i) to (iii) below: (i) A multispecific antigen-binding molecule comprising a sequence described in any one of (a1) to (a3) ​​of

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

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

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

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

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

[28] or [28-2].

[30] A nucleic acid encoding any one of the multispecific antigen-binding molecules of [1] to

[29] .

[31] A vector comprising the nucleic acid of

[30] .

[32] A cell containing the nucleic acid of

[30] or the vector of

[31] .

[33] A method for producing a multispecific antigen-binding molecule, comprising culturing the cell 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.

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

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

[36] The 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 [1] to

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

[38] The use of

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

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

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

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

[29] or a combination of [29a], and instructions for use.

[0014] Furthermore, more specifically, the present invention provides the following:

[0101] An injectable preparation in which a solution containing an anti-HLA-DQ2.5 antibody as an active ingredient is filled in a container, An injectable formulation, wherein the antibody comprises any one of the following (1) to (3): (1) a first antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 129, a CDR 2 of SEQ ID NO: 130, and a CDR 3 of SEQ ID NO: 131; a second antibody variable region comprising a CDR 1 of SEQ ID NO: 132, a CDR 2 of SEQ ID NO: 133, and a CDR 3 of SEQ ID NO: 134; a third antibody variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 153, a CDR 2 of SEQ ID NO: 154, and a CDR 3 of SEQ ID NO: 155; and a fourth antibody variable region comprising a CDR 1 of SEQ ID NO: 150, a CDR 2 of SEQ ID NO: 151, and a CDR 3 of SEQ ID NO: 152; (2) a first antibody variable region comprising the amino acid sequence of SEQ ID NO: 88; a second antibody variable region comprising the amino acid sequence of SEQ ID NO: 90; a third antibody variable region comprising the amino acid sequence of SEQ ID NO: 95; and a fourth antibody variable region comprising the amino acid sequence of SEQ ID NO: 100; or (3) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a first light chain comprising the amino acid sequence of SEQ ID NO: 43, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a second light chain comprising the amino acid sequence of SEQ ID NO: 61.

[0102] An injectable preparation of

[0101] , wherein the container is a syringe or cartridge.

[0103] An injectable formulation of

[0101] or

[0102] , in which the volume of bubbles in the container at 1 atmosphere and 25°C is 40 μL or less.

[0104] An injectable formulation of

[0103] , in which the volume of bubbles in the container at 1 atmosphere and 25°C is 10 μL or less.

[0105] An injectable preparation according to any one of

[0101] to

[0104] , wherein the container is a prefilled syringe.

[0106] The injectable preparation according to any one of

[0101] to

[0105] , wherein the concentration of the antibody in the solution is 0.1 mg / mL or more. [106a] An injectable preparation according to any one of

[0101] to

[0105] , wherein the concentration of the antibody in the solution is 50 mg / mL or more.

[0107] An injectable preparation according to any one of

[0101] to [106a], wherein the solution contained in a 1 mL syringe is in the range of 0.1 to 1.2 mL, or the solution contained in a 2.25 mL syringe is in the range of 0.1 to 2.5 mL.

[0108] The injectable preparation according to any one of

[0101] to

[0107] , wherein the injectable preparation in which the solution is filled in a container includes a syringe or cartridge containing the pharmaceutical preparation inside, and a stopper.

[0109] An injectable preparation according to any one of

[0101] to

[0108] , wherein the syringe or cartridge is made of glass or a cycloolefin-based resin.

[0110] An injectable preparation of

[0109] , wherein the cycloolefin resin is a cycloolefin polymer (COP) or a cycloolefin copolymer (COC).

[0111] An injectable formulation according to any one of

[0101] to

[0110] , wherein the solution comprises one or more pharmaceutically acceptable excipients, including a sugar, a sugar alcohol, a buffering agent, a preservative, a carrier, an antioxidant, a chelating agent, a natural polymer, a synthetic polymer, a cryoprotectant, a surfactant, a bulking agent, a stabilizer, or a combination thereof.

[0112] The injectable formulation according to

[0111] , wherein the surfactant is polysorbate, poloxamer 188, sodium lauryl sulfate, polyol, poly(ethylene glycol), glycerol, propylene glycol or poly(vinyl alcohol).

[0113] An injectable formulation described in

[0111] or

[0112] , wherein the surfactant is polysorbate or poloxamer 188.

[0114] An injectable preparation according to any one of

[0111] to

[0113] , wherein the concentration of the surfactant in the solution is 0.01 mg / mL or more.

[0115] An injectable preparation according to any one of

[0101] to

[0114] , wherein the pH of the solution is in the range of 5.5 to 6.5.

[0116] An injectable formulation according to any one of

[0101] to

[0115] , in which the average number of particles in a solution of an injectable formulation containing 0.01 mg / mL of a surfactant after storage at 5°C for one day is reduced compared to when the volume of bubbles in the injectable formulation is 120 μL at 25°C and 1 atmosphere.

[0117] An injectable preparation of any one of

[0101] to

[0116] , which is an injectable preparation for treating celiac disease.

[0118] A method for preparing any one of the injectable preparations of

[0101] to

[0117] , comprising: and filling a container with a solution containing the antibody as an active ingredient so that the volume of air bubbles in the resulting injectable formulation at 1 atmosphere and 25°C inside the container is 40 μL or less.

[0119] The method of

[0118] , which comprises filling the solution into a container so that the volume of air bubbles in the resulting injectable preparation at 1 atmosphere and 25°C inside the container is 10 μL or less.

[0120] A method according to

[0118] or

[0119] , wherein when filling the solution into the container, the container is stoppered by a vacuum stopper placement method or a mechanical stopper placement method. [Brief explanation of the drawings]

[0015] [Figure 1-1] Figure 1-1 shows the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1270 / L0722-F6) to the Ba / F3 cell line expressing HLA class II (all antibodies were tested at 0.05 micrograms (μg) / mL, and the control DQN0139bb (DQN0139bb-SG181) (WO2018 / 155692) and IC17dK were tested at 1 μg / mL). In the names of the gluten peptides shown, "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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and 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 the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-10] Figures 1-10 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1353 / L0681-F6) to the HLA class II-expressing Ba / F3 cell line (all antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-11] Figures 1-11 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1251 / L0605-F6) to the HLA class II-expressing Ba / F3 cell line (antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL, and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-12] Figures 1-12 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H0976 / L0591 / / DQN0385H1353 / L0681-F6) to the HLA class II-expressing Ba / F3 cell line (antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL, and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-13]Figures 1-13 show the binding results of anti-HLA-DQ antibodies (variants DQN0344H1013 / L0620 / / DQN0385H1255 / L0605-F6) to the HLA class II-expressing Ba / F3 cell line (antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5 / hCLIP, antibodies were tested at 0.313 μg / mL, and controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 1-14] Figures 1-14 show 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, and controls DQN0139bb and IC17dK were tested at 1 μg / mL). [Figure 1-15] Figure 1-15 shows the results of DQN0139bb binding to the Ba / F3 cell line expressing HLA class II (control DQN0139bb was tested at 1 μg / mL). [Figure 1-16] Figure 1-16 shows the results of IC17dK against the Ba / F3 cell line expressing HLA class II (control IC17dK was tested at 1 μg / mL). [Figure 2] Figure 2 shows the results of antibody binding to CD19+ B cells from PBMCs (antibodies were tested at 0.05 μg / mL, and controls DQN0139bb and IC17dK were tested at 1 μg / mL; (#) For HLA-DQ2.5 and HLA-DQ2.5-CLIP, antibodies were tested at 0.313 μg / mL; controls DQN0139bb and IC17dK were tested at 20 μg / mL). [Figure 3-1] FIG. 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] FIG. 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]FIG. 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] FIG. 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] Figures 3-5 show the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / ω2 gliadin-dependent Jurkat T cell activation. [Figure 3-6] Figures 3-6 show 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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]FIG. 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] Figures 4-6 show the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / BC hordein-dependent Jurkat T cell activation. [Figure 4-7] Figures 4-7 show the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.5 / γ1 gliadin-dependent Jurkat T cell activation. [Figure 4-8] Figures 4-8 show 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] FIG. 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] FIG. 5-1 shows HLA-DQ2.2 / α1a gliadin-dependent Jurkat T cell activation mediated by 33-mer gliadin. [Figure 5-2] FIG. 5-2 shows HLA-DQ2.2 / α2 gliadin-dependent Jurkat T cell activation mediated by 33-mer gliadin. [Figure 5-3] FIG. 5-3 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.2 / α1a gliadin-dependent Jurkat T cell activation. [Figure 5-4] FIG. 5-4 shows the inhibitory effect of anti-HLA-DQ antibodies on HLA-DQ2.2 / α2 gliadin-dependent Jurkat T cell activation. [Figure 6]Figure 6 shows photographs of the air bubbles in the syringe when they were (a) 120 μL, (b) 40 μL, and (c) 10 μL. [Figure 7] FIG. 7 is a diagram showing an example of the configuration of a syringe. DETAILED DESCRIPTION OF THE INVENTION

[0016] Description of Aspects The techniques and procedures described or referred to herein are generally well understood and routinely employed by those skilled in the art using conventional methodologies, such as those widely used in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. 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)。.

[0017] An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0018] "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0019] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0020] The term "antigen-binding portion" or "antigen-binding domain" refers to a portion of an antibody that comprises an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding portion / domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen-binding portion / domain contains both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0021] 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, such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting HLA-DQ2.5. In one embodiment, the degree of binding of the anti-HLA-DQ2.5 antigen-binding molecule (antibody) to an unrelated antigen is less than about 10% of the binding of the antibody to HLA-DQ2.5 or HLA-DQ2.5 / gluten peptide complexes, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody having "binding activity" for HLA-DQ2.5 or an HLA-DQ2.5 / gluten peptide complex has an affinity of ≦1 micromolar (μM), ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0022] As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding site or any molecule that has antigen-binding activity, and may further 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 derived from living organisms; for example, they may be polypeptides produced from artificially designed sequences. They may be naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc. In addition, scaffold molecules containing a known stable three-dimensional structure, such as an α / β barrel, as a scaffold (wherein a portion of the molecule becomes the antigen-binding site) are also an embodiment of the antigen-binding molecules described herein. In some embodiments, the "antigen-binding molecule" is an antibody. As used herein, the terms "antigen-binding molecule" and "antibody" are used in the broadest sense and encompass various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired antigen-binding activity. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody.

[0023] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0024] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competition assay, or conversely, the reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0025] "Autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against an individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, and specifically 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, inflammatory responses such as celiac disease, inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; 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 associated with leukocyte leakage; and the central nervous system (CNS). Inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; 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.

[0026] The term "celiac disease" refers to a genetic autoimmune disorder caused by damage to the small intestine when gluten is ingested in foods. Symptoms of celiac disease include, but are not limited to, gastrointestinal problems 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.

[0027] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0028] 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 major 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0029] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0030] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (residues Gly446-Lys447) 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 is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0031] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0032] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0033] As used herein, the term "gluten" refers collectively to a complex of storage proteins called prolamins found in wheat and other related grains. In the intestinal lumen, gluten is broken down into so-called gluten peptides. Gluten peptides include, but are not limited to, gliadin from wheat, hordein from barley, secalin from rye, and avenin from oat.

[0034] In celiac disease, gluten peptides are antigenic peptides recognized by T cells and are responsible for the disease. Immunodominance is a phenomenon in which an immune response is primarily driven 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 known as "α1a gliadin") and α2 gliadin (both of which are included in the 33-mer gliadin sequence), as well as ω1 gliadin, ω2 gliadin, and BC hordein (a total of five 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 seven 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.

[0035] As used herein, the phrase "substantially no binding activity" refers to the activity of an antibody to bind to a non-target antigen at a binding level that includes non-specific or background binding but not specific binding. In other words, such an antibody "has no specific / significant binding activity" for the non-target antigen. Specificity can be measured by any method described herein or known in the art. The non-specific or background binding level may be zero, close to zero but not zero, or so low that it can be technically ignored by those skilled in the art. For example, if a person skilled in the art cannot detect or observe any significant (or relatively strong) signal for binding between an antibody and a non-target antigen in an appropriate binding assay, the antibody can be said to have "substantially no binding activity" or "no specific / significant binding activity" for the non-target antigen. Alternatively, "substantially no binding activity" or "no specific / significant binding activity" can be rephrased as "does not specifically / significantly / substantially bind" (to the non-target antigen). Sometimes the phrase "no binding activity" has substantially the same meaning in the art as the phrases "substantially no binding activity" or "no specific / significant binding activity."

[0036] As used herein, "HLA-DR / DP" refers to "HLA-DR and HLA-DP" or "HLA-DR or HLA-DP." These HLAs are MHC class II molecules encoded by corresponding haplotype alleles at the MHC class II locus in humans. "HLA-DQ" collectively refers to HLA-DQ isoforms, including HLA-DQ2.5, HLA-DQ7.5, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, and HLA-DQ8. In the present invention, in addition to HLA-DQ2.5, HLA-DQ molecules include HLA-DQ molecules of known subtypes (isoforms), such as, but not limited to, 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.

[0037] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to "cells" (including the progeny of such cells) into which exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0038] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0039] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. The term "human antibody framework" may also be used to refer to such a framework. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup 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 according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0040] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and 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 comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0041] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (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) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). In one embodiment, the HVR residues include those set forth herein. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0042] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules.

[0043] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 human.

[0044] In the present invention, when evaluating the binding of anti-HLA-DQ2.5 antibodies 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.

[0045] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0046] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0047] An "isolated nucleic acid encoding an anti-HLA-DQ2.5 antigen-binding molecule (antibody)" (also simply referred to as a "nucleic acid encoding an anti-HLA-DQ2.5 antigen-binding molecule (antibody)") 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 carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0048] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants 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 include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0049] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0050] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy 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 domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0051] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed 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. Nucleic acid molecules are often described by the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases containing derivatized sugar or phosphate backbone linkages or chemically modified residues. 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 can be unmodified or modified. For example, mRNA can be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject 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).

[0052] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0053] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may 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 vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be said that a given amino acid sequence A has or contains a certain percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y. where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, 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 will not equal 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 immediately preceding paragraph.

[0054] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation / composition is administered.

[0055] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation / composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0056] As used herein, the term "HLA-DQ2.5," unless otherwise indicated, 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). The term encompasses "full-length," unprocessed HLA-DQ2.5, as well as any form of HLA-DQ2.5 resulting from processing in cells. The term also encompasses naturally occurring variants of HLA-DQ2.5, such as splice variants and allelic variants. An exemplary HLA-DQ2.5 amino acid sequence is publicly available in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) accession code 4OZG and IPD-IMGT / database.

[0057] As used herein, "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.

[0058] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0059] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0061] Amino acid modification The antigen-binding molecules (or antibodies) of the present invention may contain one or more modifications. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0062] The antigen-binding molecules (or antibodies) of the present invention may contain amino acid substitutions. Conservative substitutions are shown in Table 1-1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1-1 under the heading "Exemplary Substitutions," and as further described below with respect to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest, and the products may be screened for the desired activity, e.g., antigen binding.

[0063] (Table 1-1) TIFF2023058542000001.tif165170

[0064] As used herein, expressions indicating amino acid modifications may be appropriately expressed by indicating the one-letter or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position. For example, the modifications N100bL or Asn100bLeu, used when substituting an amino acid contained in an antibody variable region, indicate a substitution of Leu for Asn at position 100b (according to Kabat numbering). That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution. Similarly, the modifications P238D or Pro238Asp, used when substituting an amino acid in the Fc region contained in an antibody constant region, indicate a substitution of Asp for Pro at position 238 (according to EU numbering). That is, the numbers indicate amino acid positions according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0065] 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., peptide) or epitope. In some embodiments, the antigen-binding molecule (antibody) has at least a first antigen-binding moiety / domain capable of binding to one or more antigens (e.g., peptides) and a second antigen-binding moiety / domain capable of binding to one or more antigens (e.g., peptides). Some or all of the antigens bound by the first antigen-binding moiety / domain may be different from some or all of the antigens bound by the second antigen-binding moiety / domain. Alternatively, some of the antigens bound by the first antigen-binding moiety / domain may be the same as some of the antigens bound by the second antigen-binding moiety / domain.

[0066] 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) has specificity for 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 molecules normally bind to two antigens or epitopes ("bispecific antigen-binding molecule (antibody)"; used herein to have the same meaning as "dual-specific antigen-binding molecule (antibody)"), but may also have specificity for more antigens or epitopes (e.g., three or more types of antigens).

[0067] As used herein, terms such as "multispecific" and "bispecific" refer to the specificity of one antigen-binding domain / region being different from that of another. In other words, these terms refer to two or more specificities in a single antigen-binding molecule. For example, in a "bispecific" antigen-binding molecule (antibody), a first antigen-binding moiety / domain can bind to a first group of complexes formed by HLA-DQ2.5 and gluten peptides, and a second antigen-binding moiety / domain can 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 are not identical. That is, some complexes may be included in both groups. Terms such as "multispecific" and "bispecific" can encompass this situation. The same applies to the first and second groups of complexes to which the first / second antigen-binding moiety / domain do not bind.

[0068] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Preferred embodiments of the "multispecific antigen-binding molecules" of the present invention include multispecific antibodies. When an Fc region with reduced Fcγ receptor binding activity is used as the multispecific antibody Fc region, an Fc region derived from the multispecific antibody may be appropriately used. Bispecific antibodies are particularly preferred as the multispecific antibodies of the present invention. In this case, the bispecific antibody is an antibody with two different specificities. IgG-type bispecific antibodies can be secreted from hybrid hybridomas (quadromas) produced by fusing two types of IgG antibody-producing hybridomas (Milstein et al., Nature (1983) 305, 537-540).

[0069] A multispecific antigen-binding molecule (antibody) may comprise at least two antigen-binding moieties / domains. A bispecific antigen-binding molecule (antibody) may comprise a first antigen-binding moiety / domain and a second antigen-binding moiety / domain. A bispecific antigen-binding molecule (or bispecific antibody) may comprise a first antigen-binding moiety / domain and a second antigen-binding moiety / domain. The first antigen-binding moiety / domain may comprise a first antibody variable region and a second antibody variable region. The first antibody variable region associates with a second antibody variable region. The association between the first antibody variable region and the second antibody variable region enables the first antigen-binding moiety / domain to bind to a first antigen / epitope. Similarly, the second antigen-binding moiety / domain may comprise a third antibody variable region and a fourth antibody variable region. The third antibody variable region associates with a fourth antibody variable region. The association between the third antibody variable region and the fourth antibody variable region enables the second antigen-binding moiety / domain to bind to a 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 a "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 a "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 a "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 a "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 a first antigen / epitope. The second heavy chain (H chain) variable region (VH) associates with the second light chain (L chain) variable region (VL) for binding to a second antigen / epitope. The association (also referred to as "interaction") between the variable regions (i.e., between VH and VL) depends on the structure (e.g., amino acid residues) at the VH / VL interface, as known in the art. In the present invention, preferably, the bispecific antigen-binding molecule (antibody) can bind to two or more gluten peptides (or a complex formed by HLA-DQ2.5 and a gluten peptide).In some embodiments, the bispecific antigen-binding molecule (antibody) comprises a first antigen-binding moiety / domain (comprising a first antibody variable region and a second antibody variable region (described above)) that binds to one or more complexes formed by HLA-DQ2.5 and gluten peptides, and a second antigen-binding moiety / domain (comprising a third antibody variable region and a fourth antibody variable region (described above)) that binds to one or more complexes formed by HLA-DQ2.5 and gluten peptides. 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 gluten peptide members in the complex to which the first antigen-binding moiety / domain binds and the gluten peptide members in the complex to which the second antigen-binding moiety / domain bind may overlap, but may not be completely identical. The gluten peptides in the complex to which the first / second antigen-binding moiety / domain bind may be selected from any gluten peptide described herein. Preferably, the first / second antigen-binding moiety / domain is capable of binding to one type of gluten peptide, or to two or more types of gluten peptides.

[0070] In the context of the present 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 substituted for "antigen-binding molecule" where applicable.

[0071] In one aspect, the invention is based in part on the binding of anti-HLA-DQ2.5 antigen binding molecules (antibodies) to HLA-DQ2.5, which presents gluten peptides to T cells. In certain embodiments, antibodies that bind to HLA-DQ2.5 are provided.

[0072] In one aspect, the present invention provides antigen-binding molecules or antibodies that have binding activity to HLA-DQ2.5 or one or more complexes formed by HLA-DQ2.5 and gluten peptides. In certain embodiments, the anti-HLA-DQ2.5 antigen-binding molecules (antibodies) have the following functions / characteristics:

[0073] The anti-HLA-DQ2.5 antigen-binding molecule (antibody) has binding activity to HLA-DQ2.5 in the form of a complex with gluten peptides (i.e., 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 gluten peptides (i.e., HLA-DQ2.5 / gluten peptide complex).

[0074] The anti-HLA-DQ2.5 antigen-binding molecule (antibody) has substantially no binding activity to non-target antigens, such as HLA-DQ5.1 / DQ6.3 / DQ7.3 / DQ7.5 / DQ8 / DR / DP, i.e., the anti-HLA-DQ2.5 antigen-binding molecule (antibody) does not substantially bind to non-target antigens. For example, the anti-HLA-DQ2.5 antigen-binding molecule (antibody) has no specific binding activity to HLA-DR / DP or no significant binding activity to HLA-DR / DP. In other words, the antibody does not specifically bind to HLA-DR / DP or does not significantly bind to HLA-DR / DP. Similarly, 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, i.e., 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) have no 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. That is, 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 no binding activity") are preferred to prevent any substantial inhibitory effects on these non-target MHC class II molecules and to improve antibody PK in celiac disease patients with HLA-DQ2.5. The property of "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 "substantially has no binding activity" to a specific antigen may have an MFI (mean fluorescence intensity) value that is 250% or less, preferably 200% or less, and more preferably 150% or less of the MFI value of a negative control under the measurement conditions described herein.

[0075] In one aspect, with regard to bispecific antigen-binding molecules (antibodies), an anti-HLA-DQ2.5 antigen-binding molecule (antibody) that "has substantially no binding activity" to a specific antigen has an MFI value of 2% or less, 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.

[0076] Anti-HLA-DQ2.5 antigen-binding molecules (antibodies) have binding activity to HLA-DQ2.5 in a complex with gluten peptides described herein. Herein, the complex formed between an HLA-DQ2.5 molecule and a gluten peptide is referred to as a "complex formed by HLA-DQ2.5 and gluten peptide," an "HLA-DQ2.5 / gluten peptide complex," or an "HLA-DQ2.5 / gluten peptide." It can also be referred to, for example, as "HLA-DQ2.5 loaded with gluten peptides," "gluten peptide-loaded HLA-DQ2.5," "HLA-DQ2.5 bound to gluten peptides," "HLA-DQ2.5 in a complex with gluten peptides," and "complex of HLA-DQ2.5 and gluten peptides." The above language (e.g., "complex formed by HLA-DQ2.5 and... [peptide]") also applies to peptides such as: a 33mer gliadin peptide, an α1 gliadin peptide (also referred to as an "α1a gliadin peptide"), an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, an hordein 1 peptide, an hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26mer gliadin peptide, a 14mer gliadin peptide, ...14mer gliadin peptide, a 14mer gliadin peptide, a 14mer gliadin peptide, a 14mer gliadin peptide, a 14mer gliadin peptide, a 14mer gliadin peptide 1 peptide, CLIP (hCLIP) peptide, Hepatitis B virus 1 (HBV1) peptide, Salmonella peptide, Mycobacterium bovis (M. bovis) peptide, Thyroperoxidase (TPO) peptide, etc.

[0077] On the other hand, anti-HLA-DQ2.5 antigen-binding molecules (antibodies) have substantially no binding activity to "irrelevant" peptides. As used herein, "irrelevant" peptides include peptides that have been reported to be presented on HLA-DQ2.5 but are unrelated to celiac disease or unrelated to the present invention, i.e., peptides that are not gluten peptides of interest. For example, irrelevant peptides include, but are not limited to, CLIP (hCLIP) peptides, hepatitis B virus 1 (HBV1) peptides, Salmonella peptides, Mycobacterium bovis (M. bovis) peptides, and thyroperoxidase (TPO) peptides. These characteristics ("substantially no binding activity") are preferred in order to prevent any substantial inhibitory effect on HLA-DQ2.5 in complexes with these non-target MHC class II molecules and irrelevant peptides, and to improve antibody PK in celiac disease patients. The property "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 that is 300% or more, preferably 500% or more, and more preferably 1000% or more of the MFI value of a negative control under the measurement conditions described herein.

[0078] In one aspect, with regard to bispecific antigen-binding molecules (antibodies), anti-HLA-DQ2.5 antigen-binding molecules (antibodies) that have "binding activity" for a specific antigen have an MFI value of 3% or more, preferably 6% or more, preferably 10% or more, and more preferably 20% or more, under the measurement conditions described herein, where the MFI value of IC17dK is 0% and the MFI value of DQN0139bb is 100%.

[0079] When specifically referring to the specificity of binding, the term "binding activity" can be rephrased as "specific binding activity." The anti-HLA-DQ2.5 antigen-binding molecules (antibodies) of the present invention exhibit a binding activity of 5×10 to one or more complexes formed by HLA-DQ2.5 and gluten peptides described herein. -7 M or less, preferably 4 x 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 x 10 -8 M or less, preferably 8 x 10 -8 M or less, preferably 7 x 10 -8 M or less, preferably 6 × 10 -8 M or less, preferably 5 × 10 -8 M or less, preferably 4 x 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 x 10 -9 M or less, preferably 8 x 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 x 10 -9 M or less, preferably 3 × 10 -9 M or less, preferably 2 × 10 -9 It has a dissociation constant (Kd) of M or less.

[0080] 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-mentioned reduced Fcγ receptor binding activity, without being limited to its structure. In the present invention, each of the above-mentioned domains can be directly linked by a peptide bond. For example, when F(ab')2 is used as the domain containing the antibody variable regions (1) and (2), and these Fc regions are used as the domain containing an Fc region with reduced Fcγ receptor binding activity (3), the polypeptide formed by linking the antibody variable region-containing domains (1) and (2) with the Fc region-containing domain (3) via a peptide bond will form an antibody structure. Such antibodies can be produced by purifying them from the hybridoma culture medium described above, or by purifying them from the culture medium of a desired host cell stably harboring a polynucleotide encoding the antibody-constituting polypeptide.

[0081] Examples of preferred antibody heavy chain variable regions of the present invention contained in antibody variable regions having binding activity for HLA-DQ2.5 in the form of a complex with gluten peptides include any of the antibody heavy chain variable regions described herein, or antibody heavy chain variable regions having CDR sequences whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the CDR1, CDR2, and CDR3 amino acid sequences contained in the heavy chain variable regions described herein, or antibody heavy chain variable regions that are functionally equivalent to the above-mentioned variable regions.

[0082] Examples of preferred antibody variable regions of the present invention that have T cell receptor complex binding activity include antibody variable regions that have binding activity to HLA-DQ2.5 in the form of a complex with gluten peptides. Examples of antibody heavy chain variable regions contained in such antibody variable regions include the antibody heavy chain variable regions described herein, antibody heavy chain variable regions whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the CDR1, CDR2, and CDR3 amino acid sequences contained in the antibody heavy chain variable regions described herein, and antibody heavy chain variable regions that are functionally equivalent to the above-mentioned variable regions.

[0083] In the present invention, the phrase "functionally equivalent" means, when used as a multispecific antigen-binding molecule, that the binding affinity to the antigen is equivalent, or alternatively, that the neutralizing activity against cells expressing HLA-DQ2.5 / gluten peptides (or tissues containing these cells) is equivalent. Binding affinity and neutralizing activity can be measured based on the description herein. The cells used for activity measurement may be any desired cell line expressing HLA-DQ2.5 / gluten peptides (or any desired tissues containing these cells), and any suitable cell line can be used. With respect to antibody constant regions, this phrase can also mean that the reduction in Fcγ receptor binding activity is equivalent.

[0084] For example, an antibody heavy chain variable region functionally equivalent to an antibody heavy chain variable region described herein (i.e., the original heavy chain variable region) means that this region has the same binding affinity when combined with an antibody light chain variable region described herein that forms a pair with the original heavy chain, or alternatively, that the region, when used in a multispecific antigen-binding molecule, has the same neutralizing activity against cells expressing HLA-DQ2.5 / gluten peptides (or tissues containing these cells). Furthermore, an antibody light chain variable region functionally equivalent to an antibody light chain variable region described herein (i.e., the original light chain variable region) means that this region has the same binding affinity when combined with an antibody heavy chain variable region described herein that forms a pair with the original light chain, or alternatively, that the region, when used in a multispecific antigen-binding molecule, has the same neutralizing activity against cells expressing HLA-DQ2.5 / gluten peptides (or tissues containing these cells).

[0085] The term "equivalent" does not necessarily mean the same degree of activity, and the activity may be enhanced. Specifically, with respect to antigen-binding affinity, examples include a case where the value obtained by comparison with the binding affinity of an antibody variable region serving as a control (parent KD value) (KD value / parent KD value) is 1.5 or less. The value of 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 examples include a value of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , or 10 -6 More specifically, in the present invention, the value of KD value / parent KD value is preferably 10 -6 ~1.5×10 -0 , more preferably 10 -6 ~10 -1 , and even more preferably 10 -6 ~10 -2 , and even more preferably 10 -6 ~10 -3 is.

[0086] With respect to a portion / domain containing an antibody variable region having binding activity to HLA-DQ2.5 / gluten peptide, the KD value for HLA-DQ2.5 / gluten peptide is, for example, 2×10 -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 x 10 -9 It can be M or less.

[0087] In the present invention, "functionally equivalent" antibody variable regions are not particularly limited, as long as they are antibody H chain and / or L chain variable regions 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. Methods 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 are methods for introducing mutations into proteins.For example, those skilled in the art can prepare variable regions functionally equivalent to the antibody variable regions having the above-described functions by appropriately introducing mutations into the amino acid sequence using a method 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).

[0088] When modifying amino acid residues, amino acids are preferably mutated into different amino acids that preserve the characteristics of the amino acid side chain.Examples of the characteristics of amino acid side chains are: 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-containing side chains (S, T, and Y), amino acids containing sulfur atom-containing side chains (C and M), amino acids containing carboxylic acid-containing and amide-containing 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 one-letter codes 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, D.F. et al., Proc. Natl. Acad. Sci. USA; (1984) 81: 5662-6; Zoller, M.J. and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984) 224: 1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6409-13). The variable regions of the present invention containing such amino acid modifications have an amino acid sequence identity of 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% with the CDR sequence, FR sequence, or amino acid sequence of the entire variable region before the modification.As used herein, sequence identity is defined as the percentage of residues identical to those in the original amino acid sequence of the heavy or light chain variable region, determined after aligning the sequences and, if necessary, introducing appropriate gaps to maximize sequence identity. Amino acid sequence identity can be determined by the following method.

[0089] Furthermore, "functionally equivalent antibody variable regions" can be obtained from nucleic acids that hybridize under stringent conditions with nucleic acids containing nucleotide sequences encoding the amino acid sequences of the variable regions in Tables 1 to 3 above. Stringent hybridization conditions for isolating nucleic acids that hybridize under stringent conditions with nucleic acids containing nucleotide sequences encoding the amino acid sequences of the variable regions include, for example, 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. Post-hybridization washing conditions include, for example, washing with 0.5xSSC (1xSSC 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 0.2xSSC and 0.1% SDS at 60°C, even more preferably washing with 0.2xSSC and 0.1% SDS at 62°C, even more preferably washing with 0.2xSSC and 0.1% SDS at 65°C, and even more preferably washing with 0.1xSSC and 0.1% SDS at 65°C. The sequence of the isolated nucleic acid can be determined by known methods described below. The overall nucleotide sequence homology of the isolated nucleic acid is at least 50% or more, preferably 70% or more, and more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more) sequence identity.

[0090] Nucleic acids that hybridize under stringent conditions to a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of a variable region can also be isolated by using a gene amplification method such as polymerase chain reaction (PCR) using primers synthesized based on information about the nucleotide sequence encoding the variable region amino acid sequence, instead of the above-mentioned method using hybridization techniques.

[0091] The identity of one nucleotide sequence 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 based on BLAST, parameters are set, for example, as score=100 and wordlength=12. On the other hand, parameters used to analyze an amino acid sequence according to BLASTX 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 techniques for such analyses are 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).

[0092] The Fc region contained in the multispecific antigen-binding molecules of the present invention is not particularly limited, as long as it is an Fc region with reduced Fcγ receptor-binding activity, and examples of preferred Fc regions of the present invention include combinations of Fc region moieties described herein.

[0093] Examples of preferred multispecific antigen-binding molecules of the present invention include bispecific antibodies comprising a first antibody variable region having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide and a second antibody variable region having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide. Examples of such bispecific antibodies include bispecific antibodies comprising the H and L chains described herein, and bispecific antibodies containing an Fc region that binds to an epitope that overlaps with the epitope bound by the above-mentioned antibodies and has reduced Fcγ receptor binding activity.

[0094] Whether an antibody recognizes an epitope that overlaps with that 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 techniques such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer (FRET), and fluorescence microtiter assay technology (FMAT®). The amount of antibody bound to the antigen is indirectly correlated with the binding ability of a 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, an appropriately labeled antibody and the antibody to be evaluated are added to the antigen simultaneously, and the resulting bound antibody is detected using the label. The amount of antibody bound to the antigen can be easily determined by labeling the antibody in advance. The label is not particularly limited, and the labeling method is selected according to the assay technique used. Specifically, labeling methods include fluorescent labeling, radioactive labeling, enzyme labeling, and the like.

[0095] For example, fluorescently labeled antibody and unlabeled antibody or test antibody are added simultaneously to beads on which HLA-DQ2.5 / gluten peptides are immobilized, and the labeled antibody is detected by fluorescent microassay techniques.

[0096] As used herein, "antibodies that bind to overlapping epitopes" refers to the concentration at which unlabeled antibody reduces the amount of bound labeled antibody by 50% (IC 50 ) at a concentration that is typically 100-fold higher, preferably 80-fold higher, more preferably 50-fold higher, even more preferably 30-fold higher, and even more preferably 10-fold higher.

[0097] Multispecific antigen-binding molecules having antigen-binding sites of antibodies that bind to epitopes that overlap with the epitopes bound by the above-mentioned antibodies can exhibit superior binding activity or neutralizing activity.

[0098] The multispecific antigen-binding molecules of the present invention are produced by the same methods for producing recombinant antibodies as described herein.

[0099] In certain embodiments, any one or more amino acids of the anti-HLA-DQ2.5 antigen binding molecules (antibodies) provided above are substituted in any of the heavy and / or light chain constant and / or variable regions or domains.

[0100] In certain embodiments, the substitutions provided herein are conservative substitutions.

[0101] Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0102] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0103] 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 been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which 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).

[0104] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0105] Chimeric and Humanized Antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0106] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0107] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0108] Human framework regions that can be used for humanization include, but are not limited to, 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 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 mature (somatically mutated) 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 (see Baca et al., J. Biol. Chem. 272:10678-10684 (1997)). and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0109] 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 the HVR of 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 the HVR of any of the above embodiments and further comprises the FR1, FR2, FR3, or FR4 sequence shown herein. Herein, the "human framework" may also be referred to as a "humanized framework," focusing on the fact that the antibody is humanized.

[0110] In some embodiments, the multispecific antigen-binding molecule of the present invention comprises: (i) a first antigen-binding portion having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide; and (ii) a second antigen-binding moiety having binding activity for HLA-DQ2.5 in the form of a complex with a gluten peptide; Including, The antigen-binding molecule binds to two or more complexes of HLA-DQ2.5 and gluten peptides, At least one of the gluten peptides in the complex to which the first antigen-binding moiety binds is different from at least one of the gluten peptides 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 of 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 and / or light chain constant and / or variable regions of the first and / or second antigen-binding moieties in the multispecific antigen-binding molecule are modified.

[0111] In some embodiments, in the multispecific antigen-binding molecule, one or more amino acids in the heavy chain and / or light chain of the first antigen-binding moiety and / or the second antigen-binding moiety in the multispecific antigen-binding molecule 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, the gluten peptide is an immunodominant peptide associated with celiac disease. In some embodiments, the gluten peptide is selected from the group consisting of a 33mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26mer gliadin peptide. In some embodiments, the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or all of a 33mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, a γ2 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26mer gliadin peptide. In some embodiments, the gluten peptide is selected from the group consisting of a 33mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26mer gliadin peptide. In some embodiments, the gluten peptides are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or all of a 33mer gliadin peptide, an α1 gliadin peptide, an α2 gliadin peptide, a γ1 gliadin peptide, an ω1 gliadin peptide, an ω2 gliadin peptide, a BC hordein peptide, an α3 gliadin peptide, an α1b gliadin peptide, a γ4a gliadin peptide, a γ4b gliadin peptide, an avenin 1 peptide, an avenin 2 peptide, an avenin 3 peptide, a hordein 1 peptide, a hordein 2 peptide, a secalin 1 peptide, a secalin 2 peptide, and a 26mer gliadin peptide. In some embodiments, the multispecific antigen-binding molecule has substantially no binding activity to gluten peptides per se or to gluten peptides per se. In this context, the terms "per se" and "per se" refer to the state in which the gluten peptides do not form a complex with HLA-DQ2.5. In some embodiments, the multispecific antigen-binding molecule has substantially no binding activity to HLA-DQ2.5 in a complex with an irrelevant peptide, wherein the irrelevant peptide is at least one peptide selected from the group consisting of a CLIP (hCLIP) peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, and a thyroperoxidase peptide. In some embodiments, the multispecific antigen-binding molecule has substantially no binding activity to HLA-DQ2.5 in a complex with an irrelevant peptide, wherein the irrelevant peptide is all of a CLIP (hCLIP) peptide, a Hepatitis B virus 1 peptide, a Salmonella peptide, a Mycobacterium bovis peptide, and a thyroperoxidase peptide. In some embodiments, the antigen-binding molecule has enhanced binding activity to the complex formed by HLA-DQ2.5 and gluten peptide compared to before the humanization and modification. In this context, "enhanced binding activity" means that the antigen-binding molecule binds to the complex formed by HLA-DQ2.5 and gluten peptide more strongly than the original antibody before the modification, i.e., humanization and modification. In some embodiments, the antigen-binding molecule has enhanced cross-reactivity to gluten peptides compared to before the 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 against more gluten peptides than the original antibody before the modification, i.e., humanization and modification.

[0112] 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 to the amino acid sequence of the 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 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference (i.e., original) sequence, but the anti-HLA-DQ2.5 antigen binding molecule (antibody) comprising 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 relative to the reference (i.e., original) sequence. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-HLA-DQ2.5 antigen binding molecule (antibody) comprises a VH sequence disclosed herein, or a sequence comprising a post-translational modification thereof. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 disclosed herein, (b) HVR-H2 disclosed herein, and (c) HVR-H3 disclosed herein. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0113] The amino acids contained in the amino acid sequences of the present invention may be post-translationally modified (for example, modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the amino acid sequences of the present invention.

[0114] 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 the light chain variable domain (VL) disclosed herein. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference (i.e., original) sequence, but the anti-HLA-DQ2.5 antigen binding molecule (antibody) comprising the 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 relative to the reference (i.e., original) sequence. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-HLA-DQ2.5 antigen binding molecule (antibody) comprises a VL sequence disclosed herein or a sequence comprising a post-translational modification thereof. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 disclosed herein; (b) HVR-L2 disclosed herein; and (c) HVR-L3 disclosed herein. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0115] In another aspect, an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is provided, which comprises a VH sequence according to any of the embodiments provided above and a VL sequence according to any of the embodiments provided above. In one embodiment, the molecule / antibody comprises a sequence comprising a VH sequence disclosed herein or a post-translational modification thereof, and a sequence comprising a VL sequence disclosed herein or a post-translational modification thereof. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0116] In a further aspect, the present invention provides antigen-binding molecules (antibodies) that bind to the same epitope as the anti-HLA-DQ2.5 antigen-binding molecules (antibodies) provided herein. For example, in certain embodiments, molecules / antibodies are provided that bind to the same epitope as any of the molecules / antibodies described herein. In certain embodiments, molecules / antibodies are provided that bind to an epitope within a fragment of HLA-DQ2.5 consisting of approximately 8 to 17 amino acids, or within a complex formed by HLA-DQ2.5 and a gluten peptide. In this context, the gluten peptide may be any of the gluten peptides described herein.

[0117] 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 the complex formed by HLA-DQ2.5 and gluten peptides. For example, in certain embodiments, a molecule / antibody is provided that competes with any of the molecules / antibodies described herein for binding to HLA-DQ2.5 or the complex formed by HLA-DQ2.5 and gluten peptides. In this context, the gluten peptide may be any of the gluten peptides described herein.

[0118] 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), including 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, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody, or any other antibody class or isotype defined herein.

[0119] In further aspects, the anti-HLA-DQ2.5 antigen-binding molecules (antibodies) according to any of the above embodiments may incorporate any of the following properties, either alone or in combination:

[0120] Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity of ≦1 micromolar (μM), ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0121] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured using the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 micrograms (μg) / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 microliters (μL) / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0122] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized thereon. 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 injection at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of protein binding. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μL / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0123] 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 below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0124] Diabodies are antibody fragments 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 tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0125] A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0126] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0127] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0128] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. 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 WO 94 / 29351.

[0129] Fc area As used herein, the term "Fc region" or "Fc domain" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that 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 is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0130] Fc receptors The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are 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 yet to be identified, are encompassed by the term "FcR" herein.

[0131] 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 in regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0132] 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 or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides containing mutant Fc regions. WO 2000 / 42072 (Presta) describes antibody mutants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0133] Fcγ receptor An Fcγ receptor refers to a receptor capable of binding to the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody, and includes all members of a family of proteins substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), e.g., the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), e.g., the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), e.g., the 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. Fcγ receptors include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors may be derived 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. Preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16).Whether an Fcγ receptor has binding activity to the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be assessed by the above-mentioned FACS and ELISA formats, as well as by 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).

[0134] On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to an antibody Fc domain to form an Fc / Fc ligand complex. The molecule may be derived 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 lectin, mannose receptor, 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), a family of Fc receptors homologous to Fcγ receptors. Fc ligands also include unidentified molecules that bind to Fc.

[0135] Fcγ receptor binding activity The reduction in binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB can be assessed using the FACS and ELISA formats described above, as well as ALPHA screen (amplified luminescence proximity homogeneous assay) and surface plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0136] ALPHA screens are performed using ALPHA technology, based on the principles described below, which uses two types of beads: donor beads and acceptor beads. Luminescence signals are detected only when molecules linked to donor beads biologically interact with molecules linked to acceptor beads and when these two beads are placed in close proximity. When excited by a laser beam, a photosensitizer within the donor bead converts 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 light emission. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen generated by the donor bead does not reach the acceptor bead, and the chemiluminescent reaction does not occur.

[0137] 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 a competing antigen-binding molecule or antibody containing a mutant Fc domain, the Fcγ receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, resulting in a signal at 520-620 nm. The antigen-binding molecule or antibody containing an untagged mutant Fc domain competes with the antigen-binding molecule or antibody containing the wild-type Fc domain for interaction with the Fcγ receptor. Relative binding affinity can be measured by quantifying the decrease in fluorescence resulting from competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies, using sulfo-NHS-biotin are known. Suitable methods for adding a GST tag to an Fcγ receptor include fusing a polypeptide encoding the Fcγ receptor with GST in frame, expressing the gene using cells transfected with a vector carrying the fused gene, and then purifying the gene using a glutathione column. The induced signal can be analyzed by fitting to a one-site competition model based on nonlinear regression analysis, preferably using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0138] One of the substances used to observe their interaction is immobilized on a thin gold layer of a sensor chip as a ligand. When light is shone on the back of the sensor chip so that total reflection occurs at the interface between the gold layer and the glass, the intensity of the reflected light is partially reduced at a specific site (SPR signal). The other substance used to observe their interaction is injected onto the surface of the sensor chip as an analyte. 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 chip. This 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 above shift (i.e., the change in mass on the sensor chip surface) is plotted on the vertical axis, and the change in mass over time is displayed as actual 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 preferably used in the BIACORE method. An example of such an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0139] Fc region with reduced Fcγ receptor binding activity As used herein, "reduced Fcγ receptor-binding activity" means, for example, that the competitive activity of a 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, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the competitive activity of a control antigen-binding molecule or antibody, based on the above-mentioned analytical methods.

[0140] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be suitably used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in RefSeq Accession Nos. AAC82527.1, AAB59393.1, CAA27268.1, and AAB59394.1. Furthermore, when antigen-binding molecules or antibodies containing Fc domain mutants of a specific isotype of antibody are used as test substances, the effect of the mutations on Fcγ receptor binding activity is evaluated using an antigen-binding molecule or antibody containing an Fc domain of the same isotype as a control. As described above, antigen-binding molecules or antibodies containing Fc domain mutants determined to have reduced Fcγ receptor binding activity are suitably prepared.

[0141] Such known mutants include, for example, a mutant having a deletion of amino acids 231A to 238S (EU numbering) (WO2009 / 011941), as well as the mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0142] Specifically, preferred antigen-binding molecules or antibodies include those containing an Fc domain with at least one amino acid mutation (e.g., substitution) selected from the amino acid positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering) that form the Fc domain of an antibody of a particular isotype. The antibody isotype from which the Fc domain is derived is not particularly limited, and an appropriate Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0143] In the present invention, SG181 may be used as an Fcγ receptor silencing Fc that weakens Fc binding to Fcγ receptors. In some embodiments, SG181.S3n (SEQ ID NO: 101) and SG181.S3p (SEQ ID NO: 102) may be used as heavy chain constant region sequences. These heavy chain constant region sequences may be included in the antigen binding molecules or antibodies of the present invention to reduce Fcγ receptor binding.

[0144] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which any amino acid at positions 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody has been substituted with an amino acid at the corresponding position (EU numbering) in a corresponding IgG2 or IgG4.

[0145] In some embodiments, the multispecific antigen-binding molecules of the present invention further comprise an Fc domain that exhibits reduced binding affinity to a human Fcγ receptor compared to a native human IgG1 Fc domain. In some embodiments, in the multispecific antigen-binding molecules of the present invention, the Fc domain comprises Arg at positions 235 and 236, and the amino acid positions are numbered according to EU numbering.

[0146] Regulation of H / L chain association and other properties Another aspect of the present invention relates to an antigen-binding molecule in which the association between the heavy chain and the light chain is regulated, a method for producing an antigen-binding molecule in which the association between the heavy chain and the light chain is regulated, and a method for regulating the association between the heavy chain and the light chain in an antigen-binding molecule.

[0147] The antigen-binding molecules of the present invention relate to antigen-binding molecules in which the association between the heavy chain and the light chain is regulated, the heavy and light chains constituting the antigen-binding molecule are a combination of heavy and light chains of interest, and the amino acid residues at given positions in the heavy chain constant region (CH1) and the light chain constant region (CL) are amino acid residues that are electrically repulsive to each other (have the same charge).

[0148] In the present invention, by making the amino acid residues at given positions in the CH1 and CL of undesirable combinations of heavy and light chains to be amino acid residues that are electrically repulsive to each other (i.e., have the same charge), the formation of undesirable combinations of heavy and light chains can be prevented by utilizing this charge repulsion, and as a result, desirable combinations of heavy and light chains can be formed.

[0149] In the present invention, the phrases "modulating association" and "association is modulated" refer to modulation to achieve desired association conditions, more specifically modulation to prevent undesired association between heavy and light chains.

[0150] In the present invention, the term "interface" generally refers to an association surface resulting from association (interaction), and the amino acid residues forming the interface are usually one or more amino acid residues contained in the polypeptide region involved in the association, more preferably amino acid residues that come close to each other during the association and are involved in the interaction. More specifically, this interaction includes, for example, cases where amino acid residues come close to each other during the association so as to form hydrogen bonds, electrostatic interactions, or salt bridges.

[0151] In the present invention, the phrase "amino acid residues forming the interface" more specifically refers to amino acid residues contained in the polypeptide region that forms the interface. For example, the polypeptide region that forms the interface refers to a polypeptide region responsible for selective binding between molecules, for example, in 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 a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region.

[0152] In a preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding molecule has electrically repulsive (similarly charged) amino acid residues at given locations in the CH1 and CL of undesirable combinations of heavy and light chains before association modulation.

[0153] By modifying the amino acid residues in the antigen-binding molecule to make them electrically repulsive (having the same charge), it is believed that the association of these amino acid residues is inhibited by the repulsive force of the charges.

[0154] Therefore, in the above-described antigen-binding molecules, the amino acid residues to be modified are preferably those that are close to each other during association in the polypeptide region that forms the interface.

[0155] The amino acid residues that are close to each other during association can be determined, for example, by analyzing the three-dimensional structure of the polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during polypeptide association. The amino acid residues that are close to each other at the interface are preferred targets for "modification" in the antigen-binding molecules of the present invention.

[0156] Some amino acids are known to be charged. Generally, lysine (K), arginine (R), and histidine (H) are known to be positively charged amino acids. Aspartic acid (D), glutamic acid (E), and the like 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), and the like are known to be uncharged or nonpolar amino acids.

[0157] Therefore, amino acids that are electrically repulsive to each other (have the same charge) in the present invention refer to the following: (1) an amino acid, one of which is a positively charged amino acid and the other amino acid is also a positively charged amino acid, and (2) Amino acids in which one of the amino acids is a negatively charged amino acid and the other amino acid is also a negatively charged amino acid.

[0158] Examples of amino acid modifications include changing uncharged or nonpolar amino acids to positively charged amino acids, changing uncharged or nonpolar amino acids to negatively charged amino acids, changing positively charged amino acids to negatively charged amino acids, and changing negatively charged amino acids to positively charged amino acids.Furthermore, the amino acid modifications of the present invention also include changing uncharged or nonpolar amino acids to different uncharged or nonpolar amino acids, changing positively charged amino acids to different positively charged amino acids, and changing negatively charged amino acids to different negatively charged amino acids.

[0159] In the present invention, amino acid modification includes making one modification in each of the heavy and light chains, or making multiple modifications to each of the heavy and light chains. In addition, the number of modifications made to the heavy and light chains may be the same or different.

[0160] The amino acid modification in the present invention includes multiple modifications to positively charged amino acids on either the heavy chain or the light chain, and multiple modifications to negatively charged amino acids on the other chain.Furthermore, multiple modifications to positively charged amino acids and multiple modifications to negatively charged amino acids can be made on the same heavy chain or light chain.In these modifications, modifications to uncharged or nonpolar amino acids and modifications to uncharged or nonpolar amino acids can also be suitably combined.

[0161] In the modifications of the present invention, for example, amino acids on one of the chains can be used unmodified, in which case both the heavy and light chains need not be modified, but only one of the chains may be modified.

[0162] 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 constant regions of IgK(κ), IgL1, IgL2, IgL3, IgL6, and IgL7(λ). The light chain constant region of the antigen-binding molecule of the present invention is not particularly limited; when multiple types of light chains are used, the light chains may be of different types, such as κ and λ. Several allotype sequences obtained by genetic polymorphism are described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, as human IgK(κ) constant regions and human IgL7(λ) constant regions, and any of these may be used in the present invention.

[0163] 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 that strengthen or weaken the binding between the antigen-binding molecule and Fcγ receptor ("FcγR"), modifications that strengthen or weaken the binding between the antigen-binding molecule and FcRn, and modifications that strengthen or weaken the cytotoxic activity (e.g., ADCC activity and CDC activity) of the antigen-binding molecule. In addition, modifications that improve the heterogeneity of the antigen-binding molecule, and modifications that improve non-immunogenicity and / or pharmacokinetics may also be included.

[0164] Furthermore, heterogeneity in the C-terminal sequences of IgG antibodies heavy chains has been reported, resulting from amidation of the C-terminal carboxyl group due to deletion of the C-terminal amino acid lysine residue or 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 heterogeneity of the heavy chain C-terminus. Chimeric and humanized antibodies that use human-derived sequences are expected to be useful when administered to humans, such as for therapeutic purposes, because their antigenicity in the human body is reduced.

[0165] 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, and an example of such a heteromeric multimer is a multispecific antibody.

[0166] A preferred example of the multispecific antibody of the present invention is a bispecific antibody. Accordingly, a preferred embodiment 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).

[0167] To more precisely describe the "bispecific antibody" of a preferred embodiment of the antigen-binding molecule of the present invention, the above-mentioned "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 can be defined 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 can be defined as the second L chain. Usually, the first L chain and the first H chain are derived from the same antibody that binds to a specific antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody that binds to a specific antigen (or epitope). Herein, an L chain-H chain pair formed by a first H chain and L chain is referred to as a first pair, and an L chain-H chain pair formed by a second H chain and L chain is referred to as a second pair. The antigen (or epitope) used to produce the antibody from which the second pair is derived is preferably different from the antigen used to produce the antibody from which the first pair is derived. 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 chains and L chains 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 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, a chemotherapeutic agent, or a cell-derived toxin. However, when it is desired to produce antibodies having pairs formed by specific combinations 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.

[0168] A more detailed description is provided below for the case of an IgG-type bispecific antibody 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 can be applied to other antibodies as well.

[0169] If one desires to obtain a bispecific antibody that will recognize one epitope via a first CH1-A and a first CL-A and bind to another epitope via a second CH1-B and a second CL-B, theoretically, if one expresses each of the four types of chains to produce the antibody, ten types of antibody molecules could potentially be produced.

[0170] 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.

[0171] An example is modifying the amino acid residues forming the interface between CH1-A and CL-B to positively charged amino acid residues, and modifying the amino acid residues forming the interface between CH1-B and CL-A to 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. In this way, unintended association between CH1-A and CL-B and between CH1-B and CL-A are inhibited because the amino acid residues forming the interface have the same charge. 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. Furthermore, the intended association between CH1-A and CL-A is promoted 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 promoted 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.

[0172] In another example, when the amino acid residues forming the interface between CL-A and CH1-B are mutually uncharged or nonpolar amino acids, the amino acid residues forming the interface between CH1-A and CL-B are modified to positively charged amino acid residues. As a result of this modification, unintended association between CH1-A and CL-B is inhibited because both amino acid residues forming the interface are positively charged. On the other hand, because the amino acid residues forming the interface are not mutually electrically repulsive, the intended association between CH1-A and CL-A and between CH1-B and CL-B is thought to occur more easily than when the amino acids are electrically repulsive. As a result, antibodies having the intended association between CH1-A and CL-A and between CH1-B and CL-B can be efficiently obtained. On the other hand, in this example, when the amino acid residues forming the interface between CL-A and CH1-B are not mutually uncharged or nonpolar amino acids, they can be modified to become mutually uncharged or nonpolar amino acids.

[0173] In another example, when the amino acid residues forming the interface between CL-B and CH1-B are uncharged or nonpolar in CH1-B, one of the amino acid residues forming the interface between CH1-A and CL-A is modified to a positively charged amino acid residue, while the other is modified to a negatively charged amino acid residue; and the amino acid residues forming the interface between CL-B and CH1-B in CL-B are modified to have the same charge as 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 have a combination of positive and negative charges, while 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 or nonpolar amino acids in CH1-B, they may be modified to become uncharged or nonpolar amino acids.

[0174] In addition, the use of the association modulation of the present invention makes it possible to inhibit the association between CH1 (CH1-A and CH1-B) or the association between CL (CL-A and CL-B).

[0175] Those skilled in the art will be able to suitably determine the types of amino acid residues that are accessed during association at the CH1 and CL interface in a desired polypeptide for which modulation of association according to the present invention is desired.

[0176] Furthermore, those skilled in the art can also suitably obtain sequences that can be used as CH1 or CL of antibodies in organisms such as humans, monkeys, mice, rabbits, etc., by using public databases, etc. More specifically, amino acid sequence information for CH1 or CL can be obtained by the means described in the Examples below.

[0177] For example, with respect to the bispecific antibodies described in the Examples below, specific examples of amino acid residues that are close together (facing or contacting) at the interface between CH1 and CL upon 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 facing (contacting) CL; - a glutamine (Q) at position 175 according to EU numbering in CH1 and a threonine (T) or serine (S) at position 131 according to Kabat numbering in the opposing (contacting) CL; - a glutamine (Q) at position 175 according to EU numbering in CH1, and a serine (S) or threonine (T) at position 131 and a 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 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.

[0178] In the present invention, numbers written in EU numbering are indicated according to EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242). In the present invention, the phrases "amino acid residue at position X according to EU numbering" and "amino acid at position X according to EU numbering" (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 practicing the methods of the present invention.

[0179] In one aspect, the present invention provides an antigen-binding molecule in which the association between a heavy chain and a light chain is regulated, wherein one or two or more sets of amino acid residues in the heavy chain and the light chain of the antigen-binding molecule selected from the group consisting of the sets of amino acid residues shown in the following (a) to (c) are amino acid residues that are electrically repulsive to each other: (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) the amino acid residues at positions 147 and 175 in CH1 according to EU numbering and the amino acid residues at positions 131 and 160 in CL 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.

[0180] In the above-described antigen-binding molecules, the "mutually electrically repulsive amino acid residues" or "amino acid residues having 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).

[0181] In the above-described antigen-binding molecule, specific examples of the set of amino acid residues that are electrically repulsive to each other include the following sets of amino acid residues: (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.

[0182] In some embodiments, in the multispecific antigen-binding molecule, one, two, three, or all of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (d) in the heavy chain and the light chain of the antigen-binding molecule are amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) that is position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) that is position 131 according to Kabat numbering; (b) the amino acid residue in CH1 that is position 175 according to EU numbering and the amino acid residue in CL that is position 160 according to Kabat numbering; (c) the amino acid residue in CH1 that is position 175 according to EU numbering, and the amino acid residues in CL that are positions 131 and 160 according to Kabat numbering; (d) Amino acid residues in CH1 that are positions 147 and 175 according to EU numbering, and amino acid residues in CL that are positions 131 and 160 according to Kabat numbering.

[0183] The present invention provides antigen-binding molecules in which one or two or more sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a1) to (c2) in the heavy chain and light chain of the antigen-binding molecule are amino acid residues that are electrically repulsive to each other: (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, which are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively; (c2) Amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively, and amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively.

[0184] In the above-described antigen-binding molecules, specific examples of amino acid residues that are electrically repulsive to each other include the following amino acid residues: (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, which are glutamic acid (E) or aspartic acid (D), respectively, and the amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are glutamic acid (E) or aspartic acid (D), respectively; (c2) amino acid residues contained in CH1 at positions 147 and 175 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively, and amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively; (d1) 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 positions 131 and 160 according to EU numbering, which are 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 amino acid residues contained in CL at positions 131 and 160 according to EU numbering, which are lysine (K), histidine (H), or arginine (R), respectively.

[0185] In addition to the above, a technique for inhibiting undesired CH1 / CL association by introducing charge repulsion at the interface between CH1 and CL (WO 2013 / 065708) can be further applied to the antigen-binding molecules of the present invention. More specifically, the present invention provides antigen-binding molecules having a CH1 and a CL, wherein one or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in (a) to (d) below are electrically repulsive to each other: (a) the amino acid residue contained in the heavy chain constant region (CH1) at position 147 according to EU numbering, and the amino acid residue contained in the light chain constant region (CL) at position 160 according to EU numbering; (b) an amino acid residue contained in CH1 at position 147 according to EU numbering, and an 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.

[0186] Further, techniques for introducing electrical repulsion into the interface of the second heavy chain constant region (CH2) or the third heavy chain constant region (CH3) to suppress undesired association between heavy chains, techniques for introducing electrical repulsion into 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 techniques for modifying amino acid residues forming a hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to charge polar amino acids to suppress unintended association between heavy chains and light chains can be applied to the antigen-binding molecules of the present invention (see WO 2006 / 106905).

[0187] In a method for suppressing unintended association between heavy chains by introducing electrical repulsion into the CH2 or CH3 interface, examples of amino acid residues that contact other heavy chain constant region interfaces include positions 356 (EU numbering) and 439 (EU numbering), 357 (EU numbering) and 370 (EU numbering), and 399 (EU numbering) and 409 (EU numbering) in the CH3 region. For the numbering of antibody constant regions, reference may be made 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 shown.

[0188] 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 amino acid residue sets (1) to (3) may be engineered to be electrically repulsive to each other: (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.

[0189] Furthermore, the antibody may be an antibody having a set of amino acid residues in a second heavy chain CH3 region that is distinct from the aforementioned first heavy chain CH3 region, the set of amino acid residues being selected from the amino acid residue sets shown in (1) to (3) above, and the set of one to three amino acid residues corresponding to the set of amino acid residues shown in (1) to (3) above that are electrically repulsive to each other in the first heavy chain CH3 region are not electrically repulsive to the corresponding amino acid residues in the first heavy chain CH3 region.

[0190] The amino acid residues described in (1) to (3) above are close to each other during association. Those skilled in the art will be able to identify the positions corresponding to the amino acid residues described in (1) to (3) above for the desired heavy chain CH3 region or heavy chain constant region by homology modeling using commercially available software, and will be able to appropriately modify the amino acid residues at those positions.

[0191] In the above-described antigen-binding molecules, "electrically repulsive," "having the same charge," or "carrying the same charge" means, for example, that any two or more amino acid residues have amino acid residues contained in any one of the groups (X) and (Y) described herein.

[0192] In a preferred embodiment of the aforementioned antigen-binding molecule, the first heavy chain CH3 region and the second heavy chain CH3 region may be cross-linked by a disulfide bond.

[0193] In the present invention, the amino acid residues to be "modified" are not limited to those in the antigen-binding molecule variable region or antibody constant region. Those skilled in the art can identify amino acid residues that form interfaces in polypeptide variants or heteromeric multimers by homology modeling using commercially available software, and modify the amino acid residues at those sites to regulate association. 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 search is conducted for proteins that have been determined to have a three-dimensional structure highly homologous to the protein. Next, this three-dimensional structure is used as a template to construct the structure of a protein with an unknown structure, and the structure is further optimized by molecular dynamics or the like to predict the three-dimensional structure of the unknown protein.

[0194] In order to introduce electrical repulsion into the interface between the heavy chain variable region and the light chain variable region to suppress undesired association of the heavy chain and the light chain, examples of amino acid residues that 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 the Kabat numbering in the VH (FR2 region) and glutamine (Q) at position 38 according to the Kabat numbering in the opposing (contacting) VL (FR2 region). Further preferred examples are leucine (L) at position 45 according to the Kabat numbering in the VH (FR2) and proline (P) at position 44 according to the 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.

[0195] These amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. 2003; 16: 113-120). Therefore, the association of the variable regions of antigen-binding molecules can be adjusted for VH-VL associations of antigen-binding molecules other than those shown in the Examples by modifying amino acid residues corresponding to the above-mentioned amino acid residues.

[0196] In some embodiments, in the multispecific antigen-binding molecule, two or more amino acid residues forming the interface between the heavy chain variable region and the light chain variable region are amino acid residues that are electrostatically repulsive to each other.

[0197] A specific example is an antigen-binding molecule in which two or more amino acid residues forming the interface between VH and VL are mutually electrically repulsive amino acid residues. More specifically, examples include antigen-binding molecules having one or two amino acid residue sets selected from the group consisting of the amino acid residue sets shown in (a) or (b) below: (a) the amino acid residue at position 39 of the VH according to the Kabat numbering system and the amino acid residue at position 38 of the VL according to the Kabat numbering system; 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.

[0198] In some embodiments, in the multispecific antigen-binding molecule, the mutually electrostatically repulsive 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 that is position 39 according to the Kabat numbering system, and the amino acid residue in the light chain variable region that is position 38 according to the Kabat numbering system; (b) The amino acid residue in the heavy chain variable region that is position 45 according to Kabat numbering, and the amino acid residue in the light chain variable region that is position 44 according to Kabat numbering.

[0199] The amino acid residues described in (a) or (b) above are close to each other during assembly. Those skilled in the art will be able to find positions in the desired VH or VL corresponding to the amino acid residues described in (a) or (b) above by homology modeling using commercially available software, and appropriately modify the amino acid residues at those positions.

[0200] In some embodiments, in the multispecific antigen-binding molecule, the amino acid residues that are electrostatically repulsive to one another 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).

[0201] In a method for modifying amino acid residues forming the hydrophobic core at the VH / VL interface to charge-carrying polar amino acids to suppress unintended association of the heavy and light chains, preferred examples of amino acid residues capable of forming the hydrophobic core at the VH / VL interface include leucine (L) at position 45 according to the Kabat numbering in VH (FR2) and proline (P) at position 44 according to the Kabat numbering in the opposing VL (FR2). The numbering of these positions was determined based on Kabat et al. (Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, NIH).

[0202] Generally, the term "hydrophobic core" refers to the portion formed by the collection of hydrophobic amino acid side chains inside an assembled polypeptide. Examples of hydrophobic amino acids include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In addition, amino acid residues other than hydrophobic amino acids (e.g., tyrosine) can participate in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the hydrophilic amino acid side chains are exposed to the outside, becomes the driving force for promoting the association of water-soluble polypeptides. When the hydrophobic amino acids of two different domains are present on the molecular surface and exposed to water molecules, the entropy will increase and the free energy will increase. Therefore, the two domains will associate with each other to reduce free energy and become stable, and the hydrophobic amino acids at the interface will be buried in the interior of the molecule to form a hydrophobic core.

[0203] When polypeptide association occurs, modifying the hydrophobic amino acids that form the hydrophobic core to polar, charged amino acids is believed to inhibit the formation of the hydrophobic core; consequently, peptide association is believed to be inhibited.

[0204] Those skilled in the art will be able to identify the presence or absence of a hydrophobic core, as well as the site (region) where it is formed, by analyzing the amino acid sequence of a desired antigen-binding molecule. That is, the antigen-binding molecules of the present invention are characterized in that amino acid residues that can form a hydrophobic core at the interface have been modified to have charged amino acid residues. 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 contained in VL at position 44 according to Kabat numbering.

[0205] Preferred examples of charged amino acid residues in the 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).

[0206] Generally, the amino acid residues described in (1) and (2) above in humans and mice are as follows, respectively: (1) leucine (L), and (2) Proline (P). Therefore, in a preferred embodiment of the present invention, these amino acid residues are modified (e.g., substituted with charged amino acids). Furthermore, the types of amino acid residues described above in (1) and (2) are not necessarily limited to the aforementioned amino acid residues, but may be other amino acids equivalent to these amino acid residues.

[0207] Other known techniques can be applied to the antigen-binding molecules of the present invention. For example, to promote the association between a first VH (VH1) and a first VL (VL1) and / or a second VH (VH2) and a second VL (VL2), an amino acid side chain in one variable region of the H chain can be replaced with a larger side chain (knob), and an amino acid side chain in the opposing variable region of the other H chain can be replaced with a smaller side chain (hole), thereby positioning the knob in the hole, promoting the association between VH1 and VL1 and / or VH2 and VL2; and consequently, further suppressing the association between VH1 and VL2 and / or VH2 and VL1.

[0208] For example, in the case of human IgG1, modifications Y349C and T366W are made to make the amino acid side chains in the CH3 region of one H chain larger (knob), and modifications D356C, T336S, L368A, and Y407V are made to make the amino acid side chains in the CH3 region of the other H chain smaller.

[0209] In some embodiments, in the multispecific antigen-binding molecule, the Fc domain is composed of a first Fc region subunit and a second Fc region subunit that are capable of stable association. In some embodiments, in the multispecific antigen-binding molecule, the Fc domain comprises (e1) or (e2) below: (e1) a first Fc region subunit comprising Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407, and a second Fc region comprising 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).

[0210] For example, knob-into-hole technology has been described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created at the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0211] Furthermore, other known techniques can be applied to the antigen-binding molecules of the present invention. A portion of the CH3 of one H chain of the antigen-binding molecule is altered to a sequence derived from IgA that corresponds to that portion, and the complementary portion of the CH3 of the other H chain is introduced with a sequence derived from IgA that corresponds to that portion, thereby enabling complementary association of the CH3 using a chain-exchange-engineered domain CH3, thereby enabling efficient preparation of target antigen-binding molecules (Protein Engineering Design & Selection, 23: 195-202, 2010).

[0212] Other known techniques can also be applied to the antigen-binding molecules of the present invention. For example, when producing bispecific antibodies, target bispecific antibodies can be prepared by altering the variable regions of two types of heavy chains to different amino acids to impart different isoelectric points, and then utilizing the difference in isoelectric points for purification by ion exchange chromatography (WO 2007 / 114325).

[0213] A technique in which the amino acid residue at position 435 according to EU numbering, which is the site involved in the binding between IgG and Protein A, is modified to an amino acid with a different binding strength to Protein A, such as Arg, may also be used in combination with the above-mentioned technique for the antigen-binding molecules of the present invention. By using this technique, 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 technique can also be used independently without being combined with the above-mentioned technique.

[0214] The modifications of the present invention can be used on antigen-binding molecules, such as those 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 promote the 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).

[0215] Multiple, for example, two or more of the above-mentioned known techniques can be used in combination for the antigen-binding molecules of the present invention. Furthermore, the antigen-binding molecules of the present invention may be prepared based on antibodies that have been modified by the above-mentioned known techniques.

[0216] 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) modifying nucleic acids encoding CH1 and CL so that one or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (c) are amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) that is position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) that is position 131 according to Kabat numbering; (b) the amino acid residue in CH1 that is position 175 according to EU numbering and the amino acid residue in CL that is position 160 according to Kabat numbering; (c) the amino acid residue in CH1 that is position 175 according to EU numbering, and the amino acid residues in CL that are positions 131 and 160 according to Kabat numbering; (d) amino acid residues in CH1 that are positions 147 and 175 according to EU numbering, and amino acid residues in CL that are positions 131 and 160 according to Kabat numbering; (2) introducing the modified nucleic acid into a host cell and culturing the host cell so that the nucleic acid is expressed; and (3) recovering the antigen-binding molecule from the cell culture of the host cells.

[0217] In addition, the present invention relates to a production method comprising modifying a nucleic acid in the aforementioned step (1) so that the mutually electrically repulsive amino acid residues are selected from among the amino acid residues contained in either of the aforementioned groups (X) and (Y).

[0218] Furthermore, the present invention relates to a production method comprising modifying a nucleic acid in the above-mentioned step (1) so that two or more amino acid residues forming the interface between VH and VL are amino acid residues that are electrically repulsive to each other. Preferably, the mutually electrically repulsive amino acid residues are, for example, any set of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) and (b): (a) the amino acid residue at position 39 of the VH according to the Kabat numbering system and the amino acid residue at position 38 of the VL according to the Kabat numbering system; 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.

[0219] The aforementioned amino acid residues that are electrically repulsive to each other are preferably selected from amino acid residues contained in either of the aforementioned sets (X) and (Y).

[0220] In addition, the present invention provides a method for modulating the association between the heavy and light chains of an antigen-binding molecule. A preferred embodiment of the method for modulating association of the present invention is a method for modulating the association between the heavy and light chains of an antigen-binding molecule, comprising the step of modifying a nucleic acid so that one or more sets of amino acid residues selected from the group consisting of the following sets of amino acid residues (a) to (c) are amino acid residues that are electrostatically repulsive to each other: (a) the amino acid residue in the heavy chain constant region (CH1) which is position 175 according to EU numbering, and the amino acid residue in the light chain constant region (CL) which is position 131 according to EU numbering; (b) the amino acid residue in CH1 that is position 175 according to EU numbering, and the amino acid residue in CL that is position 160 according to EU numbering; (c) the amino acid residue in CH1 that is at position 175 according to EU numbering, and the amino acid residues in CL that are at positions 131 and 160 according to EU numbering; (d) Amino acid residues in CH1 that are positions 147 and 175 according to EU numbering, and amino acid residues in CL that are positions 131 and 160 according to EU numbering.

[0221] In addition, the present invention relates to a method for regulating association, which comprises modifying a nucleic acid in the aforementioned step (1) so that the mutually electrostatically repulsive amino acid residues are selected from amino acid residues contained in the aforementioned group of either (X) or (Y).

[0222] Furthermore, the present invention relates to a method for regulating association, which comprises modifying a nucleic acid in the above-mentioned step (1) so that two or more amino acid residues forming the VH-VL interface are amino acid residues that are electrostatically repulsive to each other, wherein the mutually electrostatically repulsive amino acid residues are preferably, for example, any one of the sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) and (b): (a) the amino acid residue in VH that is position 39 according to Kabat numbering, and the amino acid residue in VL that is position 38 according to Kabat numbering; (b) The amino acid residue in VH that is position 45 according to Kabat numbering, and the amino acid residue in VL that is position 44 according to Kabat numbering.

[0223] According to the method for modulating assembly of the present invention, the desired bispecific antibody can be preferentially and efficiently obtained as previously described, i.e., the desired heteromeric multimer in the form of the bispecific antibody can be efficiently formed from a mixture of monomers.

[0224] The phrase "modifying a nucleic acid" in the above-mentioned methods of the present invention refers to modifying a nucleic acid so that it corresponds to the amino acid residue introduced by the "modification" of the present invention. More specifically, this refers to modifying a nucleic acid encoding an original (pre-modification) amino acid residue to form a nucleic acid encoding the amino acid residue to be introduced by the modification. Usually, this means performing genetic engineering or mutation treatment that will result in the insertion, deletion, or substitution of at least one nucleotide in the original nucleic acid so that a codon encoding the desired 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 introduced by the modification. Such nucleic acid modifications 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 molecules of the present invention. Furthermore, vectors carrying nucleic acids are also included in the present invention.

[0225] In some embodiments, the Fc domain of a multispecific antigen-binding molecule consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises 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 no more than one Fc domain.

[0226] In one embodiment described herein, the Fc domain of the multispecific antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further specific embodiment, the Fc domain is a human IgG1 Fc region.

[0227] In some embodiments, the present disclosure provides multispecific antigen-binding molecules further comprising an Fc domain that exhibits reduced binding affinity for human Fcγ receptors compared to native human IgG1 Fc domains, and the Fc domain further exhibits stronger FcRn-binding affinity for human FcRn compared to native human IgG1 Fc domains.

[0228] In some embodiments, the present disclosure provides multispecific antigen-binding molecules further comprising an Fc domain that exhibits reduced binding affinity to a human Fcγ receptor compared to a native human IgG1 Fc domain, wherein the first and / or Fc region subunit comprise Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, wherein the amino acid positions are numbered according to EU numbering.

[0229] In some embodiments, in the multispecific antigen-binding molecule, the Fc domain further exhibits stronger FcRn-binding affinity for human FcRn compared to native human IgG1 Fc domain. In some embodiments, in the multispecific antigen-binding molecule, the first and / or Fc region subunit comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, wherein the amino acid positions are numbered according to EU numbering.

[0230] IgG-type bispecific antibodies are secreted by introducing into cells the genes for the L and H chains that make up the two desired IgG types (i.e., a total of four genes) and coexpressing them. However, these methods theoretically produce only 10 combinations of IgG H and L chains. Therefore, it is difficult to purify IgG containing the desired H and L chain combination from the 10 types of IgG. Furthermore, theoretically, the secreted amount of IgG containing the desired combination would be significantly reduced, thus necessitating large-scale culture, which would further increase production costs.

[0231] Therefore, techniques for promoting association between H chains and between L chains and H chains with desired combinations can be applied to the multispecific antigen-binding molecules of the present invention. For example, a technique for suppressing undesired heavy chain association by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain can be applied to multispecific antibody association (WO2006 / 106905).

[0232] In the present invention, the amino acid residues that are modified are not limited to the above-mentioned amino acid residues in antibody variable region or antibody constant region.Those skilled in the art can identify the amino acid residues that form the interface in mutant polypeptide or heteromultimer by using commercially available software, such as homology modeling; then, the amino acid residues at these positions can be modified to adjust the association.

[0233] Other known techniques can also be used for assembling the multispecific antibodies of the present invention. Fc region-containing polypeptides containing different amino acids can be efficiently assembled with each other by replacing an amino acid side chain present in one of the antibody heavy chain Fc regions with a larger side chain (knob) and an amino acid side chain present in the corresponding Fc region of the other heavy chain with a smaller side chain (hole), allowing the knob to be positioned within the hole. The knob-into-hole method is discussed elsewhere herein.

[0234] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. Association of polypeptides with different sequences can be efficiently induced by complementary association of CH3s using a chain-exchange engineered domain CH3 produced by modifying a portion of one of the antibody H chain CH3s to a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the other H chain CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibodies.

[0235] In addition, there are known techniques for producing antibodies 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 modulating 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; and techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain, as described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 Techniques for producing bispecific antibodies using two bacterial cell lines each expressing one of the chains of an antibody comprising a single H chain and a single L chain, such as those described by S. K. (2013), may be used to form multispecific antibodies.

[0236] Alternatively, even if the desired multispecific antibody cannot be efficiently formed, it can be obtained by separating and purifying it from the produced antibodies. For example, a method has been reported in which the difference in isoelectric point is achieved by introducing amino acid substitutions into the variable regions of two types of H chains, thereby enabling the purification of two types of homomeric forms and the desired heteromeric antibody by ion exchange chromatography (WO2007114325). To date, methods for purifying heteromeric antibodies have been reported in which protein A is used to purify a heterodimeric antibody comprising a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A (WO98050431 and WO95033844). Furthermore, heterodimeric antibodies can be efficiently purified by themselves by using H chains that contain substitutions of amino acid residues at positions 435 and 436 (EU numbering), which are the IgG-Protein A binding site, with amino acids such as Tyr and His that result in different Protein A affinities, or by using H chains with different Protein A affinities obtained according to the method of Reference Example 5 to alter the interaction between each H chain and Protein A, and then using a Protein A column.

[0237] Furthermore, Fc regions with improved C-terminal heterogeneity can be suitably used as the Fc regions of the present invention. More specifically, the present invention provides Fc regions produced by deleting glycine at position 446 and lysine at position 447 (EU numbering) from the amino acid sequences of two polypeptides that constitute the Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0238] A combination of multiple, for example, two or more, of these techniques can be used. Furthermore, these techniques can be applied appropriately and separately to the two H chains to be associated. Furthermore, these techniques can be used in combination with the above-mentioned Fc region having reduced binding activity to Fcγ receptors. Furthermore, the antigen-binding molecules of the present invention may be molecules produced separately based on the antigen-binding molecules subjected to the above-mentioned modifications so as to have the same amino acid sequence.

[0239] In some embodiments, the multispecific antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xii): (i) glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) glutamic acid or lysine at position 131 (Kabat numbering) in the light chain constant region; (iv) glutamic acid or lysine at position 160 (Kabat numbering) in the light chain constant region; (v) arginine at position 235 (EU numbering) in the heavy chain constant region; (vi) arginine at position 236 (EU numbering) in the heavy chain constant region; (vii) lysine at position 356 (EU numbering) in the heavy chain constant region; (viii) leucine at position 428 (EU numbering) in the heavy chain constant region; (ix) alanine at position 434 (EU numbering) in the heavy chain constant region; (x) arginine at position 438 (EU numbering) in the heavy chain constant region; (xi) glutamic acid at position 439 (EU numbering) in the heavy chain constant region; (xii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region. In some embodiments, the multispecific antigen-binding molecule is a bispecific antibody comprising: a first heavy chain comprising a lysine at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), a glutamic acid at position 439 (EU numbering), and a glutamic acid at position 440 (EU numbering); a first light chain containing glutamic acid at position 131 (Kabat numbering) and glutamic acid at position 160 (Kabat numbering); a second heavy chain comprising a glutamic acid at position 147 (EU numbering), a glutamic acid at position 175 (EU numbering), an arginine at position 235 (EU numbering), an arginine at position 236 (EU numbering), a lysine at position 356 (EU numbering), a leucine at position 428 (EU numbering), an alanine at position 434 (EU numbering), an arginine at position 438 (EU numbering), and a glutamic acid at position 440 (EU numbering); and A second light chain containing a lysine acid at position 131 (Kabat numbering) and a lysine at position 160 (Kabat numbering). In some embodiments, in the multispecific antigen binding molecule: the first heavy chain further comprises a glutamic acid at position 419 (EU numbering) and a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering); and The second heavy chain further comprises a lysine at position 196 (EU numbering), a proline at position 445 (EU numbering), and amino acid deletions at positions 446 and 447 (EU numbering). In some embodiments, in the multispecific antigen binding molecule: the first heavy chain further comprises a glycine at position 16 (Kabat numbering), an alanine at position 32 (Kabat numbering), a valine at position 35a (Kabat numbering), an alanine at position 50 (Kabat numbering), a lysine at position 61 (Kabat numbering), a glutamic acid at position 64 (Kabat numbering), a threonine at position 73 (Kabat numbering), a glutamic acid at position 95 (Kabat numbering), and a valine at position 102 (Kabat numbering); the first light chain further comprises a glutamic acid at position 28 (Kabat numbering), a tyrosine at position 55 (Kabat numbering), a glutamic acid or tyrosine at position 56 (Kabat numbering), a glutamic acid at position 92 (Kabat numbering), a valine at position 94 (Kabat numbering), and an alanine at position 95a (Kabat numbering); the second heavy chain further comprises a glutamic acid at position 28 (Kabat numbering), an alanine or glutamic acid at position 30 (Kabat numbering), a glutamic acid at position 31 (Kabat numbering), a tryptophan at position 32 (Kabat numbering), a phenylalanine at position 34 (Kabat numbering), and a methionine at position 35 (Kabat numbering), a serine at position 35a (Kabat numbering), a serine at position 50 (Kabat numbering), a glutamic acid or glycine at position 61 (Kabat numbering), a glutamic acid at position 64 (Kabat numbering), and a glutamic acid at position 65 (Kabat numbering); and The second light chain further contains a threonine at position 25 (Kabat numbering), a lysine at position 54 (Kabat numbering), a glutamic acid at position 56 (Kabat numbering), a leucine at position 67 (Kabat numbering), a glutamine at position 79 (Kabat numbering), and a lysine at position 94 (Kabat numbering).

[0240] Library-derived antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are 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 described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0241] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0242] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0243] Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0244] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, usually attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., 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 biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0245] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "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, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0246] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected 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 US2005 / 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.).

[0247] In a preferred embodiment, the above-mentioned antibodies may have their first heavy chain CH3 region and second heavy chain CH3 region cross-linked by a disulfide bond.

[0248] Multispecific antigen-binding molecules prepared as described herein may be purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size-exclusion chromatography, etc. The actual conditions used to purify a particular protein will 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, a matrix with Protein A or Protein G can be used for affinity chromatography purification of the multispecific antigen-binding molecules of the present invention. 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, high-pressure liquid chromatography, etc.

[0249] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a 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-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0250] antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies 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 homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if two or more polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0251] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0252] Recombination methods and constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-HLA-DQ2.5 antigen-binding molecule (antibody) described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, a method for producing an anti-HLA-DQ2.5 antigen-binding molecule (antibody) is provided, comprising culturing a host cell containing nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-HLA-DQ2.5 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0253] For recombinant production of anti-HLA-DQ2.5 antigen-binding molecules (antibodies), nucleic acids encoding the antibodies (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 may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0254] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0255] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0256] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing 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 transformation of Spodoptera frugiperda cells.

[0257] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0258] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0259] 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 assays known in the art.

[0260] Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, and the like.

[0261] In another aspect, a competitive assay can be used to identify antibodies that compete with, for example, any of the above-mentioned antibodies for binding to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complexes). In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the above-mentioned antibody. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0262] 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 to be 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 hybridoma supernatant. As a control, immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complexes), excess unbound antibody is removed and the amount of label bound to the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complexes) is measured. If the amount of label bound to the immobilized HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complexes) is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complexes). See Harlow and Lane (1988) Antibodies: A Laboratory Manual, ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0263] 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, for example, 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 allowed to bind to the biotinylated antigen, and the cells are subjected to cell sorting and culture for selection. Specific binding of the cells to the antigen can be evaluated by any appropriate method, such as ELISA. This method can also be used to evaluate the lack of cross-reactivity with non-target antigens. To isolate the selected antibody or determine its sequence, for example, RNA is purified from the cells, and DNA encoding the antibody region is prepared by reverse transcription of the RNA and PCR amplification. Furthermore, the cloned antibody gene can be expressed in appropriate cells, and the antibody can be purified from the culture supernatant for further analysis.

[0264] To test whether an anti-HLA-DQ2.5 antigen-binding molecule (antibody) binds to an antigen of interest (e.g., a complex formed by HLA-DQ2.5 and a gluten peptide, such as those described herein), any method for assessing binding can be used. For example, when using a FACS-based cell sorting method, cells expressing the antigen are incubated with a test antibody, followed by the addition of an appropriate secondary antibody against the test antibody (i.e., the primary antibody). Binding between the antigen and the test antibody is detected by FACS analysis, for example, using a colorimetric / fluorescent label attached to the secondary antibody (e.g., as described herein). Alternatively, any of the measurement methods described in the "Antibody Affinity" section herein can be used. For example, measuring Kd using a BIACORE surface plasmon resonance assay can be used to assess binding between a test antibody and an antigen of interest described herein.

[0265] In certain embodiments, the methods of the present invention further comprise the steps of testing whether an antibody has neutralizing activity against the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR (or the interaction between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and HLA-DQ2.5-restricted CD4+ T cells); and selecting an antibody having the neutralizing activity. In certain embodiments, the methods of the present invention further comprise the steps of testing whether an antibody has neutralizing activity against the binding between HLA-DQ2.2 (or HLA-DQ2.2 / gluten peptide complex) and TCR (or the interaction between HLA-DQ2.2 (or HLA-DQ2.2 / gluten peptide complex) and HLA-DQ2.2-restricted CD4+ T cells); and selecting an antibody having the 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 peptides. Neutralizing activity can be evaluated, for example, as described herein. Briefly, beads, such as yellow particles coated with streptavidin, are appropriately prepared for immobilization on a plate, and soluble HLA-DQ bound to the peptide is added to the beads. The plate is washed and blocked, and an antibody is added thereto and incubated. When evaluating the binding between HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex) and TCR, for example, D2 TCR tetramer-PE can be added and incubated. The binding between these two can be evaluated based on the colorimetric / fluorescent labeling of TCR bound to HLA-DQ2.5 (or HLA-DQ2.5 / gluten peptide complex).

[0266] In some embodiments, the multispecific antigen binding molecule blocks the interaction between the HLA-DQ2.5 / gluten peptide complex and HLA-DQ2.5 / gluten peptide-restricted CD4+ T cells. In some embodiments, the multispecific antigen binding molecule blocks the interaction between the HLA-DQ2.2 / gluten peptide complex and HLA-DQ2.2 / gluten peptide-restricted CD4+ T cells. In this context, 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.

[0267] In some embodiments, the multispecific antigen-binding molecule has substantially no binding activity for HLA-DP, HLA-DR, HLA-DQ5.1, HLA-DQ6.3, HLA-DQ7.3, HLA-DQ7.5, and HLA-DQ8.

[0268] In some embodiments, the antigen-binding molecules of the present invention have enhanced binding activity to the complex formed by HLA-DQ2.5 and gluten peptides. In this context, the gluten peptide may be any of the gluten peptides described above. The degree of enhancement may be determined by comparing the binding activity to the complex formed by HLA-DQ2.5 and an unrelated peptide, or to cells that do not have the target complex, such as HLA-DQ2.5-positive PBMC B cells and / or Ba / F3 cells expressing HLA-DQ2.5 or HLA-DQ2.2.

[0269] 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 term "arm" or "half-antibody" refers to a portion of an antibody comprising 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first arm / half-antibody and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second arm / half-antibody.

[0270] In some embodiments, for the bispecific antibodies of the invention, the first arm / half antibody is derived from DQN0344xx (DQN0344Hx / DQN0344Lx) described herein, and the second arm / half antibody is derived from DQN0385ee (DQN0385He / DQN0385Le) described herein. The sequence ID numbers (SEQ ID NOs) for the VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and full-length heavy (H) chains and light (L) chains of the bispecific antibodies of the invention are shown in Tables 2-3 to 2-6 (below). In some embodiments, the antibodies of the present invention comprise an HCDR1 comprising the sequence of SEQ ID NO: 129 or 164; an HCDR2 comprising the sequence of SEQ ID NO: 130 or 165; and an HCDR3 comprising the sequence of SEQ ID NO: 131 or 166. In some embodiments, the antibodies of the present invention comprise an LCDR1 comprising the sequence of SEQ ID NO: 132 or 167; an LCDR2 comprising the sequence of SEQ ID NO: 133 or 168; and an LCDR3 comprising the sequence of SEQ ID NO: 134 or 169. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO:88 or 89. In some embodiments, an antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 105 or 162. In some embodiments, an antibody of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:90 or 91. In some embodiments, an antibody of the invention comprises a light chain constant region comprising the sequence of SEQ ID NO:106. In some embodiments, the antibody of the present invention comprises a (full-length) heavy chain comprising 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 comprising the sequence of SEQ ID NO: 43 or 46. In some embodiments, an antibody of the present invention comprises an HCDR1 comprising the sequence of SEQ ID NO: 135, 144, 147, 153, 156, or 159; an HCDR2 comprising the sequence of SEQ ID NO: 136, 145, 148, 154, 157, or 160; and an HCDR3 comprising the sequence of SEQ ID NO: 137, 146, 149, 155, 158, or 161. In some embodiments, the antibodies of the present invention comprise an LCDR1 comprising the sequence of SEQ ID NO: 138, 141, or 150; an LCDR2 comprising the sequence of SEQ ID NO: 139, 142, or 151; and an LCDR3 comprising the sequence of SEQ ID NO: 140, 143, or 152. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 92, 93, 94, 95, 96, or 97. In some embodiments, an antibody of the invention comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 104 or 163. In some embodiments, an antibody of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 98, 99, or 100. In some embodiments, an antibody of the invention comprises a light chain constant region comprising the sequence of SEQ ID NO:107. In some embodiments, an antibody of the invention comprises a (full-length) heavy chain comprising the sequence of SEQ ID NO: 53, 54, 57, 58, 59, 60, 62, 63, 64, 65, 66, or 67. In some embodiments, the antibody of the present invention comprises a (full-length) light chain comprising the sequence of SEQ ID NO: 55, 56, or 61.

[0271] Specific sequences of the full-length H and L chains of the arm / half antibodies (included in the bispecific antibodies of the present invention) are shown in Table 1-2.

[0272] Table 1-2: Full-length heavy and light chains of bispecific antibodies The H (or L) chain comprises, from N- to C-terminus, HCDR1, HCDR2, and HCDR3 (or LCDR1, LCDR2, and LCDR3), which are underlined in the table. TIFF2023058542000002.tif226151TIFF2023058542000003.tif232151TIFF20230585420 00004.tif230151TIFF2023058542000005.tif223151TIFF2023058542000006.tif108151

[0273] In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, The present invention provides a multispecific antigen-binding molecule, wherein the first antigen-binding portion comprises any one of the following (a1) to (a3): (a1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, and CDR 3 of SEQ ID NO: 131, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, 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, CDR 3 of SEQ ID NO: 166, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (a3) A first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 to a second antibody variable region described in (a1) or (a2). In some embodiments, in the multispecific antigen-binding molecule comprising a second antigen-binding portion, the second antigen-binding portion comprises any one of the following (b1) to (b8): (b1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (b2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (b4) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b5) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b6) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (b7) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (b8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 to a fourth antibody variable region described in any one of (b1) to (b7). In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, The first antigen-binding portion is selected from the following (c1) to (c3): (c1) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 129, CDR 2 of SEQ ID NO: 130, CDR 3 of SEQ ID NO: 131, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 132, CDR 2 of SEQ ID NO: 133, CDR 3 of SEQ ID NO: 134; (c2) a first antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 164, CDR 2 of SEQ ID NO: 165, CDR 3 of SEQ ID NO: 166, and a second antibody variable region comprising CDR 1 of SEQ ID NO: 167, CDR 2 of SEQ ID NO: 168, CDR 3 of SEQ ID NO: 169; and (c3) a first amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a 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 to a second antibody variable region described in (c1) or (c2). including one of the following: The second antigen-binding portion is selected from the following (d1) to (d8): (d1) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 138, CDR 2 of SEQ ID NO: 139, CDR 3 of SEQ ID NO: 140; (d2) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 135, CDR 2 of SEQ ID NO: 136, CDR 3 of SEQ ID NO: 137, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d3) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 144, CDR 2 of SEQ ID NO: 145, CDR 3 of SEQ ID NO: 146, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; (d4) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 147, CDR 2 of SEQ ID NO: 148, CDR 3 of SEQ ID NO: 149, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d5) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 153, CDR 2 of SEQ ID NO: 154, CDR 3 of SEQ ID NO: 155, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d6) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 156, CDR 2 of SEQ ID NO: 157, CDR 3 of SEQ ID NO: 158, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 150, CDR 2 of SEQ ID NO: 151, CDR 3 of SEQ ID NO: 152; (d7) a third antibody variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 159, CDR 2 of SEQ ID NO: 160, CDR 3 of SEQ ID NO: 161, and a fourth antibody variable region comprising CDR 1 of SEQ ID NO: 141, CDR 2 of SEQ ID NO: 142, CDR 3 of SEQ ID NO: 143; and (d8) A third amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity 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 to a fourth antibody variable region described in any one of (d1) to (d7). The present invention provides a multispecific antigen-binding molecule comprising any one of: In some embodiments, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion comprising a first and a second antibody variable region and a second antig...

Claims

[Claim 1] The invention described herein.

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