Method for producing and / or concentrating recombinant antigen-binding molecules

Abstract

This invention provides a method for producing novel antigen-binding molecules that have the activity to regulate interactions between antigen molecules. [Solution] A method for producing an antibody preparation, comprising the steps of contacting an antibody solution with a reducing agent and, following the contact step, re-oxidizing the antibody, wherein the antibody comprises a first antigen-binding domain and a second antigen-binding domain linked to each other by at least one disulfide bond, the at least one disulfide bond being formed between amino acid residues at EU numbering position 191 in the CH1 region of each of the first and second antigen-binding domains, and the antibody solution comprises two structural isoforms of antibodies differing only by the at least one disulfide bond.

Description

[Technical Field]

[0001] The present invention relates to an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain that can be linked to one another via at least one disulfide bond formed between two antigen-binding domains, and to a method for producing such an antigen-binding molecule. More specifically, the present invention relates to a method for increasing or concentrating an antibody protein in a preferred form, and to a method for removing disulfide heterogeneity from recombinant antibody proteins. [Background technology]

[0002] Antibodies are proteins that bind specifically to antigens with high affinity. A wide variety of molecules, from small molecules to proteins, are known to act as antigens. Since the development of monoclonal antibody production techniques, antibody modification techniques have advanced, making it easier to obtain antibodies that recognize specific molecules. Furthermore, antibody modification techniques have expanded beyond simply modifying the protein itself to include fields aiming to add new functions through conjugation with small molecules. For example, cysteine-modified antibodies, which contain free cysteine ​​amino acids in their heavy or light chains, are used in medical applications as antibody-drug conjugates (ADCs) (Patent Document 1).

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low incidence of side effects. Antibodies not only bind to antigens and exhibit agonist and antagonist activity, but also induce cytotoxic activity (also called effector function) by effector cells, such as ADCC (Antibody-Dependent Cell Cytotoxicity), ADCP (Antibody-Dependent Cell Phagocytosis), and CDC (Complement-Dependent Cytotoxicity). Pharmaceuticals for cancer, immunological diseases, chronic diseases, and infectious diseases have been developed utilizing these functions of antibodies (Non-Patent Literature 1).

[0004] For example, drugs utilizing agonist antibodies against costimulatory molecules that promote the activation of cytotoxic T cells have been developed as anticancer agents (Non-Patent Literature 2). In recent years, it has become clear that inhibitory antibodies against immune checkpoints that have antagonist activity against co-inhibitory molecules are useful as anticancer agents, and antibody drugs that inhibit the interaction of CTLA4 / CD80 and PD-1 / PD-L1, such as ipilimumab, nivolumab, pembrolizumab, and atezolizumab, have been successively launched (Non-Patent Literature 1).

[0005] However, since such antibodies may not fully exert the expected effects in their natural IgG form, second-generation antibody drugs have been developed in which the function of natural IgG antibodies is artificially enhanced or added, or weakened or deleted, according to the intended use of the antibody. Examples of second-generation antibody drugs include antibodies with enhanced or deleted effector function (Non-Patent Literature 3), antibodies that bind to antigens in a pH-dependent manner (Non-Patent Literature 4), and antibodies that bind to two or more antigens with a single molecule (antibodies that bind to two antigens are generally called "bispecific antibodies") (Non-Patent Literature 5).

[0006] Bispecific antibodies are expected to become more effective pharmaceuticals. For example, antibodies with enhanced antitumor activity have been developed that crosslink cytotoxic T cells and cancer cells by binding to a protein expressed on the cell membrane of T cells as one antigen and to a cancer antigen as the other antigen (Non-Patent Documents 7, 8, and 2). Examples of bispecific antibodies that have been reported include molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules in which antigen-binding sites have been added to the N-terminus or C-terminus of the antibody (DVD-Ig and scFv-IgG), molecules in which one Fab region binds to two antigens (Two-in-one IgG), molecules in which the loop portion of the CH3 region has been manipulated to form a new antigen-binding site (Fcab) (Non-Patent Document 9), and molecules in which Fab-Fab are connected in series (Non-Patent Document 10).

[0007] On the other hand, antibodies with effector function tend to act on normal cells with low expression of the target antigen, making them prone to side effects. Therefore, attempts are being made to make the effector function of antibody drugs exerted specifically on target tissues. For example, antibodies whose binding activity changes upon binding to cellular metabolites (Patent Document 3), antibodies that exhibit antigen-binding ability after being cleaved by proteases (Patent Document 4), and a technology to control the crosslinking between antibody-mediated chimeric antigen receptor T cells (CAR-T cells) and cancer cells by adding a compound (ABT-737) (Non-Patent Document 11) have been reported.

[0008] Obtaining agonist antibodies can be difficult depending on the target, and various methods have been developed, particularly for membrane proteins such as G protein-coupled receptors (Non-Patent Literature 12). Therefore, there is a need for a simple method to enhance the agonist effect of antibodies against such targets. Existing methods include cross-linking anti-DR4 (Death Receptor 4) or anti-DR5 (Death Receptor 5) antibodies (Non-Patent Literature 13), multimerizing nanobodies of anti-DR5 (Death Receptor 5) antibodies (Non-Patent Literature 14), converting anti-thrombopoietin receptor antibodies into covalent dibobodies of sc(Fv)2 (Non-Patent Literature 15), changing the IgG subclass of anti-CD40 antibodies (Non-Patent Literature 16), hexamerizing anti-CD20 antibodies (Non-Patent Literature 17), and producing spherical antibody-like molecules (Patent Literature 5). Furthermore, methods using bispecific antibodies have been reported, including a method that combines two appropriate anti-erythropoietin antibodies with different epitopes as bispecific antibodies (Non-Patent Document 18), a method that combines antibodies for guide and effector functions as bispecific antibodies (Non-Patent Document 19), and a method that conjugates multiple antibody fragments with different epitopes into which Cys residues have been introduced (Non-Patent Documents 20, 21, and 6). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2016 / 040856 [Patent Document 2] International Publication No. 2008 / 157379 [Patent Document 3] International Publication No. 2013 / 180200 [Patent Document 4] International Publication No. 2009 / 025846 [Patent Document 5] International Publication No. 2017 / 191101

Patent document 6

Non-licensed literature

[0010] [Non-licensed document 1] Nature Reviews Drug Discovery (2018) 17, 197-223 [Non-licensed document 2] Clinical and Experimental Immunology (2009) 157, 9-19 [Non-licensed document 3] Current Pharmaceutical Biotechnology (2016) 17, 1298-1314

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0011] The object of the present invention is to provide a novel antigen-binding molecule (e.g., an IgG antibody) having activity to modulate the interaction between two or more antigen molecules, and / or a method for producing or using such an antigen-binding molecule. More specifically, the present invention solves the problem that conventional antibodies (e.g., wild-type IgG) have uncontrolled mobility of two antigen-binding domains (e.g., two Fab arms) by introducing one or more manipulated disulfide bonds between two antigen-binding domains (two Fabs) of an antibody through the introduction of mutations into the heavy chain and / or light chain. Specifically, by introducing one or more thiol-containing amino acids (e.g., cysteine ​​and methionine) into each of the two antigen-binding domains (two Fabs) of an antibody, such an antibody can form one or more disulfide bonds between the two antigen-binding domains (two Fabs). [Means for solving the problem]

[0012] The antigen-binding molecule of the present invention comprises a first antigen-binding domain and a second antigen-binding domain that can be “linked” to each other via at least one disulfide bond between the two antigen-binding domains. This at least one disulfide bond can be “formed” between the two antigen-binding domains, for example, between amino acid residues not located within a hinge region. The terms “can be linked” and “can be formed” include cases where the disulfide bond is already formed, and cases where the disulfide bond is not formed but can be formed later under appropriate conditions.

[0013] In one non-limiting aspect, one or more manipulated disulfide bonds between two Fabs of an IgG antibody allow control of the mobility, distance, and / or cell binding orientation (i.e., cis or trans) of the two Fab arms, thereby improving the activity and / or safety of the IgG antibody compared to the corresponding wild-type IgG antibody without one or more manipulated disulfide bonds. In another non-limiting aspect, one or more manipulated disulfide bonds between two Fabs of IgG improve the agonist activity of the IgG antibody compared to the corresponding wild-type IgG antibody without one or more manipulated disulfide bonds. In addition, in another non-limiting aspect, one or more manipulated disulfide bonds between two Fabs of IgG improve the resistance of the IgG antibody to protease digestion compared to the corresponding wild-type IgG antibody without one or more manipulated disulfide bonds.

[0014] While preparing antibodies capable of forming one or more manipulated disulfide bonds between two Fabs of the antibody, the inventors have further discovered that multiple structural isoforms of the same antibody (same sequence) but with different disulfide structures, specifically isoforms having "paired cysteine" and isoforms having "free or unpaired cysteine" (i.e., two types of structural isoforms), can arise during recombinant antibody production in mammalian cells. Therefore, another aspect of the present invention aims to provide efficient and easy production, purification, and analysis of antibodies having one or more manipulated disulfide bonds between two Fabs of the antibody. More specifically, the present invention describes a method for increasing the structural uniformity and relative abundance of antibodies in the "paired cysteine" form, i.e., antibodies having one or more manipulated disulfide bonds formed between two Fabs of the antibody. In other words, the present invention describes a method for reducing the relative abundance of antibodies in the form of "free or unpaired cysteine," that is, antibodies that do not have the manipulated disulfide bond formed between the two Fabs of the antibody.

[0015] As will be described in more detail below, in some embodiments of the present invention, the addition of a reducing agent can promote the formation of one or more manipulated disulfide bonds in the antibody, thereby resulting in structural homogeneity of the molecule.

[0016] More specifically, the present invention provides the following: [1] (i) A method for producing an antibody preparation, (ii) a method for purifying an antibody having a desirable three-dimensional structure, or (iii) a method for improving the uniformity of an antibody preparation, The method comprises contacting an antibody preparation with a reducing reagent, wherein the antibody comprises a first antigen-binding domain and a second antigen-binding domain that can be linked to each other via at least one disulfide bond, and the at least one disulfide bond can be formed between amino acid residues that are not in a hinge region. [2] (i) a method for producing an antibody preparation, (ii) a method for purifying an antibody preparation, or (iii) a method for improving the uniformity of an antibody preparation, The method comprising isolating a fraction of an antibody having a desired three-dimensional structure via one or more chromatographic steps selected from the group consisting of reversed-phase chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, and electrophoresis; wherein the antibody having the desired three-dimensional structure is characterized by having at least one disulfide bond formed between amino acid residues not in a hinge region. [2A] The method according to [2], wherein one or more chromatographic steps are ion exchange chromatography (IEC) and / or hydrophobic interaction chromatography (HIC), or mixed-mode chromatography of IEC and HIC. [3] The method according to any one of [1] to [2A], wherein the antibody preparation comprises two or more structural isoforms that differ only by at least one disulfide bond formed between amino acid residues that are not in the hinge region. [3A] The method according to [3], wherein the antibody preparation comprises two structural isoforms that differ only by at least one disulfide bond formed between amino acid residues not located in the hinge region. [3B] The method according to any one of [1] to [3A], which preferentially enriches or increases a population of antibody structural isoforms having at least one disulfide bond formed between amino acid residues not located within a hinge region. [3C] The method according to any one of [1] to [3B] for producing a homogeneous antibody preparation having the antibody having at least one disulfide bond formed between amino acid residues not in a hinge region in a molar ratio of at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%. [3D] The method according to any one of [1] to [3C], wherein each of the first antigen-binding domain and the second antigen-binding domain includes a hinge region or does not include a hinge region. [3E] The method according to any one of [1] to [3D], wherein the amino acid residue not located within the hinge region is an introduced or manipulated cysteine. [3F] The method according to any one of [1] to [3E], wherein the at least one disulfide bond is an interchain disulfide bond. [3I] The method according to any one of [1] to [3F], wherein the at least one disulfide bond is an engineered disulfide bond that is not present in wild-type IgG. [4] The method according to any one of [1] to [3J], wherein the at least one disulfide bond is formed between the CH1 region, CL region, VL region, VH region, and / or VHH region of the first antigen-binding domain and the second antigen-binding domain. [5] The method according to any one of [1] to [4], wherein the at least one disulfide bond is formed between the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain. [5.1] The method according to [5], wherein the at least one disulfide bond is formed between antigen-binding domains at any one of the EU numbering positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, and 201 to 214 in the CH1 region. [5.2] The at least one disulfide bond is located in the CH1 region at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165 The method according to [5], wherein an antigen-binding domain is formed at any one of the following positions: 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, and 214. [5.3] The method according to [5], wherein the at least one disulfide bond is formed between antigen-binding domains at any one of EU numbering positions 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 in the CH1 region. [5.4] The method according to [5], wherein the at least one disulfide bond is formed between antigen-binding domains at any one of EU numbering positions 135, 136, or 191 in the CH1 region. [5.5] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123. [5.6] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140. [5.7] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 148, 149, and 150. [5.8] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. [5.9] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178. [5.10] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197. [5.11] The method according to [5], wherein the at least one disulfide bond is formed between amino acid residues in a first antigen-binding domain and a second antigen-binding domain selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214. [5.12] The method according to [5], wherein the difference in the positions of amino acid residues in the first antigen-binding domain and the second antigen-binding domain is 3 amino acids or less. [5.13] The method according to [5], wherein at least one disulfide bond linking two antigen-binding domains is formed by linking an amino acid residue at EU numbering position 135 in the CH1 region of the first antigen-binding domain with an amino acid residue at any one of EU numbering positions 132 to 138 in the CH1 region of the second antigen-binding domain. [5.14] The method according to [5], wherein at least one disulfide bond linking two antigen-binding domains is formed by linking an amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain with an amino acid residue at any one of EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain. [5.15] The method according to [5], wherein at least one disulfide bond linking two antigen-binding domains is formed by linking an amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with an amino acid residue at any one of EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain. [5.16] The method according to [5], wherein one disulfide bond is formed between two antigen-binding domains at EU numbering position 135 in the CH1 region. [5.17] The method according to [5], wherein one disulfide bond is formed between two antigen-binding domains at EU numbering position 136 in the CH1 region. [5.18] The method according to [5], wherein one disulfide bond is formed between two antigen-binding domains at EU numbering position 191 in the CH1 region. [5A] The method according to [5], wherein the subclass of the CH1 region is γ1, γ2, γ3, γ4, α1, α2, μ, δ, or ε. [6] The method according to [5] to [5A], wherein one disulfide bond is formed between the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and the second antigen-binding domain. [6A] The method according to [6], wherein one, two or more additional disulfide bonds are formed between the first antigen-binding domain and the second antigen-binding domain via amino acid residues at the following positions according to EU numbering in each of the CH1 regions of the first antigen-binding domain and the second antigen-binding domain: (a) Between amino acid residues at any of the positions 131-138, 194, and 195 in each of the two antigen-binding domains; (b) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (c) Between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (d) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (e) Between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (f) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (g) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (h) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (i) Between the amino acid residue at position 138 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (j) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (k) Between the amino acid residue at position 132 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (l) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (m) Between the amino acid residue at position 134 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (n) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (o) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (p) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; and (q) Between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains. [6B] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more inversely charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6C] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more negatively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6D] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more negatively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6E] Either the first or second antigen-binding domain contains the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 is glutamic acid (E) or aspartic acid (D); (b) The amino acid residue at position 137 is glutamic acid (E) or aspartic acid (D); (c) The amino acid residue at position 138 is glutamic acid (E) or aspartic acid (D). It includes one, two or more of the following; and The other antigen-binding domain of the first and second antigen-binding domains has the following amino acid residues in each CH1 region (according to EU numbering): (d) The amino acid residue at position 193 is lysine (K), arginine (R), or histidine (H); (e) The amino acid residue at position 194 is lysine (K), arginine (R), or histidine (H); and (f) The amino acid residue at position 195 is lysine (K), arginine (R), or histidine (H). The method according to [6] or [6A], comprising one, two or more of the above. [6F-1] Either the first or second antigen-binding domain contains the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 is lysine (K), arginine (R), or histidine (H); (b) The amino acid residue at position 137 is lysine (K), arginine (R), or histidine (H); (c) The amino acid residue at position 138 is lysine (K), arginine (R), or histidine (H). Includes one or more of the following; and Of the two antigen-binding domains, the other antigen-binding domain in each CH1 region contains the following amino acid residues (according to EU numbering): (d) The amino acid residue at position 193 is glutamic acid (E) or aspartic acid (D); (e) The amino acid residue at position 194 is glutamic acid (E) or aspartic acid (D); and (f) The amino acid residue at position 195 is glutamic acid (E) or aspartic acid (D). The method according to [6] or [6A], comprising one or more of the above. [6F-2] The method according to [6] or [6A], wherein each of the first and second antigen-binding domains contains one of the specific combinations of charge mutations (according to EU numbering) in each CH1 region listed in Table 7, Table 82, or Table 85. [6G] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more hydrophobic amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more hydrophobic amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6H] The method according to [6G], wherein the hydrophobic amino acid residue is alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp). [6I] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one "knob" amino acid residue at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more "hole" amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6J] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more “whole” amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one “knob” amino acid residue at positions 193-195 (EU numbering) in each CH1 region. [6K] The method according to [6I] or [6J], wherein the "knob" amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "whole" amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (T), or serine (S). [6L] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more aromatic amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6M] The method according to [6] or [6A], wherein either the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more aromatic amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [6N-1] The method according to [6L] or [6M], wherein the aromatic amino acid residue is selected from the group consisting of tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe); and the positively charged amino acid residue is selected from the group consisting of lysine (K), arginine (R), or histidine (H). [6N-2] The method according to [6] or [6A], wherein each of the first and second antigen-binding domains contains one of the specific combinations of hydrophobic amino acid mutations (according to EU numbering) in each CH1 region listed in Table 10. [7] The method according to any one of [1] to [4], wherein the at least one disulfide bond is formed between the CL region of the first antigen-binding domain and the CL region of the second antigen-binding domain. [7.1] The method according to [7], wherein at least one amino acid residue on which a disulfide bond is formed between two antigen-binding domains is located at one of the following positions in the CL region: Kabat numbering positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213. [7.2] The method according to [7], wherein the amino acid residues that form at least one disulfide bond between two antigen-binding domains are located in the CL region at positions selected from the group consisting of Kabat numbering positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213. [7.3] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at Kabat numbering position 126 in the CL region. [7.4] The method according to [7], wherein at least one disulfide bond linking two antigen-binding domains is formed by linking an amino acid residue in the CL region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. [7.5] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at a position independently selected from the group consisting of Kabat numbering positions 108, 109, 110, 111, and 112. [7.6] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at a position independently selected from the group consisting of Kabat numbering positions 151, 152, 153, 154, 155, and 156. [7.7] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at a position independently selected from the group consisting of Kabat numbering positions 184, 185, 186, 187, 188, 189, and 190. [7.8] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at a position independently selected from the group consisting of Kabat numbering positions 200, 201, 202, and 203. [7.9] The method according to [7], wherein the amino acid residue on which at least one disulfide bond is formed between the two antigen-binding domains is located at a position independently selected from the group consisting of Kabat numbering positions 208, 209, 210, 211, 212, and 213. [7.10] The method according to [7] to [7.9], wherein the difference in the positions of the amino acid residues in which at least one disulfide bond is formed between the two antigen-binding domains is within 3 amino acids. [7.11] The method according to [7], wherein at least one of the bonds linking two antigen-binding domains is formed by linking amino acid residues at Kabat numbering position 126 in the CL region of the two antigen-binding domains to each other. [8] The method according to any one of [1] to [4], wherein at least one of the disulfide bonds is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. [8.1] The method according to [8], wherein the amino acid residue in the CH1 region is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. [8.2] The method according to [8], wherein at least one disulfide bond linking two antigen-binding domains is formed by linking an amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with an amino acid residue at Kabat numbering position 126 in the CL region of the second antigen-binding domain. [8A] The method according to [7] to [8], wherein the subclass of the CL region is κ or λ. [9] The method according to any one of [1] to [4], wherein the at least one disulfide bond is formed between the variable region of the first antigen-binding domain and the second antigen-binding domain. [9.1] The method according to [9], wherein at least one amino acid residue on which a disulfide bond is formed between antigen-binding domains is located within the VH region. [9.2] The method according to [9], wherein the amino acid residue on which at least one disulfide bond is formed between antigen-binding domains is located at a position selected from the group consisting of Kabat numbering positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b in the VH region. [9.3] The method according to [9], wherein at least one amino acid residue in which a disulfide bond is formed between antigen-binding domains is located within the VL region. [9.4] The method according to [9], wherein at least one amino acid residue on which a disulfide bond is formed between antigen-binding domains is located at a position selected from the group consisting of Kabat numbering positions 21, 27, 58, 77, 100, 105, and 107 in the VL region (subclass κ). [9.5] The method according to [9], wherein at least one amino acid residue on which a disulfide bond is formed between antigen-binding domains is located at a position selected from the group consisting of Kabat numbering positions 6, 19, 33, and 34 in the VL region (subclass λ). [9A] The method according to [4], wherein at least one amino acid residue in which a disulfide bond is formed between two antigen-binding domains is located within the VHH region. [9B] The method according to [9A], wherein the amino acid residues on which at least one disulfide bond is formed between antigen-binding domains are located at positions selected from the group consisting of Kabat numbering positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 in the VHH region.

[10] A method according to any one of [1] to [9B], characterized by one or more of the following: (a) The at least one disulfide bond restricts the antigen-binding orientation of the two antigen-binding domains to cis-antigen binding (i.e., binding to two antigens on the same cell), or restricts the binding of the two antigen-binding domains to two antigens that are spatially close to each other; (b) The at least one disulfide bond holds the first antigen-binding domain and the second antigen-binding domain in a more spatially close position to each other compared to the same corresponding antibody that does not have the at least one disulfide bond; (c) The at least one disulfide bond reduces the mobility and / or mobility of the first antigen-binding domain and the second antigen-binding domain compared to the same antibody that does not have the at least one disulfide bond; (d) The at least one disulfide bond increases the antibody's resistance to protease cleavage compared to the corresponding same antibody that does not have the at least one disulfide bond; (e) The at least one disulfide bond enhances or reduces the interaction between two antigen molecules bound by the antigen-binding molecule compared to the corresponding same antibody that does not have the at least one disulfide bond; (f) The method produces an antibody preparation that is more uniform than the same antibody preparation that has not been treated by the method; (g) The method produces an antibody preparation in which the biological activity is increased compared to the same antibody that has not been treated by the method; (h) The method produces an antibody in which the activity of holding two antigen molecules in spatially close proximity is enhanced compared to the same antibody that has not been treated by the method; (i) The method produces an antibody whose stability is enhanced compared to the same antibody that has not been treated by the method; and (j) The method preferentially concentrates antibodies having at least one disulfide bond formed outside the hinge region, and the preferentially concentrated form has pharmaceutically desirable properties selected from any of (a) to (i) above, compared to a preparation not treated by the method.

[11] The method according to any one of [1] to

[10] , wherein each of the first and second antigen-binding domains has a Fab, Fab', scFab, Fv, scFv, or VHH structure. [11A] The method according to

[11] , comprising a Fab and a hinge region, wherein the first and second antigen-binding domains each form an F(ab')2 structure.

[12] The method according to any one of [1] to [11A], wherein the antigen-binding molecule further comprises an Fc region. [12A] The method according to

[12] , wherein the Fc region is an Fc region in which binding activity to FcγR is reduced compared to that of the Fc region of a wild-type human IgG1 antibody.

[13] The method according to any one of [1] to [12A], wherein the antibody is an IgG antibody, preferably an IgG1, IgG2, IgG3, or IgG4 antibody.

[14] The method according to any one of [1] to

[13] , wherein both the first and second antigen-binding domains bind to the same antigen. [14A] The method according to any one of [1] to

[13] , wherein both the first and second antigen-binding domains bind to the same epitope on the antigen. [14B] The method according to any one of [1] to

[13] , wherein each of the first and second antigen-binding domains binds to a different epitope on the antigen. [14C] The method according to any one of [1] to

[13] , wherein each of the first and second antigen-binding domains binds to a different antigen. [14D] The method according to any one of [1] to

[13] , wherein the first and second antigen-binding domains both have the same amino acid sequence. [14E] The method according to any one of [1] to

[13] , wherein each of the first and second antigen-binding domains has a different amino acid sequence. [14F] The method according to any one of [1] to [14E], wherein at least one of the two antigens to which the first and second antigen-binding domains bind is a soluble protein. [14G] The method according to any one of [1] to [14E], wherein at least one of the two antigens to which the first and second antigen-binding domains bind is a membrane protein. [14H] The method according to any one of [1] to [14G], which has activity to modulate the interaction between two antigen molecules. [14I] The method according to [14H], which can enhance or reduce the interaction between two antigen molecules compared to the same corresponding antibody that does not have at least one disulfide bond. [14J] The method according to any one of [14H] to [14I], wherein the two antigen molecules are ligands and their receptors, and the antibody has the activity to promote the activation of the receptor by the ligands. [14K] The method according to any one of [14H] to [14I], wherein the two antigen molecules are an enzyme and its substrate, and the antigen-binding molecule has the activity to promote the catalytic reaction between the enzyme and the substrate. [14L] The method according to any one of [14H] to [14I], wherein both antigen molecules are proteins present on the cell surface, and the antibody has the activity to promote interaction between a cell expressing the first antigen and a cell expressing the second antigen. [14M] The method according to any one of [14L], wherein the cell expressing the first antigen is a cytotoxic cell, the cell expressing the second antigen is its target cell, and the antibody promotes damage to the target cell by the cytotoxic cell. [14N] The method according to [14M], wherein the cytotoxic cells are T cells, NK cells, monocytes, or macrophages. [14O] The method according to [14N], wherein the antibody having at least one disulfide bond enhances or diminishes the activation of two antigen molecules compared to the same corresponding antibody that does not have at least one disulfide bond. [14P] The method according to any one of

[14] to [14O], wherein the antigen molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-bound receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[15] The method according to any one of

[14] to [14P], wherein the first antigen-binding domain and the second antigen-binding domain can each bind to CD3.

[16] The method according to any one of [1] to

[15] , wherein the pH of the reducing reagent that is brought into contact with the antibody is approximately 3 to approximately 10. [16A] The method according to

[16] , wherein the pH of the reducing reagent that is brought into contact with the antibody is about 6, 7, or 8. [16B] The method according to

[16] , wherein the pH of the reducing reagent that is brought into contact with the antibody is approximately 7. [16C] The method according to

[16] , wherein the pH of the reducing reagent that is brought into contact with the antibody is approximately 3.

[17] The method according to any one of [1] to [16B], wherein the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and Na2SO3. [17A] The method according to

[17] , wherein the reducing agent is TCEP.

[18] The method according to any one of

[17] to [17A], wherein the concentration of the reducing agent is approximately 0.01 mM to approximately 100 mM.

[19] The method according to

[18] , wherein the concentration of the reducing agent is about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 mM, preferably about 0.01 mM to 25 mM.

[20] The method according to any one of [1] to

[19] , wherein the contact step is carried out for at least 30 minutes. [20A] The method according to any one of [1] to

[19] , wherein the contact step is carried out over a period of approximately 2 to 48 hours. [20B] The method according to any one of [1] to

[19] , wherein the contact step is carried out for approximately 2 hours or approximately 16 hours.

[21] The method according to any one of [1] to [20B], wherein the contact step is carried out at a temperature of about 20°C to 37°C, preferably 23°C, 25°C, or 37°C, more preferably 23°C.

[22] The method according to any one of [1] to

[21] , wherein the antibody is at least partially purified before the step of contacting it with the reducing agent. [22A] The method according to

[22] , wherein the antibody is partially purified by affinity chromatography (preferably protein A chromatography) before contact.

[23] The method according to any one of [1] to

[22] , wherein the antibody concentration is approximately 1 mg / ml to approximately 50 mg / ml. [23A] The method according to

[23] , wherein the antibody concentration is approximately 1 mg / ml or approximately 20 mg / ml.

[24] The method according to any one of [1] to

[23] , further comprising isolating a fraction of a contact antibody having a desired three-dimensional structure. [24A] The method according to

[24] , wherein the method for isolation is selected from the group consisting of reversed-phase chromatography HPLC, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, dialysis, and electrophoresis. [24B] The method according to

[24] , wherein the method for isolation is ion exchange chromatography (IEC) and / or hydrophobic interaction chromatography (HIC). [24C] The method according to any one of [1] to [24B], further comprising the step of removing a reducing agent, preferably by dialysis, more preferably by chromatography.

[25] A preparation of IgG antibodies prepared by any one of the methods of [1] to [24B], comprising a homogeneous population of IgG antibodies having at least one disulfide bond in addition to the hinge region.

[26] A preparation of an IgG antibody prepared by any one of the methods of [1] to

[25] , comprising an IgG antibody having at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%, in molar ratios of at least 50%, 60%, 70%, 80%, 90% of which have at least one disulfide bond other than the hinge region.

[27] The preparation according to

[25] or

[26] , further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[28] A pharmaceutical composition comprising a homogeneous population of antibodies as defined in

[25] and a pharmaceutically acceptable carrier, excipient, or diluent.

[0017] In another aspect, the present invention also provides: [1] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more binding sites. [2] The antigen-binding molecule according to [1], wherein at least one of the bonds linking the two antigen-binding domains is a covalent bond. [3] The antigen-binding molecule described in [2], wherein a covalent bond is formed by direct crosslinking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. [4] The antigen-binding molecule described in [3], wherein the crosslinked amino acid residue is cysteine. [5] The antigen-binding molecule described in [4], wherein the covalent bond formed is a disulfide bond. [6] The antigen-binding molecule described in [2], wherein a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain via a crosslinking agent. [7] The antigen-binding molecule described in [6], wherein the crosslinking agent is an amine-reactive crosslinking agent. [8] The antigen-binding molecule described in [7], wherein the crosslinked amino acid residue is lysine. [9] The antigen-binding molecule according to [1], wherein at least one of the bonds linking the two antigen-binding domains is a non-covalent bond.

[10] The antigen-binding molecule described in [9], wherein the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond.

[11] The antigen-binding molecule described in

[10] , wherein an ionic bond is formed between an acidic amino acid and a basic amino acid.

[12] The antigen-binding molecule described in

[11] , wherein the acidic amino acid is aspartic acid (Asp) or glutamic acid (Glu), and the basic amino acid is histidine (His), lysine (Lys), or arginine (Arg).

[13] An antigen-binding molecule according to any one of [1] to

[12] , wherein at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is an artificially introduced mutant amino acid residue.

[14] The antigen-binding molecule according to

[13] , wherein the mutated amino acid residue is a cysteine ​​residue.

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

[14] , wherein at least one of the first and second antigen-binding domains has the activity to bind to an antigen on its own.

[16] An antigen-binding molecule described in any one of [1] to

[15] , wherein both the first and second antigen-binding domains are of the same type.

[17] An antigen-binding molecule according to any one of [1] to

[16] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking amino acid residues located at the same positions on the first antigen-binding domain and the second antigen-binding domain to each other.

[18] An antigen-binding molecule according to any one of [1] to

[16] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking amino acid residues located at different positions on the first antigen-binding domain and the second antigen-binding domain to each other.

[19] An antigen-binding molecule according to any one of [1] to

[18] , wherein at least one of the first and second antigen-binding domains contains an antibody fragment that binds to a specific antigen.

[20] The antigen-binding molecule described in

[19] , wherein the antibody fragment is Fab, Fab', scFab, Fv, scFv, or a single-domain antibody.

[21] An antigen-binding molecule according to

[19] or

[20] , wherein at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is present within the antibody fragment.

[22] The antigen-binding molecule according to

[21] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the constant region.

[23] The antigen-binding molecule described in

[22] , wherein the constant region is of human origin.

[24] The antigen-binding molecule according to

[22] or

[23] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the CH1 region.

[25] The antigen-binding molecule described in

[24] , wherein the subclass of the CH1 region is γ1, γ2, γ3, γ4, α1, α2, μ, δ, or ε.

[26] The antigen-binding molecule according to

[24] or

[25] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at one of the following positions in the EU numbering of the CH1 region: 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, or 218 to 219.

[27] The amino acid residues that serve as the starting point for binding between antigen-binding domains are located at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, and 167 in the CH1 region. An antigen-binding molecule as described in

[26] , located in any of the groups selected from the group consisting of 174th, 176th, 177th, 178th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 201st, 203rd, 205th, 206th, 207th, 208th, 211th, 212th, 213th, 214th, 218th, and 219th positions.

[28] The antigen-binding molecule according to

[27] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at EU numbering position 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 of the CH1 region.

[29] The antigen-binding molecule according to

[28] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at EU numbering position 135, 136, or 191 of the CH1 region.

[30] An antigen-binding molecule according to any one of

[24] to

[29] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CH1 region of the second antigen-binding domain.

[31] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123.

[32] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140.

[33] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 148, 149, and 150.

[34] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167.

[35] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178.

[36] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197.

[37] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.

[38] The antigen-binding molecule according to

[30] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 218 and 219.

[39] An antigen-binding molecule according to any one of

[30] to

[38] , wherein the difference in the positions of the amino acid residues in the first antigen-binding domain and the second antigen-binding domain is 3 amino acids or less.

[40] The antigen-binding molecule according to

[39] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residue at EU numbering position 135 in the CH1 region of the first antigen-binding domain with any one amino acid residue from EU numbering positions 132 to 138 in the CH1 region of the second antigen-binding domain.

[41] The antigen-binding molecule according to

[39] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain with any one amino acid residue at EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain.

[42] The antigen-binding molecule according to

[39] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with any one amino acid residue from EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain.

[43] The antigen-binding molecule according to

[40] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residues at EU numbering position 135 in the CH1 region of the two antigen-binding domains.

[44] The antigen-binding molecule according to

[41] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residues at EU numbering position 136 in the CH1 region of the two antigen-binding domains.

[45] The antigen-binding molecule according to

[42] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residues at EU numbering position 191 in the CH1 region of the two antigen-binding domains. [45A] The antigen-binding molecule described in

[42] , wherein a single disulfide bond is formed between amino acid residues at EU numbering position 191 in each CH1 region of the first antigen-binding domain and the second antigen-binding domain. [45B] The antigen-binding molecule described in [45A], wherein one, two or more additional disulfide bonds are formed between the first antigen-binding domain and the second antigen-binding domain via amino acid residues at the following positions according to EU numbering in each of the CH1 regions of the first and second antigen-binding domains: (a) Between amino acid residues at any of the positions 131-138, 194, and 195 in each of the two antigen-binding domains; (b) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (c) Between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (d) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (e) Between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (f) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (g) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (h) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (i) Between the amino acid residue at position 138 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (j) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (k) Between the amino acid residue at position 132 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (l) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (m) Between the amino acid residue at position 134 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (n) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (o) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (p) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; and (q) Between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains. [45C] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more inversely charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45D] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more negatively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45E] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more negatively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain contains one, two or more positively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45F] Either the first or second antigen-binding domain contains the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 is glutamic acid (E) or aspartic acid (D); (b) The amino acid residue at position 137 is glutamic acid (E) or aspartic acid (D); (c) The amino acid residue at position 138 is glutamic acid (E) or aspartic acid (D). It includes one, two or more of the following; and Of the two antigen-binding domains, the other antigen-binding domain in each CH1 region contains the following amino acid residues (according to EU numbering): (d) The amino acid residue at position 193 is lysine (K), arginine (R), or histidine (H); (e) The amino acid residue at position 194 is lysine (K), arginine (R), or histidine (H); and (f) The amino acid residue at position 195 is lysine (K), arginine (R), or histidine (H). An antigen-binding molecule as described in [45A] or [45B], comprising one, two, or more of the above. [45G-1] Either the first or second antigen-binding domain contains the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 is lysine (K), arginine (R), or histidine (H); (b) The amino acid residue at position 137 is lysine (K), arginine (R), or histidine (H); (c) The amino acid residue at position 138 is lysine (K), arginine (R), or histidine (H). Includes one or more of the following; and Of the two antigen-binding domains, the other antigen-binding domain in each CH1 region contains the following amino acid residues (according to EU numbering): (d) The amino acid residue at position 193 is glutamic acid (E) or aspartic acid (D); (e) The amino acid residue at position 194 is glutamic acid (E) or aspartic acid (D); and (f) The amino acid residue at position 195 is glutamic acid (E) or aspartic acid (D). An antigen-binding molecule as described in [45A] or [45B], comprising one or more of the above. [45G-2] An antigen-binding molecule as described in [45A] or [45B], wherein each of the first and second antigen-binding domains contains one of the specific combinations of charge mutations (according to EU numbering) in each CH1 region listed in Table 7, Table 82, or Table 85. [45H] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more hydrophobic amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more hydrophobic amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45I-1] The antigen-binding molecule according to [45H], wherein the hydrophobic amino acid residue is alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp). [45I-2] The method according to [45A] or [45B], wherein each of the first and second antigen-binding domains contains one of the specific combinations of hydrophobic amino acid mutations (according to EU numbering) in each CH1 region listed in Table 10. [45J] The antigen-binding molecule described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one "knob" amino acid residue at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain contains one, two or more "whole" amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45K] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more "whole" amino acid residues at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one "knob" amino acid residue at positions 193-195 (EU numbering) in each CH1 region. [45L] The antigen-binding molecule according to [45J] or [45K], wherein the "knob" amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "whole" amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (T), or serine (S). [45M] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more aromatic amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45N] An antigen-binding molecule as described in [45A] or [45B], wherein either the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain contains one, two or more aromatic amino acid residues at positions 193-195 (EU numbering) in each CH1 region. [45O] The antigen-binding molecule according to [45M] or [45N], wherein the aromatic amino acid residue is selected from the group consisting of tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe); and the positively charged amino acid residue is selected from the group consisting of lysine (K), arginine (R), or histidine (H).

[46] The antigen-binding molecule according to

[22] or

[23] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the CL region.

[47] The antigen-binding molecule described in

[46] , wherein the subclass of the CL region is κ or λ.

[48] ​​The antigen-binding molecule according to

[46] or

[47] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at one of the following positions in the CL region: Kabat numbering positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, or 208 to 213.

[49] The antigen-binding molecule according to

[48] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located in any of the group selected from the group consisting of Kabat numbering positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213 of the CL region.

[50] The antigen-binding molecule described in

[49] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at position 126 of the Kabat numbering in the CL region.

[51] An antigen-binding molecule according to any one of

[46] to

[50] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking an amino acid residue in the CL region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain.

[52] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 108, 109, 110, 111, and 112.

[53] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128.

[54] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbering positions 151, 152, 153, 154, 155, and 156.

[55] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 184, 185, 186, 187, 188, 189, and 190.

[56] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 195 and 196.

[57] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbering positions 200, 201, 202, and 203.

[58] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbering positions 208, 209, 210, 211, 212, and 213.

[59] An antigen-binding molecule according to any one of

[51] to

[58] , wherein the difference in the positions of the amino acid residues in the first antigen-binding domain and the second antigen-binding domain is 3 amino acids or less.

[60] The antigen-binding molecule according to

[59] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residues at Kabat numbering position 126 in the CL region of the two antigen-binding domains.

[61] An antigen-binding molecule according to any one of

[24] to

[29] and

[46] to

[50] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain.

[62] The antigen-binding molecule according to

[61] , wherein the amino acid residue in the CH1 region is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128.

[63] The antigen-binding molecule according to

[62] , wherein at least one of the bonds linking the two antigen-binding domains is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with the amino acid residue at Kabat numbering position 126 in the CL region of the second antigen-binding domain.

[64] The antigen-binding molecule according to

[21] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within a variable region.

[65] The antigen-binding molecule according to

[64] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the VH region.

[66] The antigen-binding molecule according to

[65] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located at any of the group selected from the Kabat numbering positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b of the VH region.

[67] The antigen-binding molecule according to

[64] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the VL region.

[68] The antigen-binding molecule according to

[67] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located in any of the group selected from the group consisting of Kabat numbering positions 21, 27, 58, 77, 100, 105, and 107 of the VL region (subclass κ).

[69] The antigen-binding molecule according to

[67] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located in one of the groups selected from the group consisting of Kabat numbering positions 6, 19, 33, and 34 of the VL region (subclass λ).

[70] The antigen-binding molecule according to

[64] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within the VHH region.

[71] The antigen-binding molecule according to

[70] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located in any of the group selected from the group consisting of Kabat numbering positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 of the VHH region.

[72] An antigen-binding molecule according to any one of [1] to

[18] , wherein at least one of the first and second antigen-binding domains comprises a non-antibody protein or a fragment thereof that binds to a specific antigen.

[73] The antigen-binding molecule described in

[72] , wherein a non-antibody protein is either a ligand or a receptor that specifically binds to each other.

[74] An antigen-binding molecule according to any one of [1] to

[73] , wherein the antigen-binding domain includes a hinge region.

[75] The antigen-binding molecule described in

[74] , wherein at least one of the cysteine ​​residues present in the hinge region of the wild type is replaced with another amino acid residue.

[76] The antigen-binding molecule according to

[75] , wherein the cysteine ​​residue is located at EU numbering positions 226 and / or 229 of the hinge region.

[77] An antigen-binding molecule as described in

[74] or

[76] , wherein at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the hinge region.

[78] The antigen-binding molecule according to

[77] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located in any of the group selected from the group consisting of EU numbering positions 216, 218, and 219 of the hinge region.

[79] An antigen-binding molecule according to any one of [1] to

[78] , wherein the first antigen-binding domain and the second antigen-binding domain are linked to each other by two or more binding sites.

[80] The antigen-binding molecule described in

[79] , wherein at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is an amino acid residue present in the wild-type sequence.

[81] The antigen-binding molecule according to

[80] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is located within a hinge region.

[82] The antigen-binding molecule according to

[81] , wherein the amino acid residue that serves as the starting point for binding between antigen-binding domains is a cysteine ​​residue in the hinge region.

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

[80] to

[82] , wherein at least one of the bonds linking the two antigen-binding domains is a disulfide bond formed by cross-linking of cysteine ​​residues located within the hinge region.

[84] The antigen-binding molecule according to

[83] , wherein the cysteine ​​residue is located at EU numbering positions 226 and / or 229 of the hinge region.

[85] An antigen-binding molecule according to any one of

[79] to

[84] , wherein at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the antibody fragment and at least one is located within the hinge region.

[86] The antigen-binding molecule according to

[85] , wherein the first and second antigen-binding domains each include a Fab and a hinge region, and the antigen-binding molecule containing the two antigen-binding domains is F(ab')2.

[87] An antigen-binding molecule described in any one of [1] to

[86] , wherein the antigen-binding domain includes an Fc region.

[88] An antigen-binding molecule as described in

[87] , wherein one or more amino acid mutations that promote multimerization of the Fc region are introduced into the Fc region.

[89] The antigen-binding molecule according to

[88] , wherein the amino acid mutation that promotes multimerization is an amino acid mutation at at least one position selected from the group consisting of EU numbering positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447.

[90] The antigen-binding molecule described in

[88] or

[89] , wherein the polymerization is hexamerization.

[91] An antigen-binding molecule described in any one of

[87] to

[90] , which is a full-length antibody.

[0018] In another aspect, the present invention also provides:

[92] An antigen-binding molecule described in any one of [1] to

[91] , wherein both the first and second antigen-binding domains bind to the same antigen.

[93] The antigen-binding molecule according to

[92] , wherein the first and second antigen-binding domains both bind to the same epitope on the antigen.

[94] The antigen-binding molecule according to

[92] , wherein each of the first and second antigen-binding domains binds to a different epitope on the antigen.

[95] An antigen-binding molecule according to any one of [1] to

[91] , wherein each of the first and second antigen-binding domains binds to a different antigen.

[96] The antigen-binding molecule described in

[93] , wherein both the first and second antigen-binding domains have the same amino acid sequence.

[97] An antigen-binding molecule according to any one of

[93] to

[95] , wherein each of the first and second antigen-binding domains has a different amino acid sequence.

[98] An antigen-binding molecule according to any one of [1] to

[91] , wherein at least one of the two antigens to which the first and second antigen-binding domains bind is a soluble protein.

[99] An antigen-binding molecule according to any one of [1] to

[91] , wherein at least one of the two antigens to which the first and second antigen-binding domains bind is a membrane protein. In another aspect, the present invention also provides:

[100] An antigen-binding molecule described in any one of [1] to

[99] , which has the activity to control the interaction between two antigen molecules.

[101] An antigen-binding molecule according to

[100] that can enhance or reduce the interaction between two antigen molecules compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule according to

[100] only in that the number of bonds between the two antigen-binding domains is one less.

[102] The antigen-binding molecule according to

[100] or

[101] , wherein the two antigen molecules are a ligand and its receptor, and the molecule has the activity to promote the activation of the receptor by the ligand.

[103] The antigen-binding molecule according to

[100] or

[101] , wherein the two antigen molecules are an enzyme and its substrate, and the antigen-binding molecule has the activity to promote the catalytic reaction of the enzyme with respect to the substrate.

[104] The antigen-binding molecule according to

[100] or

[101] , wherein both antigen molecules are proteins present on the cell surface, and the antigen-binding molecule has the activity to promote interaction between a cell expressing a first antigen and a cell expressing a second antigen.

[105] The antigen-binding molecule according to

[104] , wherein the cell expressing the first antigen is a cell having cytotoxic activity, the cell expressing the second antigen is its target cell, and the antigen-binding molecule promotes damage to the target cell by the cell having cytotoxic activity.

[106] The antigen-binding molecule described in

[105] , wherein the cytotoxic cell is a T cell, NK cell, monocyte, or macrophage.

[107] An antigen-binding molecule according to any one of [1] to

[99] , having the activity to control the activation of two antigen molecules that are activated by association with each other.

[108] An antigen-binding molecule as described in

[107] , which enhances or reduces the activation of two antigen molecules compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule described in

[107] only in that there is one fewer bond between the two antigen-binding domains.

[109] An antigen-binding molecule according to

[107] or

[108] , wherein the antigen molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-bound receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[110] An antigen-binding molecule according to any one of [1] to

[99] , which has the activity of holding two antigen molecules in spatially close proximity.

[111] An antigen-binding molecule according to

[110] , which can hold two antigen molecules in closer proximity to each other than a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule according to

[110] only in that there is one fewer bond between the two antigen-binding domains.

[112] An antigen-binding molecule according to any one of [1] to

[99] , wherein the two antigen-binding domains are located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced.

[113] An antigen-binding molecule according to

[112] , wherein the two antigen-binding domains are located in closer proximity to each other and / or the mobility of the two antigen-binding domains is reduced compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule according to

[112] only in that there is one fewer bond between the two antigen-binding domains.

[114] An antigen-binding molecule described in any one of [1] to

[99] that is resistant to protease cleavage.

[115] An antigen-binding molecule as described in

[114] , wherein the antigen-binding molecule has increased resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differs from the antigen-binding molecule described in

[114] only in that there is one fewer bond between the two antigen-binding domains.

[116] The antigen-binding molecule according to

[115] , wherein the proportion of full-length molecules remaining after protease treatment is increased compared to the control antigen-binding molecule.

[117] The antigen-binding molecule according to

[115] or

[116] , wherein the proportion of a specific fragment generated after protease treatment is reduced compared to the control antigen-binding molecule.

[118] An antigen-binding molecule described in any one of [1] to

[99] , wherein, when treated with a protease, a dimer of the antigen-binding domain or a fragment thereof is cleaved.

[119] An antigen-binding molecule as described in

[118] , wherein when a control antigen-binding molecule is treated with the protease, monomers of the antigen-binding domain or a fragment thereof are cleaved, wherein the control antigen-binding molecule differs from the antigen-binding molecule described in

[118] only in that there is one fewer bond between the two antigen-binding domains.

[120] An antigen-binding molecule as described in

[118] or

[119] , wherein the protease cleaves the hinge region.

[121] An antigen-binding molecule described in any one of the following:

[101] to

[106] ,

[108] to

[109] ,

[111] ,

[113] ,

[115] to

[117] , or

[119] to

[120] , wherein the bond is formed at one less site than the mutated amino acid residue.

[122] The antigen-binding molecule described in

[121] , wherein the mutated amino acid residue is a cysteine ​​residue.

[0019] In another aspect, the present invention also provides:

[123] A pharmaceutical composition comprising an antigen-binding molecule described in any one of [1] to

[122] and a pharmaceutically acceptable carrier.

[0020] In another aspect, the present invention also provides:

[124] A method for controlling the interaction between two antigen molecules, comprising: (a) To provide an antigen-binding molecule containing two antigen-binding domains, (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together, (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules.

[125] A method for controlling the activity of two antigen molecules that are activated by association with each other, comprising: (a) To provide an antigen-binding molecule containing two antigen-binding domains, (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together, (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules.

[126] A method for holding two antigen molecules in spatially close proximity, comprising: (a) To provide an antigen-binding molecule containing two antigen-binding domains, (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together, (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules.

[127] A method for positioning two antigen-binding domains in spatially close proximity and / or reducing the mobility of two antigen-binding domains, comprising: (a) To provide an antigen-binding molecule containing two antigen-binding domains, and (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together.

[128] A method for increasing the resistance of an antigen-binding molecule to protease cleavage, comprising: (a) To provide an antigen-binding molecule containing two antigen-binding domains, and (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together.

[0021] In another aspect, the present invention also provides:

[129] A method for producing an antigen-binding molecule having activity to control the interaction between two antigen molecules, comprising: (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain, (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides express, and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites.

[130] A method for producing an antigen-binding molecule having activity to control the activation of two antigen molecules that are activated by association with each other, comprising: (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain, (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides express, and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites.

[131] A method for producing an antigen-binding molecule having the activity of holding two antigen molecules in spatially close proximity, comprising: (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain, (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides express, and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites.

[132] A method for producing an antigen-binding molecule in which two antigen-binding domains are located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced, comprising: (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain, (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides express, and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites.

[133] A method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain, (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides express, and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites.

[0022] In another aspect, the present invention also provides:

[134] A method for identifying a novel set of protein molecules that are activated by association with each other, comprising: (a) To provide any two protein molecules, (b) To produce an antigen-binding molecule comprising two antigen-binding domains that bind to the two protein molecules, respectively, by the method described in any one of

[129] to

[133] , (c) Contacting the antigen-binding molecule produced in (b) with the two protein molecules, and (d) Evaluate whether the two protein molecules are activated.

[135] The method according to

[134] , wherein at least one protein molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-bound receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows unreduced SDS-PAGE gel images for analyzing OKT3 and its variant with cysteine ​​substitution (see Example 1). The two dashed lines indicate the upper and lower bands. The lower band can be considered to correspond to antibodies having one or more manipulated disulfide bonds formed between CH1 regions. [Figure 2] Figure 2 shows unreduced SDS-PAGE gel images for analyzing OKT3 variants with cysteine ​​substitution and OKT3-KiH (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 3] Figure 3 shows an unreduced SDS-PAGE gel image for analyzing the OKT3-KiH variant with cysteine ​​substitution (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 4] Figure 4 shows an unreduced SDS-PAGE gel image for analyzing the OKT3-KiH variant with cysteine ​​substitution (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 5]Figure 5 shows images of the unreduced SDS-PAGE gels (left panel) with the 2-MEA concentrations for each sample indicated, and graphs (right panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 4). 20 mg / mL of antibody was reacted by mixing with 2-MEA at various concentrations. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM 2-MEA). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 6] Figure 6 shows images of the unreduced SDS-PAGE gels (upper panel) with the 2-MEA concentrations for each sample indicated, and graphs (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 4). 20 mg / mL of antibody was reacted by mixing with 2-MEA at various concentrations. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM 2-MEA). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 7] Figure 7 shows images of the unreduced SDS-PAGE gels (left panel) with the 2-MEA concentrations for each sample indicated, and graphs (right panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 4). 1 mg / mL of antibody was reacted by mixing with 2-MEA at various concentrations. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM 2-MEA). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 8]Figure 8 shows images of the unreduced SDS-PAGE gels (upper panel) with the 2-MEA concentrations for each sample indicated, and graphs (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 4). 1 mg / mL of antibody was reacted by mixing with 2-MEA at various concentrations. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM 2-MEA). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 9] Figure 9 shows images of the unreduced SDS-PAGE gels (left panel) with the TCEP concentrations for each sample indicated, and graphs (right panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 5). 20 mg / mL of antibody was reacted by mixing with various concentrations of TCEP. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM TCEP). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 10] Figure 10 shows images of the unreduced SDS-PAGE gels (upper panel) with the TCEP concentrations for each sample indicated, and graphs (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 5). 20 mg / mL of antibody was reacted by mixing with each concentration of TCEP. ND means no bands were detected. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM TCEP). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 11]Figure 11 shows images of the unreduced SDS-PAGE gels (upper panel) with the TCEP concentrations for each sample indicated, and graphs (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for each sample (see Example 5). 1 mg / mL of antibody was reacted by mixing with each concentration of TCEP. ND means no bands were detected. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (0 mM TCEP). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 12] Figure 12 shows images of the unreduced SDS-PAGE gels (top panel) with reagent concentrations for each sample; and graphs showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for samples reacted with DTT (left) or cysteine ​​(right) (see Example 6). 20 mg / mL of antibody was reacted by mixing with each concentration of DTT or cysteine. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) for the control (no reducing agent). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 13] Figure 13 shows images of the unreduced SDS-PAGE gels (upper panel) with reagent concentrations for each sample; and graphs (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) of the samples reacted with GSH (left) or Na2SO3 (right) (see Example 6). 20 mg / mL of antibody was reacted by mixing with each concentration of GSH or Na2SO3. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (crosslinking rate or crosslinking %) of the control (no reducing agent). The numbers within the bars are the values ​​for the ratio of the lower band to the upper band (crosslinking rate or crosslinking %). [Figure 14]Figure 14 shows images of the unreduced SDS-PAGE gel (see Example 7). 20 mg / mL of antibody was reacted by mixing with 2-MEA or TCEP under conditions of pH 3, 4, and 5. The buffer pH for each sample is indicated in the figure. Lanes 3, 6, and 9: No reducing agent. Lanes 4, 7, and 10: Mixed with 1 mM 2-MEA. Lanes 5, 8, and 11: Mixed with 0.25 mM TCEP. [Figure 15] Figure 15 shows images of the unreduced SDS-PAGE gel (see Example 7). 20 mg / mL of antibody was reacted by mixing with 2-MEA or TCEP under conditions of pH 6, 7, and 8. The buffer pH for each sample is indicated in the figure. Lanes 3, 6, and 9: No reducing agent. Lanes 4, 7, and 10: Mixed with 1 mM 2-MEA. Lanes 5, 8, and 11: Mixed with 0.25 mM TCEP. [Figure 16] Figure 16 is a graph showing the ratio of the lower band to the upper band (crosslinking rate) of the antibody samples in Figures 14 and 15 (see Example 7). For each pH, ​​the leftmost (white) bar represents the ratio of the lower band to the upper band (crosslinking rate) of the control (no reducing agent treatment). The middle (shaded) bar represents the ratio of the lower band to the upper band (crosslinking rate) of the sample mixed with 1 mM 2-MEA. The rightmost (black) bar represents the ratio of the upper band to the lower band (crosslinking rate) of the sample mixed with 0.25 mM TCEP. The numbers within the bars represent the ratio of the lower band to the upper band (crosslinking rate). [Figure 17] Figure 17 shows the chromatogram of cation exchange chromatography performed on the OKT3.S191C antibody sample as described in Example 8-1. [Figure 18]Figure 18 shows gel images of unreduced SDS-PAGE analysis of OKT3.S191C antibody samples separated by cation exchange chromatography as described in Example 8-1. Lanes 5 and 10: OKT3.S191C (unfractionated). Lane 6: Mixture of RA3 and RA4. Lane 7: Mixture of RA5 and RA6. Lane 8: Mixture of RA7 and RA8. Lane 9: Mixture of RA9 and RA10. [Figure 19] Figure 19 shows the chromatogram of cation exchange chromatography performed on the OKT3.S191C0110 antibody sample as described in Example 8-2. [Figure 20] Figure 20 shows gel images of the analysis of unreduced SDS-PAGE of OKT3.S191C0110 antibody samples separated by cation exchange chromatography as described in Example 8-2. Lane 3: OKT3.S191C0110 (unfractionated). Lane 4: Mixture of RA4 and RA5. Lane 5: Mixture of RA6 and RA7. Lane 6: Mixture of RA8 and RA9. Lane 7: Mixture of RA10 and RA11. Lane 8: Mixture of RB11 and RB10. Lane 9: Mixture of RB8 and RB7. Lane 10: Mixture of RB6 and RB5. Lane 11: Mixture of RB4 and RB3. [Figure 21] Figure 21 shows an example of a modified antibody in which Fabs are crosslinked, as described in Reference Example 1. Here, the structural differences between the wild-type antibody (WT), a modified antibody in which the CH1 regions of the antibody H chain are crosslinked (HH type), a modified antibody in which the CL regions of the antibody L chain are crosslinked (LL type), and a modified antibody in which the CH1 region of the antibody H chain and the CL region of the antibody L chain are crosslinked (HL type or LH type) are schematically shown. [Figure 22] Figure 22 shows the results of measuring the CD3-mediated agonist activity of a wild-type anti-CD3ε antibody molecule (CD3-G4s) and modified antibody molecules (CD3-G4sLL, CD3-G4sHH) prepared by linking the Fab-Fab bonds of the wild-type anti-CD3ε antibody molecule (CD3-G4s) with additional disulfide bonds, as described in Reference Example 4-3. [Figure 23] Figure 23 shows the results of measuring the CD3-mediated agonist activity of wild-type anti-CD3ε antibody molecules (OKT3-G1s) and modified antibody molecules (OKT3-G1sLL, OKT3-G1sHH) prepared by linking the Fab-Fab bonds of the wild-type anti-CD3ε antibody molecule (OKT3-G1s) with additional disulfide bonds, as described in Reference Example 4-3. [Figure 24] Figure 24 shows the results of measuring the CD3 and / or CD28-mediated agonist activity for wild-type anti-CD3ε antibody molecules (CD3-G1s), anti-CD28 antibody molecules (CD28-G1s), anti-CD3ε × anti-CD28 bispecific antibody (CD3 / / CD28-G1s), and modified antibody molecules (CD3 / / CD28-G1sLL, CD3 / / CD28-G1sHH, CD3 / / CD28-G1sLH, CD3 / / CD28-G1sHL) prepared by linking the Fab-Fab of the bispecific antibody with additional disulfide bonds, as described in Reference Example 4-3. [Figure 25] Figure 25 shows the results of measuring the CD3 and / or CD28-mediated agonist activity for wild-type anti-CD3ε antibody molecules (OKT3-G1s), anti-CD28 antibody molecules (CD28-G1s), anti-CD3ε × anti-CD28 bispecific antibody (OKT3 / / CD28-G1s), and modified antibody molecules (OKT3 / / CD28-G1sHH, OKT3 / / CD28-G1sHL) prepared by linking the Fab-Fab of the bispecific antibody with an additional disulfide bond, as described in Reference Example 4-3. [Figure 26] Figure 26 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (1 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 27] Figure 27 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of anti-IL6R antibody, as described in Reference Example 5-2 (2 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 28] Figure 28 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (3 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 29] Figure 29 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (4 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 30]Figure 30 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (5 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 31] Figure 31 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (6 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 32] Figure 32 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (7 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 33]Figure 33 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody, and modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (8 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 34] Figure 34 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (1 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 35] Figure 35 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (2 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 36]Figure 36 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (3 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 37] Figure 37 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (4 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 38] Figure 38 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (5 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 39]Figure 39 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (6 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 40] Figure 40 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (7 / 10). Each antibody treated with protease was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 41] Figure 41 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (8 / 10). Each antibody treated with protease was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 42]Figure 42 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (9 / 10). Each antibody treated with protease was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 43] Figure 43 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody, and modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 6-2 (10 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using an anti-kappa chain antibody. [Figure 44] Figure 44 shows the results of protease treatment of anti-IL6R antibody (MRA) and a modified antibody (MRAL-k0.K126C) prepared by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 7-2. Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection using anti-kappa chain antibody or anti-human Fc antibody. [Figure 45] Figure 45 shows the correspondence between the molecular weight of each band obtained as a result of protease treatment of the antibody sample, as described in Reference Example 7-2, and its assumed structure. Below the structure of each molecule, it is also indicated whether the molecule can react with anti-kappa chain antibody or anti-Fc antibody (whether the band is detected in the electrophoresis in Figure 44). [Figure 46]Figure 46 shows the results of measuring the CD3-mediated agonist activity of an anti-CD3 antibody molecule (OKT3), modified antibody molecules (H_T135C, H_S136C, H_S191C, L_K126C) prepared by linking the Fab-Fab segments of the anti-CD3 antibody molecule (OKT3) with additional disulfide bonds, and an anti-KLH antibody molecule (IC17) (negative control), as described in Reference Example 13-4. [Figure 47] Figure 47 shows the results of measuring CD3-mediated agonist activity for the following: an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (OKT3_KiH) produced by introducing a Knobs-into-Holes (KiH) modification that promotes heterodimerization into the heavy chain constant region of OKT3, modified antibody molecules (H_S191C_KiH, H_S191C / V188C_KiH, H_S191C / P189C_KiH, H_S191C / S190C_KiH, H_S191C / S192C_KiH, H_S191C / L193C_KiH, H_S191C / G194C_KiH) produced by linking the Fab-Fab of the modified antibody molecules with additional disulfide bonds, as described in Reference Example 14-4, and an anti-KLH antibody (IC17) (negative control). [Figure 48]Figure 48 shows the following modified antibody molecules, as described in Reference Example 15-4: an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (H_S191C) produced by linking its Fab-Fab with an additional disulfide bond, a modified antibody molecule (OKT3_KiH) produced by introducing a Knobs-into-Holes (KiH) modification that promotes heterodimerization into the heavy chain constant region of OKT3, a modified antibody molecule (H_S191C_KiH) produced by linking its Fab-Fab with an additional disulfide bond, and a piece of OKT3_KiH. This figure shows the results of measuring CD3-mediated agonist activity for modified antibody molecules (0004 / / 0004, 0004 / / 0006) prepared by introducing a positively charged amino acid substitution in one heavy chain constant region and a negatively charged amino acid substitution in the other heavy chain constant region, modified antibody molecules (0004 / / OKT3, OKT3 / / 0004, OKT3 / / 0006) prepared by introducing a positively charged or negatively charged amino acid substitution in one heavy chain constant region of OKT3_KiH, and the anti-KLH antibody molecule (IC17) (negative control). [Figure 49] Figure 49 shows the results of measuring the CD3-mediated agonist activity of the anti-CD3 antibody molecule (OKT3), modified antibody molecules (dh1, dh2, dh3) prepared by removing the disulfide bond in the hinge region of the anti-CD3 antibody molecule (OKT3), modified antibody molecules (H_S191C_dh1, H_S191C_dh2, H_S191C_dh3) prepared by linking the Fab-Fab regions of these molecules with additional disulfide bonds, and the anti-KLH antibody molecule (IC17) (negative control), as described in Reference Example 16-4. [Figure 50]Figure 50 shows the results of measuring CD3-mediated agonist activity for the following, as described in Reference Example 20: anti-CD3 monospecific antibody molecule (OKT3-G1s), modified antibody molecule (OKT3-G1sHH) produced by linking its Fab-Fab with an additional disulfide bond, modified antibody molecule (CD3-G1sLL) produced by linking the Fab-Fab of the anti-CD3 monospecific antibody (CD3-G1s) with an additional disulfide bond, anti-CD3 biparatopic antibody molecule (CD3 / / OKT3-G1s), modified antibody molecules (CD3 / / OKT3-G1sHH, CD3 / / OKT3-G1sLH) produced by linking its Fab-Fab with an additional disulfide bond, and a combination of CD3-G1sLL and OKT3-G1s (CD3-G1sLL+OKT3-G1s). [Figure 51A] Figure 51A shows the results of measuring the agonist activity via CD3 and / or PD1 for an anti-CD3 × anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fab with an additional disulfide bond, as described in Reference Example 22-1. Figure 51A shows the agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 117-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (117), and modified antibody molecules (OKT3 / / 117-G1silentHH, OKT3 / / 117-G1silentHL, OKT3 / / 117-G1silentLL) prepared by linking its Fab-Fab with an additional disulfide bond. [Figure 51B]Figure 51B shows the results of measuring the agonist activity via CD3 and / or PD1 for an anti-CD3 × anti-PD1 bispecific antibody and a modified antibody molecule prepared by linking its Fab-Fab with an additional disulfide bond, as described in Reference Example 22-1. Figure 51B shows the agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 10-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (10), and modified antibody molecules (OKT3 / / 10-G1silentHH, OKT3 / / 10-G1silentHL) prepared by linking its Fab-Fab with an additional disulfide bond. [Figure 51C] Figure 51C shows the results of measuring the agonist activity via CD3 and / or PD1 for an anti-CD3 × anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fab with an additional disulfide bond, as described in Reference Example 22-1. Figure 51C shows the agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (CD3 / / 949-G1silent) composed of an anti-CD3 antibody (CD3) and an anti-PD1 antibody (949), and modified antibody molecules (CD3 / / 949-G1silentLH, CD3 / / 949-G1silentHH, CD3 / / 949-G1silentLL, CD3 / / 949-G1silentHL) prepared by linking its Fab-Fab with an additional disulfide bond. [Figure 51D]Figure 51D shows the results of measuring the agonist activity via CD3 and / or PD1 for an anti-CD3 × anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fab with an additional disulfide bond, as described in Reference Example 22-1. Figure 51D shows the agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), and modified antibody molecules (OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentLL) prepared by linking its Fab-Fab with an additional disulfide bond. [Figure 52] Figure 52 shows the results of measuring the CD3 and / or PD1-mediated agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), as described in Reference Example 22-2, as well as modified antibody molecules (OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentLH, OKT3 / / 949-G1silentLL) prepared by linking the Fab-Fab bonds of the anti-CD3 × anti-PD1 antibody (OKT3 / / 949-G1silent), and OKT3 / / 949-G1silentLL. [Figure 53A]Figure 53A shows the results of evaluating the T cell-dependent inhibitory effect on cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-1. When the above-mentioned CD28 / CD3 clamping bispecific antibody and GPC3 / attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells), the GPC3 / attenuated CD3 bispecific antibody brings the target cells and effector cells into close proximity, while the CD28 / CD3 clamping bispecific antibody activates the effector cells. Figure 53A shows the inhibitory effects on cancer cell proliferation when using the GPC3 / binding attenuation CD3 bispecific antibody molecule (GPC3 / attCE115) as an antibody to target T cells to cancer cells, and the GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), the KLH / CD3 clamping bispecific antibody molecule (KLH / clamp CD3), the CD28 / CD3 clamping bispecific antibody molecule (CD28 / clamp CD3), or a modified antibody molecule (CD28 / clamp CD3_HH) created by linking the Fab-Fab of these molecules with additional disulfide bonds, respectively, as antibodies to activate T cells. [Figure 53B]Figure 53B, similar to Figure 53A, shows the results of evaluating the T cell-dependent inhibitory effect on cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuation CD3 bispecific antibody are used in combination, as described in Reference Example 23-1. Figure 53B shows the inhibitory effects on cancer cell proliferation when using a modified antibody molecule (GPC3 / attCE115_LL) created by linking the Fab-Fab of a GPC3 / binding-attenuated CD3 bispecific antibody with an additional disulfide bond as an antibody for targeting T cells to cancer cells, and when using a GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), a KLH / CD3 clamping bispecific antibody molecule (KLH / clamp CD3), a CD28 / CD3 clamping bispecific antibody molecule (CD28 / clamp CD3), and a modified antibody molecule (CD28 / clamp CD3_HH) created by linking the Fab-Fab of these molecules with an additional disulfide bond as antibodies for activating T cells. [Figure 54A] Figure 54A shows the results of evaluating cytokine production from T cells when a CD28 / CD3 clamping bispecific antibody and a GPC3 / attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-2. When the above-mentioned CD28 / CD3 clamping bispecific antibody and GPC3 / attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells), the GPC3 / attenuated CD3 bispecific antibody brings the target cells and effector cells into close proximity, while the CD28 / CD3 clamping bispecific antibody activates the effector cells. Figure 54A shows the amount of IL-6 produced when using a GPC3 / attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) created by linking the Fab-Fab of a CD28 / CD3 clamping bispecific antibody with an additional disulfide bond, either alone or in combination, in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 54B] Figure 54B, similar to Figure 54A, shows the results of evaluating cytokine production from T cells when a CD28 / CD3 clamping bispecific antibody and a GPC3 / attenuated CD3 bispecific antibody were used in combination, as described in Reference Example 23-2. Figure 54B shows the amount of IL-6 produced when the GPC3 / attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) created by linking the Fab-Fab of the CD28 / CD3 clamping bispecific antibody with an additional disulfide bond were used individually or in combination, in the presence of effector cells (T cells) alone. [Figure 54C] Figure 54C, similar to Figure 54A, shows the results of evaluating cytokine production from T cells when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-2. Figure 54C shows the cancer cell proliferation inhibitory effect when the GPC3 / binding attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) created by linking the Fab-Fab of the CD28 / CD3 clamping bispecific antibody with an additional disulfide bond are used individually or in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 55A] Figure 55A is a schematic diagram illustrating the mechanism of action of T cell-dependent inhibition of cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-1 (where "ε" represents CD3ε). Figure 55A shows the mechanism of action of inhibition of cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 55B]Figure 55B is a schematic diagram illustrating the mechanism of action of T cell-dependent inhibition of cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-1 (ε in the figure represents CD3ε). Figure 55B shows the mechanism of action of inhibition of cancer cell proliferation when a modified antibody molecule, which has been modified to introduce an additional disulfide bond between the Fab-Fab of the CD28 / CD3 clamping bispecific antibody, and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 56A] Figure 56A is a schematic diagram showing the mechanism of cytokine production from T cells when a CD28 / CD3 clamping bispecific antibody and a GPC3 / attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-2 (ε in the figure represents CD3ε). Figure 56A shows the mechanism of cytokine production when a modified antibody molecule, which has been modified to introduce an additional disulfide bond between the Fab-Fab of the CD28 / CD3 clamping bispecific antibody, and a GPC3 / attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 56B] Figure 56B is a schematic diagram showing the mechanism of cytokine production from T cells when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuation CD3 bispecific antibody are used in combination, as described in Reference Example 23-2 (ε in the figure represents CD3ε). Figure 56B shows the mechanism of cytokine production when a modified antibody molecule, which has been modified to introduce an additional disulfide bond between the Fab-Fab of the CD28 / CD3 clamping bispecific antibody, is used in combination with a GPC3 / binding attenuation CD3 bispecific antibody in the presence of effector cells (T cells) alone. [Figure 57A]Figure 57A shows the results of measuring the agonist activity of the CD8 / CD28 bispecific antibody molecule (CD8 / CD28-P587) and modified antibody molecules (CD8 / CD28-P587(HH), CD8 / CD28-P587(LL), CD8 / CD28-P587(HL), CD8 / CD28-P587(LH)) prepared by linking the Fab-Fab bonds with additional disulfide bonds, as described in Reference Example 24. The anti-KLH antibody molecule (KLH-P587) was used as a negative control. The results shown are obtained using peripheral blood mononuclear cells (PBMCs) from two different donors (top: Donor A, bottom: Donor B). Figure 57A shows the percentage of divided regulatory T (Treg) cells contained in the PBMCs. [Figure 57B] Figure 57B shows the results of measuring the agonist activity of the CD8 / CD28 bispecific antibody molecule (CD8 / CD28-P587) and modified antibody molecules (CD8 / CD28-P587(HH), CD8 / CD28-P587(LL), CD8 / CD28-P587(HL), CD8 / CD28-P587(LH)) prepared by linking the Fab-Fab of the antibody via additional disulfide bonds, as described in Reference Example 24. Figure 57B also shows the percentage of divided CD8α-positive T cells in PBMCs. [Figure 58] Figure 58 shows a chromatogram obtained by cation exchange chromatography (CIEX) on an antibody sample of an OKT3 variant having a charged amino acid substitution, as described in Example 9-3. [Figure 59] Figure 59 shows chromatograms obtained by cation exchange chromatography (CIEX) on antibody samples of OKT3 variants having charged amino acid substitutions, as described in Examples 2-2 and 9-3. [Figure 60]Figure 60 is a scatter plot of the ratio of the lower band to the upper band (unreduced SDS-PAGE gel image) of the OKT3 and MRA antibody variants prepared in Example 10-1. The Y-axis represents the ratio of the lower band to the upper band of the MRA variant sample shown in Table 87, and the X-axis represents the ratio of the lower band to the upper band of the OKT3 variant sample shown in Table 87. [Figure 61A] Figure 61A shows a chromatogram obtained by cation exchange chromatography (CIEX) on an antibody sample of an OKT3 variant having a charged amino acid substitution, as described in Example 10-3. [Figure 61B] Figure 61B shows a chromatogram obtained by cation exchange chromatography (CIEX) on an antibody sample of an MRA variant having charged amino acid substitutions, as described in Example 10-3. [Figure 62A] Figure 62A is a schematic diagram illustrating the effect of additional amino acid mutations on enhancing Fab crosslinking of manipulated disulfide bonds. (Left) The G1T4.S191C variant, having a cysteine ​​substitution, for example, at S191C (EU numbering) of CH1, contains a mixture of crosslinking and non-crosslinking antibodies. (Center) The G1T4.S191C variant containing an additional amino acid mutation X (where X may be a charged amino acid, a hydrophobic amino acid, or a knob-hole amino acid) shows a higher proportion of crosslinking antibody. (Right) The amino acid position (EU numbering) at the CH1-CH1 interface where the additional amino acid mutation X (where X may be a charged amino acid, a hydrophobic amino acid, or a knob-hole amino acid) can promote crosslinking of manipulated disulfide bonds. [Figure 62B] Figure 62B is a schematic diagram showing the effect of additional mutations on the separation of crosslinked and non-crosslinked Fab by chromatography methods such as CIEX. [Modes for carrying out the invention]

[0024] I. Definition In this specification, the term “antigen-binding molecule” refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In another embodiment, the antigen-binding molecule is a non-antibody protein, a fragment thereof, or a derivative thereof.

[0025] In this specification, “antigen-binding domain” means a region that specifically binds to and is complementary to a part or all of an antigen. In this specification, an antigen-binding molecule includes an antigen-binding domain. When the molecular weight of the antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. This specific portion is called an epitope. In one embodiment, the antigen-binding domain includes an antibody fragment that binds to a specific antigen. The antigen-binding domain may be provided from one or more antibody variable domains. In one non-limiting embodiment, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include “scFv (single chain Fv)”, “single chain antibody”, “Fv”, “scFv2 (single chain Fv 2)”, “Fab”, and “Fab'”. In another embodiment, the antigen-binding domain includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the antigen-binding domain includes a hinge region.

[0026] In this specification, "specifically binds" means that one of the molecules involved in specific binding binds without showing any significant binding to any molecule other than its one or more binding partner molecules. This expression is also used when the antigen-binding domain is specific to a particular epitope among several epitopes contained in a given antigen. If the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens containing the epitope.

[0027] In this disclosure, "binding to the same epitope" means that the epitopes to which the two antigen-binding domains bind overlap to at least a portion. The degree of overlap is not limited to but is at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, particularly preferably 90%, and most preferably 100%. In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0028] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any variant antibodies that may arise (e.g., variant antibodies containing naturally occurring mutations, or variant antibodies that arise during the manufacture of the monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0029] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa and lambda.

[0030] The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0031] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0032] In one embodiment of the present invention, the constant region is preferably an antibody constant region, more preferably an antibody constant region of type IgG1, IgG2, IgG3, and IgG4, and even more preferably a human antibody constant region of type IgG1, IgG2, IgG3, and IgG4. In another embodiment of the present invention, the constant region is preferably a heavy chain constant region, more preferably an heavy chain constant region of type IgG1, IgG2, IgG3, and IgG4, and even more preferably a human heavy chain constant region of type IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the human IgG1, human IgG2, human IgG3, and human IgG4 constant regions are known. Multiple allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of them can be used in the present invention. The amino acid-modified constant region of the present invention may include other amino acid mutations or modifications, as long as it includes the amino acid mutation of the present invention.

[0033] The term "hinge region" refers to the antibody heavy chain polypeptide portion that links the CH1 and CH2 domains in the wild-type antibody heavy chain, for example, from around position 216 to 230 in the EU numbering system, or from around position 226 to 243 in the Kabat numbering system. In natural-type IgG antibodies, it is known that the cysteine ​​residue at EU numbering position 220 in the hinge region forms a disulfide bond with the cysteine ​​residue at position 214 in the antibody light chain. Furthermore, it is known that disulfide bonds are formed between the cysteine ​​residues at EU numbering position 226 and between the cysteine ​​residues at position 229 in the hinge region of two antibody heavy chains. Generally, the "hinge region" is defined as extending from position 216 to 238 (EU numbering) or from position 226 to 251 (Kabat numbering) in human IgG1. This hinge can be further divided into three distinct regions: the upper hinge, the central hinge, and the lower hinge. In human IgG1 antibodies, these regions are generally defined as follows: Upper hinge: 216th to 225th (EU numbering) or 226th to 238th (Kabat numbering), Center hinge: 226th-230th (EU numbering) or 239th-243rd (Kabat numbering), Lower hinge: 231st to 238th (EU numbering) or 244th to 251st (Kabat numbering). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond in the same position (see, for example, Brekke et al., 1995, Immunol (Table 1 of Today 16: 85-90)). Hinge regions as used herein include the wild-type hinge region and variants in which amino acid residues in the wild-type hinge region are altered by substitution, addition, or deletion. The term “disulfide bonds formed between amino acids not within the hinge region” (or “disulfide bonds formed between amino acids outside the hinge region”) means disulfide bonds formed, connected, or linked through amino acids located within any antibody region other than the “hinge region” as defined above. For example, such disulfide bonds are formed, connected, or linked through amino acids at any position in the antibody other than the hinge region (e.g., approximately positions 216–230 in the EU numbering system, or approximately positions 226–243 in the Kabat numbering system). In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located within the CH1 region, CL region, VL region, VH region, and / or VHH region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at EU numbering positions 119–123, 131–140, 148–150, 155–167, 174–178, 188–197, and 201–214 in the CH1 region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, and 164 in the CH1 region. , are formed, connected, or linked through amino acids located at positions 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, and 214 of the CH1 region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 of the CH1 region.In one preferred embodiment, such disulfide bonds are formed, connected, or linked through an amino acid located at EU numbering position 191 in the CH1 region.

[0034] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0035] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0036] An "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 to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0037] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0038] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0039] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0040] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0041] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0042] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0043] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0044] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0045] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0046] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-chain Fab (scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0047] "Contact" means to provide or expose to a solution. Antibodies, proteins, or polypeptides can be contacted with reducing reagents and can also be bound to a solid support (e.g., an affinity column or chromatography matrix). Preferably, the solution is buffered. To maximize the yield of antibodies / proteins with the desired conformation, the pH of the solution is selected to preserve the stability of the antibody / protein and be optimal for disulfide exchange. In the implementation of the present invention, the pH of the solution is preferably not strongly acidic. Therefore, some pH ranges are above pH 5, preferably about pH 6 to about pH 11, more preferably about pH 7 to about pH 10, and even more preferably about pH 6 to about pH 8. In one non-limiting embodiment of the present invention, the optimal pH was found to be about pH 7. However, the optimal pH in specific embodiments of the present invention can be readily determined experimentally by those skilled in the art.

[0048] The terms "reducing reagent" and "reducing agent" are used interchangeably. In some embodiments, the reducing agent is a free thiol. The reducing reagent is preferably composed of compounds from the group consisting of glutathione (GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-aminoethanethiol (2-MEA), TCEP (tris(2-carboxyethyl)phosphine), dithionitrobenzoate, cysteine, and Na2SO3. In some embodiments, TCEP, 2-MEA, DTT, cysteine, GSH, or Na2SO3 can be used. In some preferred embodiments, 2-MEA can be used. In some preferred embodiments, TCEP can be used.

[0049] The reducing agent may be added to the fermentation medium on which cells producing recombinant proteins are grown. In a further embodiment, the reducing agent may be added to the LC mobile phase during the LC separation step for separating the recombinant proteins. In a particular embodiment, the protein is immobilized on the stationary phase of the LC column, and the reducing agent is part of the mobile phase. In a particular embodiment, untreated IgG antibody may be eluted as a heterogeneous mixture indicated by the number of peaks. The use of a reduction / oxidation coupling reagent results in a simpler and more uniform peak pattern. It is intended that this more uniform peak of interest can be isolated as a more homogeneous preparation of IgG.

[0050] The reducing agent is present at a concentration sufficient to increase the relative proportion of the desired three-dimensional structure (e.g., an antibody in the "paired cysteine" form, having one or more manipulated disulfide bonds formed between two Fabs of the antibody, e.g., between amino acid residues not within the hinge region). The optimal absolute concentration and molar ratio of the reducing agent are determined by the concentration of total IgG and, in some situations, a specific IgG subclass. When used to prepare IgG1 molecules, they are also determined by the number and accessibility of unpaired cysteines in the protein. Generally, the concentration of free thiols derived from the reducing agent can be about 0.05 mM to about 100 mM, more preferably about 0.1 mM to about 50 mM, and even more preferably about 0.2 mM to about 20 mM. In some preferred embodiments, the concentrations of the reducing agent are 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 mM. In some preferred embodiments, 0.05 mM to 1 mM of 2-MEA can be used. In some preferred embodiments, TCEP in concentrations of 0.01 mM to 25 mM can be used.

[0051] Contact between the recombinant protein preparation and the reducing agent is carried out for a sufficient time to increase the relative proportion of the desired conformation. Any relative increase in proportion is desirable, and includes, for example, the conversion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, and even 80% or 90% of the proteins with undesirable conformations to proteins with the desired conformation. Contact may be carried out by providing the reducing agent to the fermentation medium in which the protein is being produced. Alternatively, contact may be carried out during the partial purification of the protein from the cell culture in which it is produced. In yet another embodiment, contact may be carried out after the protein has been eluted from the chromatography column but before any further processing. Essentially, contact may be carried out at any stage during antibody preparation, purification, storage, or formulation. In some embodiments, partial purification by affinity chromatography (e.g., protein A chromatography) may be performed before contact.

[0052] Contact may be performed using an antibody attached to the stationary phase of the chromatography column, with the reducing agent being part of the mobile phase; in this case, contact may be performed as part of a chromatographic purification technique. Examples of typical chromatographic refolding processes include size exclusion (SEC); solvent exchange during reversible adsorption on a protein A column; hydrophobic interaction chromatography (HIC); immobilized metal affinity chromatography (IMAC); reversed-phase chromatography (RPC); and the use of immobilized folding catalysts, such as GroE1, GroES, or other proteins with folding properties. On-column refolding is attractive because it can be easily automated using commercially available preparative chromatography systems. On-column refolding of recombinant proteins produced in microbial cells was recently outlined in (Li et al., 2004).

[0053] When the contact step is performed on partially or highly purified preparations of recombinant proteins, the contact step can be performed for short periods of time, from about 1 hour to about 4 hours, and for longer periods, from about 6 hours to about 4 days. Contact steps of about 2 to about 48 hours or about 16 hours have been shown to work well. The contact step can also be performed between other steps, for example, on a solid phase, or between any other steps in filtration or purification.

[0054] The method of the present invention can be carried out over a wide temperature range. For example, the method of the present invention has been successfully carried out at temperatures of about 4 degrees Celsius ("°C") to about 37°C, but the best results have been achieved at lower temperatures. Typical temperatures for contacting partially or completely purified preparations of recombinant proteins are about 4°C to about 25°C (ambient temperature), or preferably 23°C, but can also be carried out at lower and higher temperatures.

[0055] In addition, the method may be carried out under high pressure. Previously, high hydrostatic pressure (1000-2000 bar) combined with low non-denaturing concentrations of guanidine hydrochloride below 1 M has been used to deaggregate (solubilize) and refold multiple denatured proteins produced as inclusion bodies by Escherichia coli (E-coli), including human growth hormone, lysozyme, and β-lactamase (St John et al., Proc Natl Acad Sci USA, 96:13029-13033 (1999)). β-lactamase was refolded in high yield of active protein even without the addition of GdmHCl. Another study (Seefeldt et al., Protein Sci, 13:2639-2650 (2004)) showed that the refolding yield of the mammalian cell-produced protein bikunin obtained by high-pressure modulated refolding at 2000 bas was 70% by RP-HPLC, which was significantly higher than the 55% (by RP-HPLC) obtained by conventional guanidine hydrochloride "dilution refolding". These findings indicate that high hydrostatic pressure promotes the disruption of intermolecular and intramolecular interactions, leading to protein unfolding and deaggregation. The interaction of high pressure with proteins is analogous to the interaction between proteins and chaotropic agents. Therefore, in the method of the present invention, high pressure is intended to be used for protein unfolding instead of using chaotropic agents. Of course, in some cases, a combination of high pressure and chaotropic agents may be used.

[0056] Recombinant antibody / protein preparations can be contacted with reducing agents in various amounts as needed. For example, the method of the present invention has been successfully implemented on analytical laboratory scale (1–50 mL), preparation scale (50 mL–10 L), and production scale (10 L or more). The method of the present invention can be performed reproducibly on both small and large scales. Thus, the antibody concentration can be industrial-grade (in terms of grams) (e.g., industrial quantities of a particular IgG) or milligrams. In certain embodiments, the concentration of recombinant antibody in the reaction mixture is about 1 mg / ml to about 50 mg / ml, more specifically 10 mg / ml, 15 mg / ml, or 20 mg / ml. One molecule of recombinant IgG at these concentrations is particularly intended.

[0057] In certain embodiments, proteins produced using a culture medium containing a reducing agent are further treated in another processing step utilizing a chaotropic denaturing agent such as sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride (GuHCl). A substantial amount of the chaotropic agent is required to observe recognizable unfolding. In some embodiments, the processing step uses 0.1 M to 2 M chaotrope, which produces an effect equivalent to that of using 0.1 M to 2 M guanidine hydrochloride. In certain embodiments, oxidative refolding is achieved in the presence of approximately 1.0 M guanidine hydrochloride, or some amount of another chaotropic agent that produces the same or similar amount of refolding as 1 M guanidine hydrochloride. In some embodiments, the method uses approximately 1.5 M to 0.5 M chaotrope. The amount of chaotropic agent used is based on the structural stability of the protein in the presence of the chaotrope. A chaotrope must be present in an amount sufficient to disrupt the local tertiary and / or quaternary structures of the protein's domain interactions, but less than what is needed to completely unfold the secondary structures of the molecule and / or individual domains. To determine the point at which a protein begins to unfold by equilibrium denaturation, those skilled in the art may titrate the chaotrope into a solution containing the protein and monitor the structure by techniques such as circular dichroism or fluorescence. Other parameters exist that can be used to unfold or slightly disrupt the structure of a protein, and may be used instead of a chaotrope. Temperature and pressure are two basic parameters previously used to alter the structure of a protein and may be used instead of the chaotropic agent during contact with the redox agent. The inventors intend that any parameter shown to denature or disrupt the structure of a protein may be used by those skilled in the art instead of the chaotropic agent.

[0058] Disulfide exchange can be stopped by any method known to those skilled in the art. For example, the reducing agent may be removed, or its concentration may be reduced through a purification step, and / or it may be chemically deactivated, for example, by acidifying the solution. Typically, when the reaction is stopped by acidification, the pH of the solution containing the reducing agent is lowered to less than pH 7. In some embodiments, the pH is lowered to less than pH 6. Generally, the pH is lowered to about pH 2 to about pH 6. In some embodiments, the removal of the reducing agent may be carried out by dialysis, buffer exchange, or any chromatographic method described herein.

[0059] The term “preferentially enriched (or increased)” means an increase in the relative abundance of a desired form, an increase in the relative proportion of a desired form, or an increase in the population of a desired form (structural isoform). In some embodiments, the methods described herein increase the relative abundance of an antibody structural isoform, e.g., an antibody having at least one disulfide bond formed between amino acid residues outside the hinge region. In one embodiment, the at least one disulfide bond is formed between amino acid residues at EU numbering position 191 in each CH1 region of the first antigen-binding domain and the second antigen-binding domain. In a particular embodiment, the method produces a homogeneous antibody preparation having a uniform antibody having at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%, in molar ratios of at least 50%, 60%, 70%, 80%, 90%, and preferably at least 95%.

[0060] A “homogeneous” population of antibodies means a population of antibodies that primarily comprises a single form of the antibody, for example, at least 50%, 60%, 70%, 80%, or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100% of the antibody in a solution or composition, in a well-folded form. Similarly, a “homogeneous” population of antibodies having at least one disulfide bond formed outside the hinge region means a population of the antibody that primarily comprises a single well-folded form, for example, a population of the antibody having at least one disulfide bond formed outside the hinge region in a molar ratio of at least 50%, 60%, 70%, 80%, or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100%. In one preferred embodiment, a “homogeneous” population of antibodies contains at least one disulfide bond formed between amino acid residues at EU numbering position 191 in each CH1 region of the first antigen-binding domain and the second antigen-binding domain (i.e., “paired cysteines” at EU numbering position 191 in the CH1 region). In a preferred embodiment, the method of the present invention produces a homogeneous antibody population or a homogeneous antibody preparation by the steps described herein.

[0061] Determining whether an antibody population is homogeneous, and the relative abundance or proportion of protein / antibody structures in a mixture, can be done using a variety of analytical and / or qualitative techniques. If two structures are separated differently through separation techniques, e.g., chromatography, electrophoresis, filtration, or other purification techniques, the relative proportion of structures in the mixture can be determined using such purification techniques. For example, at least two different structures of recombinant IgG can be separated by hydrophobic interaction chromatography. Furthermore, since far-ultraviolet circular dichroism has been used to estimate the composition of a protein's secondary structure (Perczel et al., 1991, Protein Engrg. 4:669-679), such techniques can determine whether another structure of the protein exists. Yet another technique used to determine structure is fluorescence spectroscopy, which can be used to confirm the complementarity of tertiary structures assignable to tryptophan and tyrosine fluorescence. Other techniques that can be used to determine differences in three-dimensional structure, and therefore the relative proportions of three-dimensional structures, include online SEC for measuring the aggregation state, differential scanning calorimetry for measuring fusion transition (Tm) and component enthalpy, and chaotrope unfolding. Yet another technique that can be used to determine differences in three-dimensional structure, and therefore the relative proportions of three-dimensional structures, is LC / MS detection for determining protein heterogeneity.

[0062] Alternatively, if there are differences in activity between the three-dimensional structures of the antibody / protein, the relative proportions of these structures in the mixture can be determined by activity assays (e.g., ligand binding, enzymatic activity, biological activity, etc.). The biological activity of the protein can also be used. Alternatively, a binding assay can be used that expresses activity as active units / mg protein.

[0063] In some embodiments described in detail below herein, the present invention utilizes IEC chromatography to determine antibody / protein heterogeneity. In such cases, the antibody is purified to be "homogeneous" or considered "homogeneous," meaning that no polypeptide peak or fraction corresponding to any other polypeptide is detectable during analysis by IEC chromatography. In certain embodiments, the antibody is purified to be "homogeneous" or considered "homogeneous" such that polypeptide bands corresponding to other polypeptides are not detectable during analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It is recognized by those skilled in the art that multiple bands corresponding to polypeptides can be visualized by SDS-PAGE due to differential glycosylation and differential post-translational processing, etc. Most preferably, the polypeptide of the present invention is purified to be substantially homogeneous so that it is indicated by a single polypeptide band during analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, and / or (if the polypeptide is radiolabeled) by autoradiography.

[0064] In this specification, examples of conditions for SDS-PAGE analysis are as follows: A Sample Buffer Solution (×4) without 2-mercaptoethanol may be used for the preparation of electrophoretic samples. Samples may be treated for 10 minutes under conditions of sample concentration of 50 or 100 micrograms / mL and 70°C, and then subjected to non-reducing SDS-PAGE. In non-reducing SDS-PAGE, electrophoresis may be performed for 90 minutes at 125 V using a 4% SDS-PAGE gel. The gel is then stained with CBB, a gel image is acquired, and the bands may be quantified using an imaging device. In the gel image, multiple bands, for example two, i.e., an "upper band" and a "lower band," may be observed in the antibody variant sample. In this case, the molecular weight of the upper band may correspond to that of the parent antibody (unmodified). Structural changes, such as cross-linking via disulfide bonds in Fab, may be caused by cysteine ​​substitution and may result in changes in electrophoretic mobility. In this case, the lower band may be considered to correspond to an antibody having one or more manipulated disulfide bonds formed between CH1 regions. Antibody variant samples with additional cysteine ​​substitutions may show a higher ratio of the lower band to the upper band compared to the control sample. Additional cysteine ​​substitutions may enhance / promote disulfide bond crosslinking of Fab; may increase the percentage or structural uniformity of antibody preparations having manipulated disulfide bonds formed at the mutation site; may decrease the percentage of antibody preparations without manipulated disulfide bonds formed at the mutation site. In this specification, the term “ratio of the lower band to the upper band” refers to the ratio between the amount / intensity of the upper band and the amount / intensity of the lower band, which can be quantified during the SDS-PAGE test described above.

[0065] Fv (variable fragment) In this specification, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies could be prepared from the periplasmic fraction of E. coli by inserting the antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction showed VH and VL associated in a manner that allowed for binding to the antigen.

[0066] scFv, single-chain antibody, and sc(Fv)2 In this specification, the terms “scFv,” “monochain antibody,” or “sc(Fv)2” all refer to an antibody fragment that contains variable regions derived from both the heavy and light chains within a single polypeptide chain, but lacks a constant region. Generally, monochain antibodies further include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is expected to allow antigen binding. Monochain antibodies have been discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg, and Moore (ed.), Springer-Verlag, New York, 269-315 (1994). See also International Patent Application Publication WO 1988 / 001649; U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monochain antibodies are bispecific and / or can be humanized.

[0067] scFv is an antigen-binding domain in which the VH and VL components of Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). This peptide linker allows VH and VL to be kept in close proximity.

[0068] sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and VLs may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes a bispecific sc(Fv)2 that recognizes two different epitopes present in the same antigen, for example, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, sc(Fv)2 can be prepared by linking scFv with a linker such as a peptide linker.

[0069] In this specification, the antigen-binding domain of sc(Fv)2 is characterized by having two VH units and two VL units arranged in the order VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of a single-chain polypeptide. However, the order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. For example, the following configurations can also be given. [VL]-Linker-[VH]-Linker-[VH]-Linker-[VL] [VH]-Linker-[VL]-Linker-[VL]-Linker-[VH] [VH]-Linker-[VH]-Linker-[VL]-Linker-[VL] [VL]-Linker-[VL]-Linker-[VH]-Linker-[VH] [VL]-Linker-[VH]-Linker-[VL]-Linker-[VH]

[0070] Fab, F(ab')2, and Fab' "Fab" consists of a light chain and a heavy chain comprising a CH1 region and a variable region. The heavy chain of the wild-type Fab molecule cannot form disulfide bonds with other heavy-chain molecules. This specification includes not only the wild-type Fab molecule but also Fab variants in which amino acid residues in the wild-type Fab are altered by substitution, addition, or deletion. In certain embodiments, mutant amino acid residues in a Fab variant (e.g., substituted, added, or inserted cysteine ​​or lysine residues) can form disulfide bonds with other heavy-chain molecules or parts thereof (e.g., Fab molecules).

[0071] scFab is an antigen-binding domain in which the CH1 region and variable region of one light chain and one heavy chain constituting the Fab are linked by a peptide linker. This peptide linker allows the CH1 region and variable region of the light chain and heavy chain to be kept in close proximity.

[0072] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin and papain, and by digesting the immunoglobulin (monoclonal antibody) near the disulfide bond located between the hinge regions of the two H chains. For example, papain can cleave IgG upstream of the disulfide bond located between the hinge regions of the two H chains, and two homologous antibody fragments can be produced in which the L chain, containing VL (variable L chain region) and CL (constant L chain region), is bound to the H chain fragment, containing VH (variable H chain region) and CHγ1 (γ1 region in the constant H chain region), by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab'.

[0073] "F(ab')2" consists of two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that a disulfide bond is formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably prepared by partially digesting a full-length monoclonal antibody having a desired antigen-binding domain with a protease such as pepsin, and then removing the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited as long as it can restrictively cleave a full-length antibody to produce F(ab')2 by appropriately setting the reaction conditions of the enzyme, such as pH, and such proteases include, for example, pepsin and ficin.

[0074] Single-domain antibody In this specification, the term "single-domain antibody" refers to an antibody whose structure is not particularly limited, as long as the domain alone can exert antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL domains, single-domain antibodies are known to exert antigen-binding activity solely through their own domain structure without pairing with other domains. Single-domain antibodies usually have a relatively low molecular weight and exist in monomeric form. Examples of single-domain antibodies, though not limited to them, include, for example, VHH antibodies for camelids and V antibodies for sharks. NAR Examples include naturally occurring antigen-binding molecules lacking a light chain, and antibody fragments containing all or part of the VH domain or all or part of the VL domain of an antibody. Examples of single-domain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, single-domain antibodies artificially produced starting from human antibody VH or human antibody VL, as described in, for example, U.S. Patent No. 6,248,516B1. In some embodiments of the present invention, one single-domain antibody has three CDRs (CDR1, CDR2, and CDR3). Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single-domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single-domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized single-chain antibodies can also be obtained by replacing the framework sequence of a single-domain antibody obtained from an animal with a human germline sequence or a similar sequence. Humanized single-domain antibodies (e.g., humanized VHH) are one embodiment of the single-domain antibody of the present invention. Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), and synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11). (1691-1701) is one example.

[0075] "Binding activity" refers to the total strength of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, the binding activity is not strictly limited to the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when the members of the binding pair reflect a 1:1 interaction in a monovalent manner, the binding activity refers to the intrinsic binding affinity ("affinity"). When the members of the binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of molecule X for its partner Y can generally be represented by the dissociation constant (KD) or the "amount of analyte bound per unit amount of ligand". The binding activity can be measured by conventional methods known in the art, including those described herein.

[0076] As used herein, an "agonist" antigen-binding molecule or "agonist" antibody is an antigen-binding molecule or antibody that significantly enhances the biological activity of the antigen to which it binds.

[0077] As used herein, a "blocking" antigen-binding molecule or "blocking" antibody or "antagonist" antigen-binding molecule or "antagonist" antibody is an antigen-binding molecule or antibody that significantly inhibits (either partially or completely) the biological activity of the antigen to which it binds.

[0078] As used herein, the expressions "substantially reduced" or "substantially different" mean that the difference between two numerical values (usually between those related to a molecule and those related to a reference / comparative molecule) is large enough for a person skilled in the art to consider the difference between those two numerical values to be statistically significant from the perspective of the biological characteristic measured by that numerical value (e.g., the KD value).

[0079] As used herein, the terms “substantially similar” or “substantially identical” mean that the similarity between two numerical values ​​(e.g., between the antibody of the present invention and the reference / comparative antibody) is sufficiently high that a person skilled in the art would consider the difference between the two numerical values ​​to be little or no biological and / or statistically significant in terms of the biological characteristics measured by the numerical value (e.g., the KD value).

[0080] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations in which the biological activity of the active ingredients contained herein can exert its effect, and which do not contain additional elements that are toxic to an extent unacceptable to the subject to whom the preparation is administered.

[0081] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0082] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0083] II. Antigen-binding molecules In one aspect, this disclosure is based in part on the finding that antigen-binding molecules comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via one or more bindings, exhibit enhanced or reduced activity compared to control antigen-binding molecules comprising antigen-binding domains linked via no or fewer bindings. In a particular embodiment, an antigen-binding molecule is provided that has the activity of holding two or more antigen molecules in spatially close proximity. The antigen-binding molecule of this disclosure is useful, for example, in that it can control the activation of two antigen molecules that are activated by association with each other. In another particular embodiment, an antigen-binding molecule is provided that has acquired resistance to protease digestion by linkage between antigen-binding domains.

[0084] A. Exemplary antigen-binding molecules <Structure of antigen-binding molecule> In one aspect, the present disclosure provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via one or more bonds.

[0085] In one aspect of the above, at least one of the one or more bonds linking the two antigen-binding domains is a covalent bond. In a particular aspect, a covalent bond is formed by direct cross-linking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. The cross-linked amino acid residue is, for example, cysteine, and the formed covalent bond is, for example, a disulfide bond. In another specific embodiment, a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the amino acid residue to be crosslinked is, for example, lysine.

[0086] In one aspect of the above, at least one of the one or more bonds linking the antigen-binding domain is a non-covalent bond. In a particular aspect, the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond. An ionic bond is formed, for example, between an acidic amino acid and a basic amino acid. An acidic amino acid is, for example, aspartic acid (Asp) or glutamic acid (Glu), and a basic amino acid is, for example, histidine (His), lysine (Lys), or arginine (Arg).

[0087] The amino acid residues that serve as the starting point for binding between antigen-binding domains (binding that links two antigen-binding domains) are present in the first and second antigen-binding domains, respectively, and the binding between these antigen-binding domains is formed by linking these amino acid residues. In one embodiment of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is an artificially introduced mutant amino acid residue, for example, an artificially introduced cysteine ​​residue. Such mutant amino acid residues can be introduced into wild-type antigen-binding domains by methods such as amino acid substitution. When the antigen-binding domain includes, for example, an antibody fragment, the sites of amino acid residues that can serve as the starting point for binding between antigen-binding domains are disclosed herein in the constant regions (CH1 region, CL region, and hinge region) and the variable regions (VH region, VL region, and VHH region), and for example, cysteine ​​residues can be introduced into these sites.

[0088] In one aspect of the above, at least one of the first and second antigen-binding domains has antigen-binding activity on its own (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular aspect, both the first and second antigen-binding domains have antigen-binding activity on their own.

[0089] In one aspect of the above, the first and second antigen-binding domains are both antigen-binding domains of the same type. As will be described later, examples of proteins constituting antigen-binding domains include polypeptides derived from antibodies or non-antibody proteins and their fragments (e.g., Fab, Fab', scFab, Fv, scFv, single-domain antibodies, etc.). From the viewpoint of such molecular morphology, if the structures of the proteins constituting the first and second antigen-binding domains are identical, then those antigen-binding domains are judged to be of the same type.

[0090] In one embodiment of the above aspect, the linkage between the first antigen-binding domain and the second antigen-binding domain may be formed by linking amino acid residues located at the same position in the first antigen-binding domain and the second antigen-binding domain, respectively, or by linking amino acid residues located at different positions in the first antigen-binding domain and the second antigen-binding domain.

[0091] The positions of amino acid residues in antigen-binding domains can be indicated according to the Kabat numbering or EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. For example, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at the same corresponding position in each antigen-binding domain, the positions of those amino acid residues can be indicated by the same number according to the Kabat numbering or EU numbering system. Alternatively, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at different, non-corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by different numbers according to the Kabat numbering or EU numbering system.

[0092] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains includes an antibody fragment that binds to a specific antigen. In a particular embodiment, the antibody fragment is Fab, Fab', scFab, Fv, scFv, or a single-domain antibody. In a particular embodiment, at least one of the amino acid residues that initiate binding between the antigen-binding domains is present within the antibody fragment.

[0093] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the constant region. In a particular aspect, the amino acid residue is located within the CH1 region, for example, at EU numbering positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, or 218 to 219 of the CH1 region. According to certain sources, the amino acid residues in question are located at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, and 165 in the CH1 region. The amino acid residue is located in one of the groups selected from positions 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218, and 219. In a particular embodiment, the amino acid residue is located at EU numbering positions 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 in the CH1 region. In a particular embodiment, the amino acid residue is located at EU numbering positions 135, 136, or 191 in the CH1 region. In one embodiment of the above, the steady-state region is of human origin. In a particular embodiment, the subclass of the heavy chain steady-state region is one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a particular embodiment, the subclass of the CH1 region is one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[0094] In one embodiment of the above aspect, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CH1 region of the second antigen-binding domain. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 148, 149, and 150. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 218 and 219, respectively.

[0095] In one embodiment of the above aspect, the difference in the positions of the amino acid residues that serve as the starting point for binding in the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. This means that when comparing the position of the amino acid residue that serves as the starting point for binding in the CH1 region of the first antigen-binding domain and the position of the amino acid residue that serves as the starting point for binding in the CH1 region of the second antigen-binding domain using EU numbering, the difference (i.e., distance) is within 3 amino acids. In a particular embodiment, at least one binding linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 135 in the CH1 region of the first antigen-binding domain with any amino acid residue at EU numbering positions 132 to 138 in the CH1 region of the second antigen-binding domain. In a particular embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain with any amino acid residue at EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain. In certain embodiments, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 191 of the EU numbering in the CH1 region of the first antigen-binding domain with any of the amino acid residues at positions 188 to 194 of the EU numbering in the CH1 region of the second antigen-binding domain. In one exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 135 of the EU numbering in the CH1 regions of the two antigen-binding domains. In one exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 136 of the EU numbering in the CH1 regions of the two antigen-binding domains. In one exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 191 of the EU numbering in the CH1 regions of the two antigen-binding domains.

[0096] In one aspect of the above situation, at least one of the amino acid residues that serve as the starting point of the linkage between antigen-binding domains is present within the CL region, for example, at any of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, 208 to 213 of the Kabat numbering of the CL region. In certain embodiments, the amino acid residue is present at any one selected from the group consisting of positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213 of the Kabat numbering of the CL region. In certain embodiments, the amino acid residue is present at position 126 of the Kabat numbering of the CL region. In one embodiment of the above-mentioned aspect, the steady-state region is of human origin. In a particular embodiment, the subclass of the CL region is κ or λ.

[0097] In one embodiment of the above aspect, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CL region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 108, 109, 110, 111, and 112. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 151, 152, 153, 154, 155, and 156, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 184, 185, 186, 187, 188, 189, and 190, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 195 and 196, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 200, 201, 202, and 203, respectively. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 208, 209, 210, 211, 212, and 213.

[0098] In one embodiment of the above aspect, the difference in the positions of the amino acid residues that serve as the starting point for binding in the first antigen-binding domain and the second antigen-binding domain (i.e., the distance between them) is within 3 amino acids. This means that when comparing the positions of the amino acid residues that serve as the starting point for binding in the CL region of the first antigen-binding domain and the positions of the amino acid residues that serve as the starting point for binding in the CL region of the second antigen-binding domain using EU numbering, the difference (i.e., the distance) is within 3 amino acids. In one exemplary embodiment, at least one binding linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at Kabat numbering position 126 in the CL regions of the two antigen-binding domains.

[0099] In one embodiment of the above aspect, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a particular embodiment, the amino acid residue in the CH1 region of the first antigen-binding domain is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region of the second antigen-binding domain is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. In one exemplary embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with the amino acid residue at Kabat numbering position 126 in the CL region of the second antigen-binding domain.

[0100] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within a variable region. In a particular aspect, the amino acid residue is located within the VH region, for example, in any of the group selected from the Kabat numbering positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b of the VH region. In a particular aspect, the amino acid residue is located within the VL region, for example, in any of the group selected from the Kabat numbering positions 21, 27, 58, 77, 100, 105, and 107 of the VL region (subclass κ), and Kabat numbering positions 6, 19, 33, and 34 of the VL region (subclass λ). In a particular embodiment, the amino acid residue is located within a VHH region, for example, at any of the group selected from positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 of the VHH region's Kabat numbering.

[0101] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the non-antibody protein is either a pair of ligands and / or receptors that bind specifically to each other. Such receptors include, for example, receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0102] In one embodiment of the above aspect, the first and / or second antigen-binding domains include a hinge region. In a particular embodiment, at least one of the cysteine ​​residues present in the wild-type hinge region is substituted with another amino acid residue. Such cysteine ​​residues are, for example, located at EU numbering positions 226 and / or 229 of the wild-type hinge region. In a particular embodiment, at least one of the amino acid residues that initiate binding between the antigen-binding domains is located within the hinge region and is, for example, located at any of the group consisting of EU numbering positions 216, 218, and 219 of the hinge region.

[0103] In one aspect of the above-mentioned configuration, the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more binding sites.

[0104] In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is an amino acid residue present in the wild-type sequence, for example, a cysteine ​​residue in the wild-type hinge region. In certain embodiments, at least one binding site linking the first antigen-binding domain and the second antigen-binding domain is a disulfide bond formed by cross-linking of cysteine ​​residues present in the wild-type hinge region. Such cysteine ​​residues are located, for example, at EU numbering positions 226 and / or 229 in the wild-type hinge region.

[0105] In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the antibody fragment, and at least one is located within the hinge region. In one exemplary embodiment, the antigen-binding molecule of the present disclosure is F(ab')2, wherein both the first and second antigen-binding domains include a Fab and a hinge region.

[0106] In one aspect of the foregoing, the antigen-binding molecule of the Disclosure further comprises an Fc region and is, for example, a full-length antibody. In a particular aspect, the Fc region of the antigen-binding molecule of the Disclosure is introduced with one or more amino acid mutations that promote multimerization of the Fc region. Such amino acid mutations include, for example, amino acid mutations at at least one position selected from the group consisting of EU numbering positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447 (see, for example, WO2016 / 164480). In a particular aspect, the multimerization is hexamerization.

[0107] <Antigen to which antigen-binding molecules bind> In one aspect of the above, the first and second antigen-binding domains both bind to the same antigen. In a particular aspect, the first and second antigen-binding domains bind to the same epitope on the same antigen. In another particular aspect, each of the first and second antigen-binding domains binds to a different epitope on the same antigen. In a particular aspect, the antigen-binding molecule of the present disclosure is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets one specific antigen. In another embodiment of the aforementioned aspect, each of the first and second antigen-binding domains binds to a different antigen. In another aspect of the foregoing, the antigen-binding molecule of the Disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). In this Specification, a clamping antigen-binding molecule means an antigen-binding molecule that specifically binds to an antigen-antigen complex formed from an antigen A and an antigen-binding molecule that binds to that antigen A, thereby increasing the binding activity of the antigen-binding molecule to that antigen A (or stabilizing the antigen-antigen complex formed from the antigen A and the antigen-binding molecule that binds to that antigen A). For example, a CD3 clamping antibody can specifically bind to an antigen-antibody complex formed from CD3 and an antibody with reduced binding ability to CD3 (a CD3-attenuated antibody), thereby increasing the binding activity of the CD3-attenuated antibody to CD3 (or stabilizing the antigen-antibody complex formed from CD3 and the CD3-attenuated antibody). In certain aspects, the first and / or second antigen-binding domains in the antigen-binding molecule of the Disclosure may be antigen-binding domains derived from a clamping antigen-binding molecule (clamping antigen-binding domains). In one embodiment of the above aspect, the first and second antigen-binding domains both have the same amino acid sequence. In another embodiment, each of the first and second antigen-binding domains has a different amino acid sequence.

[0108] In one embodiment of the above aspect, at least one of the two antigens to which the first and second antigen-binding domains bind is a soluble protein or a membrane protein.

[0109] <Function of antigen-binding molecules> In one aspect of the foregoing, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one less bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (for example, a cysteine ​​residue that is not present in the wild-type Fab or hinge region). In another aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has activity to control the interaction between two antigen molecules. While not bound by any particular theory, this interaction-controlling activity is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In certain aspects, the antigen-binding molecule of the present disclosure can enhance or reduce the interaction between two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one less bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are a ligand and its receptor, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the activation of the receptor by the ligand. In another particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are an enzyme and its substrate, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the catalytic reaction of the enzyme with respect to the substrate. In another specific embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the Disclosure has the activity to facilitate interaction between a cell expressing a first antigen and a cell expressing a second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen are cytotoxic cells and their target cells, respectively, and the antigen-binding molecule of the Disclosure facilitates damage to the target cells by the cytotoxic cells. Cytotoxic cells are, for example, T cells, NK cells, monocytes, or macrophages.

[0110] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has an activity that controls the activation of two antigen molecules that are activated by association with each other. Although not bound by any particular theory, this activity that controls the activation is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can enhance or reduce the activation of two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). For example, such antigen molecules are selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, C. difficile antigens, costimulatory molecules, and cell adhesion molecules.

[0111] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has two antigen-binding domains located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced. In a particular aspect, the antigen-binding molecule of the present disclosure has two antigen-binding domains located in closer proximity and / or the mobility of the two antigen-binding domains is further reduced compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond may be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0112] In one aspect of the aforementioned aspect, the antigen-binding molecule of the Disclosure is resistant to protease cleavage. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the Disclosure only in that it has one fewer bond between its two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular aspect, the antigen-binding molecule of the Disclosure exhibits an increased proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment compared to a control antigen-binding molecule. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits a decreased proportion of specific fragments (e.g., Fab monomers) generated after protease treatment compared to a control antigen-binding molecule.

[0113] In one embodiment of the aforementioned aspect, when the antigen-binding molecule of the present disclosure is treated with a protease, a dimer of the antigen-binding domain or a fragment thereof (e.g., a crosslinked Fab dimer) is cleaved. In a particular embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the present disclosure in that it has one fewer bond between two antigen-binding domains is treated with the protease, a monomer of the antigen-binding domain or a fragment thereof is cleaved. In a further embodiment, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). In these embodiments, the protease can cleave the hinge region of the antigen-binding molecule.

[0114] In a further embodiment, the control antigen-binding molecule differs from the antigen-binding molecule of this disclosure only in that it has one fewer bond between two antigen-binding domains, and this one less bond is formed starting from a mutant amino acid residue. The mutant amino acid residue is, for example, an artificially introduced cysteine ​​residue.

[0115] <Pharmaceutical composition> In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule and a pharmaceutically acceptable carrier.

[0116] <Uses of antigen-binding molecules> In one aspect, the present disclosure provides a method for holding two antigen molecules in spatially close proximity, (a) To provide an antigen-binding molecule containing two antigen-binding domains; (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. The present invention provides a method including the following: In a particular embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bindings, in which case some or all of the one or more bindings are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The present invention also provides a method for holding two antigen molecules in spatial proximity, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in holding two antigen molecules in spatial proximity.

[0117] In another aspect, the present disclosure provides a method for controlling the interaction between two antigen molecules, (a) To provide an antigen-binding molecule containing two antigen-binding domains; (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. The present invention provides a method including the following: In a particular embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bindings, in which case some or all of the one or more bindings are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The present invention also provides a method for controlling the interaction between two antigen molecules, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in controlling the interaction between two antigen molecules.

[0118] In another aspect, the present disclosure provides a method for controlling the activity of two antigen molecules that are activated by association with each other, (a) To provide an antigen-binding molecule containing two antigen-binding domains; (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. The present invention provides a method including the following: In a particular embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bindings, in which case some or all of the one or more bindings are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The present invention also provides a method for controlling the activity of two antigen molecules that are activated by association with each other, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in controlling the activity of two antigen molecules that are activated by association with each other.

[0119] In another aspect, the present disclosure provides a method for positioning two antigen-binding domains in spatial proximity and / or reducing the mobility of the two antigen-binding domains. (a) To provide an antigen-binding molecule containing two antigen-binding domains; and (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together. The present invention provides a method including the following: In a particular embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bindings, in which case some or all of the one or more bindings are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0120] In another aspect, the present disclosure relates to a method for increasing the resistance of an antigen-binding molecule to protease cleavage, (a) To provide an antigen-binding molecule containing two antigen-binding domains; and (b) Adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains together. The present invention provides a method including the following: In a particular embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bindings, in which case some or all of the one or more bindings are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0121] The antigen-binding molecules used in these various methods may have the characteristics of antigen-binding molecules described herein.

[0122] <Method for producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having the activity of holding two antigen molecules in spatially close proximity, (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain; (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites. The present invention provides a method comprising, preferably, a step of contacting an antibody preparation with a reducing agent.

[0123] In certain embodiments, the contact with the reducing agent ("the contact step") preferentially enriches or increases a population of antibody structural isoforms having at least one disulfide bond formed between amino acid residues not within the hinge region. In certain embodiments, the method produces a homogeneous antibody preparation having the antibody having at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%, in molar ratios.

[0124] In certain embodiments, the pH of the reducing reagent in contact with the antibody is approximately 3 to approximately 10. In certain embodiments, the pH of the reducing reagent in contact with the antibody is approximately 6, 7, or 8. In some embodiments, the pH of the reducing reagent in contact with the antibody is approximately 7 or approximately 3.

[0125] In certain embodiments, the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and Na2SO3. In some preferred embodiments, the reducing agent is TCEP. In certain embodiments, the concentration of the reducing agent is approximately 0.01 mM to approximately 100 mM. In some preferred embodiments, the concentration of the reducing agent is about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 mM, preferably about 0.01 mM to 25 mM. In one preferred embodiment, the reducing agent is TCEP in a concentration of 0.01 mM to 25 mM.

[0126] In certain embodiments, the step of contacting the reducing agent is carried out for at least 30 minutes. In certain embodiments, the contact step is carried out for about 2 to about 48 hours. In some preferred embodiments, the contact step is carried out for about 2 hours or about 16 hours.

[0127] In certain embodiments, the contact step is carried out at a temperature of about 20°C to 37°C, preferably 23°C, 25°C, or 37°C, more preferably 23°C. In certain embodiments, the antibody is partially purified by affinity chromatography (preferably protein A chromatography) before contact. In certain embodiments, the antibody concentration is about 1 mg / ml to about 50 mg / ml. In some preferred embodiments, the antibody concentration is about 1 mg / ml or about 20 mg / ml.

[0128] In a particular embodiment, the contacting step preferentially enriches or increases a population of antibody structural isoforms having at least one disulfide bond formed between amino acid residues not within the hinge region. In a particular embodiment, the contacting step produces a homogeneous antibody preparation having the antibody having at least one disulfide bond formed between amino acid residues not within the hinge region in a molar ratio of at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%.

[0129] In a particular embodiment, the contact step produces a more uniform antibody preparation than the same antibody preparation that has not been treated by the method. In a particular embodiment, the contact step produces an antibody preparation having increased biological activity compared to the same antibody that has not been treated by the method. In a particular embodiment, the contact step generates an antibody that exhibits enhanced activity in holding two antigen molecules in spatially close proximity compared to the same antibody that has not been treated by the method. In a particular embodiment, the contact step produces an antibody that is more stable than the same antibody that has not been treated by the method.

[0130] In a particular embodiment, the contact step preferentially concentrates antibodies having at least one disulfide bond formed outside the hinge region, and the preferentially concentrated form has pharmaceutically desirable properties selected from any of (a) to (e) below, compared to a preparation not treated by the contact step: (a) The at least one disulfide bond restricts the antigen-binding orientation of the two antigen-binding domains to cis-antigen binding (i.e., binding to two antigens on the same cell), or restricts the binding of the two antigen-binding domains to binding to two antigens that are spatially close to each other; (b) The at least one disulfide bond holds the first antigen-binding domain and the second antigen-binding domain in a more spatially close position to each other compared to the same corresponding antibody that does not have the at least one disulfide bond; (c) The at least one disulfide bond reduces the mobility and / or mobility of the first antigen-binding domain and the second antigen-binding domain compared to the same antibody that does not have the at least one disulfide bond; (d) The at least one disulfide bond increases the antibody's resistance to protease cleavage compared to the same antibody that does not have the at least one disulfide bond; or (e) The at least one disulfide bond enhances or reduces the interaction between two antigen molecules bound by the antigen-binding molecule, compared to the corresponding same antibody that does not have the at least one disulfide bond. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0131] In another aspect, the present disclosure relates to a method for producing an antigen-binding molecule having activity to control the interaction between two antigen molecules, (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain; (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites. This provides a method that includes this. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0132] In another aspect, the present disclosure relates to a method for producing an antigen-binding molecule having activity to control the activation of two antigen molecules that are activated by association with each other, (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain; (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites. This provides a method that includes this. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0133] In another aspect, the present disclosure relates to a method for producing an antigen-binding molecule in which two antigen-binding domains are located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced. (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain; (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites. This provides a method that includes this. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0134] In another aspect, the present disclosure relates to a method for producing an antigen-binding molecule with increased resistance to protease cleavage, (a) To provide a nucleic acid encoding a polypeptide containing a first antigen-binding domain, and a nucleic acid encoding a polypeptide containing a second antigen-binding domain; (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one binding site is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by one or more binding sites. This provides a method that includes this. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0135] The antigen-binding molecules produced in these various situations may have the characteristics of antigen-binding molecules described herein.

[0136] <Screening method for antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel set of protein molecules that are activated by association with each other. (a) Provide any two protein molecules; (b) To produce an antigen-binding molecule comprising two antigen-binding domains that bind to each of the two protein molecules, respectively, by the manufacturing method of the present disclosure; (c) Contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) Evaluate whether the two protein molecules are activated. This provides a method that includes this. In a particular embodiment, at least one of the two protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0137] A. Exemplary antigen-binding molecules <Structure of antigen-binding molecule> In one aspect, the present disclosure provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via two or more bonds. In one embodiment, at least one of the first and second antigen-binding domains has antigen-binding activity on its own (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular embodiment, both the first and second antigen-binding domains have antigen-binding activity on their own.

[0138] In one aspect of the above, at least one of the first and second antigen-binding domains includes an antibody fragment that binds to a specific antigen. In a particular aspect, the first and / or second antigen-binding domains include a hinge region. The amino acid residues that initiate binding between the antigen-binding domains are located in the first and second antigen-binding domains, respectively, and the binding between the antigen-binding domains is formed by linking these amino acid residues. In a particular aspect, at least one of the amino acid residues that initiate binding between the antigen-binding domains is located within the antibody fragment. In a particular aspect, at least one of the amino acid residues that initiate binding between the antigen-binding domains is located within the hinge region. In a particular aspect, at least one of the amino acid residues that initiate binding between the antigen-binding domains is located within the antibody fragment, and at least one of said amino acid residues is located within the hinge region.

[0139] In one aspect of the above, in at least one of the first and second antigen-binding domains, there are multiple amino acid residues that serve as the starting point for binding between the antigen-binding domains, located at positions 7 amino acids or more apart from each other on the primary structure. This means that there are 6 or more amino acid residues other than those amino acid residues between any two of the above multiple amino acid residues. In a particular aspect, the combination of multiple amino acid residues that serve as the starting point for binding between the antigen-binding domains may include pairs of amino acid residues located at positions less than 7 amino acids apart on the primary structure. In a particular aspect, if the first and second antigen-binding domains are linked to each other via three or more bindings, three or more amino acid residues, including pairs of amino acid residues located at positions 7 amino acids or more apart from each other on the primary structure, can serve as the starting point for binding between the antigen-binding domains. In certain embodiments, amino acid residues located at the same position in the first antigen-binding domain and the second antigen-binding domain link together to form a bond. In certain embodiments, amino acid residues located at different positions in the first antigen-binding domain and the second antigen-binding domain link together to form a bond.

[0140] The positions of amino acid residues in antigen-binding domains can be indicated according to the Kabat numbering or EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. For example, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at the same corresponding position in each antigen-binding domain, the positions of those amino acid residues can be indicated by the same number according to the Kabat numbering or EU numbering system. Alternatively, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at different, non-corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by different numbers according to the Kabat numbering or EU numbering system.

[0141] In one aspect of the above, at least one of the two or more bonds linking the antigen-binding domains is a covalent bond. In a particular aspect, a covalent bond is formed by direct cross-linking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. The cross-linked amino acid residue is, for example, cysteine, and the formed covalent bond is, for example, a disulfide bond. At least one of the cross-linked cysteine ​​residues may be located within a hinge region. In another specific embodiment, a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the amino acid residue to be crosslinked is, for example, lysine.

[0142] In one aspect of the above, at least one of the two or more bonds linking the antigen-binding domain is a non-covalent bond. In a particular aspect, the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond.

[0143] In one embodiment of the above-mentioned aspect, the antibody fragment is Fab, Fab', scFab, Fv, scFv, or a single-domain antibody.

[0144] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the constant region. In a particular aspect, the amino acid residue is located within the CH1 region, for example, at a position selected from the group consisting of EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213, and 214 of the CH1 region. In one exemplary embodiment, the amino acid residue is located at EU numbering position 191 of the CH1 region, and the amino acid residues at EU numbering position 191 in the CH1 regions of the two antigen-binding domains are linked together to form a bond. In some embodiments of the aforementioned aspect, a disulfide bond is formed between amino acid residues at EU numbering position 191 in the CH1 region of the first antigen-binding domain and the second antigen-binding domain. In some aspects of the aforementioned plane, an additional one, two, or more disulfide bonds are formed between the first antigen-binding domain and the second antigen-binding domain via amino acid residues located at the following positions according to EU numbering in each of the CH1 regions of the first and second antigen-binding domains: (a) Between amino acid residues at any of the positions 131-138, 194, and 195 in each of the two antigen-binding domains; (b) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (c) Between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (d) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (e) Between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains; (f) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (g) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (h) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (i) Between the amino acid residue at position 138 in each of the two antigen-binding domains, and between the amino acid residue at position 194 in each of the two antigen-binding domains; (j) Between the amino acid residue at position 131 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (k) Between the amino acid residue at position 132 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (l) Between the amino acid residue at position 133 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (m) Between the amino acid residue at position 134 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (n) Between the amino acid residue at position 135 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (o) Between the amino acid residue at position 136 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; (p) Between the amino acid residue at position 137 in each of the two antigen-binding domains, and between the amino acid residue at position 195 in each of the two antigen-binding domains; and (q) Between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains. In some embodiments of the aforementioned aspects, either the first or second antigen-binding domain contains one, two or more charged amino acid residues at positions 136–138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more inversely charged amino acid residues at positions 193–195 (EU numbering) in each CH1 region. In some embodiments of the aforementioned aspects, one of the first and second antigen-binding domains contains one, two or more positively charged amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first and second antigen-binding domains contains one, two or more negatively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. In some embodiments of the aforementioned aspects, one of the first and second antigen-binding domains contains one, two or more negatively charged amino acid residues at positions 136–138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first and second antigen-binding domains contains one, two or more positively charged amino acid residues at positions 193–195 (EU numbering) in each CH1 region. In some embodiments of the aforementioned aspect, either the first or second antigen-binding domain has the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 that is glutamic acid (E) or aspartic acid (D); (b) The amino acid residue at position 137 that is glutamic acid (E) or aspartic acid (D); (c) The amino acid residue at position 138 is glutamic acid (E) or aspartic acid (D). It includes one, two or more of the following; and The other antigen-binding domain of the first and second antigen-binding domains consists of the following amino acid residues (according to EU numbering) in each CH1 region: (d) The amino acid residue at position 193 that is lysine (K), arginine (R), or histidine (H); (e) the amino acid residue at position 194 which is lysine (K), arginine (R), or histidine (H); and (f) The amino acid residue at position 195, which is lysine (K), arginine (R), or histidine (H). It includes one, two, or more of them. In some embodiments of the aforementioned aspect, either the first or second antigen-binding domain has the following amino acid residues (according to EU numbering) in each CH1 region: (a) The amino acid residue at position 136 that is lysine (K), arginine (R), or histidine (H); (b) The amino acid residue at position 137 that is lysine (K), arginine (R), or histidine (H); (c) The amino acid residue at position 138 is lysine (K), arginine (R), or histidine (H). Includes one or more of the following; and The other antigen-binding domain of the first and second antigen-binding domains consists of the following amino acid residues (according to EU numbering) in each CH1 region: (d) The amino acid residue at position 193, which is glutamic acid (E) or aspartic acid (D); (e) the amino acid residue at position 194 which is glutamic acid (E) or aspartic acid (D); and (f) The amino acid residue at position 195, which is glutamic acid (E) or aspartic acid (D) Includes one or more of the following. In some embodiments of the aforementioned aspects, one of the first and second antigen-binding domains contains one, two or more hydrophobic amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first and second antigen-binding domains contains one, two or more hydrophobic amino acid residues at positions 193-195 (EU numbering) in each CH1 region. In some aspects of the aforementioned configuration, the hydrophobic amino acid residues are alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp).

[0145] In some embodiments of the aforementioned aspect, either the first or second antigen-binding domain contains one "knob" amino acid residue at positions 136-138 (EU numbering) in each CH1 region, and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more "whole" amino acid residues at positions 193-195 (EU numbering) in each CH1 region. In some embodiments, the “knob” amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the “whole” amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (T), or serine (S).

[0146] In some embodiments of the aforementioned aspect, either the first or second antigen-binding domain contains one, two or more aromatic amino acid residues at positions 136-138 (EU numbering) in each CH1 region; and the other antigen-binding domain of the first or second antigen-binding domain contains one, two or more positively charged amino acid residues at positions 193-195 (EU numbering) in each CH1 region. In some embodiments, the aromatic amino acid residue is selected from the group consisting of tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe); and the positively charged amino acid residue is selected from the group consisting of lysine (K), arginine (R), or histidine (H). In a particular embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the hinge region, for example, at a position selected from the group consisting of EU numbering positions 216, 218, and 219 of the hinge region. In a particular embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the CL region, for example, at a position selected from the group consisting of EU numbering positions 109, 112, 121, 126, 128, 151, 152, 153, 156, 184, 186, 188, 190, 200, 201, 202, 203, 208, 210, 211, 212, and 213 of the CL region. In an exemplary embodiment, the amino acid residue is located at EU numbering position 126 of the CL region, and the amino acid residues at EU numbering position 126 in the CL regions of the two antigen-binding domains link together to form a binding. In a particular embodiment, an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain are linked to form a bond. In an exemplary embodiment, an amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and an amino acid residue at EU numbering position 126 in the CL region of the second antigen-binding domain are linked to form a bond.

[0147] In one embodiment of the above, the steady-state region is of human origin. In a particular embodiment, the subclass of the heavy-chain steady-state region is one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a particular embodiment, the subclass of the CH1 region is one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε. In a particular embodiment, the subclass of the CL region is κ or λ.

[0148] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within a variable region. In a particular aspect, the amino acid residue is located within the VH region, for example, at a position selected from the group consisting of Kabat numbering positions 8, 16, 28, 74, and 82b within the VH region. In a particular aspect, the amino acid residue is located within the VL region, for example, at a position selected from the group consisting of Kabat numbering positions 100, 105, and 107 within the VL region.

[0149] In one embodiment of the above aspect, both the first and second antigen-binding domains include a Fab and a hinge region. In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region, for example, a cysteine ​​residue in the hinge region. Examples of such cysteine ​​residues include those at EU numbering positions 226 and 229. In another specific embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region, for example, a cysteine ​​residue not present in the wild-type Fab or hinge region. Such mutant amino acid residues can be introduced into the wild-type Fab or hinge region by means of, for example, amino acid substitution. In each of the CH1 region, hinge region, CL region, VH region, and VL region, sites of amino acid residues that can initiate binding between antigen-binding domains are disclosed herein, and for example, cysteine ​​residues can be introduced into these sites. Alternatively, in another embodiment, amino acid residues present in the wild-type Fab or hinge region that may be involved in the binding between antigen-binding domains (e.g., cysteine ​​residues) may be substituted or deleted with other amino acid residues. Examples of such cysteine ​​residues include the cysteine ​​residues at EU numbering positions 220, 226, and 229 in the hinge region, and the cysteine ​​residue at position 214 in the CL region. In a particular embodiment, the antigen-binding molecule of the present disclosure is F(ab')2, wherein both the first and second antigen-binding domains include a Fab and a hinge region.

[0150] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the non-antibody protein is either a pair of ligands and / or receptors that bind specifically to each other. Examples of receptors here include receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, C. difficile antigens, costimulatory molecules, and cell adhesion molecules.

[0151] In one aspect of the foregoing, the antigen-binding molecule of the Disclosure further comprises an Fc region and is, for example, a full-length antibody. In a particular aspect, the Fc region of the antigen-binding molecule of the Disclosure is introduced with one or more amino acid mutations that promote multimerization of the Fc region. Such amino acid mutations include, for example, amino acid mutations at at least one position selected from the group consisting of EU numbering positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447 (see, for example, WO2016 / 164480). In a particular aspect, the multimerization is hexamerization.

[0152] <Antigen to which antigen-binding molecules bind> In one aspect of the above, the first and second antigen-binding domains both bind to the same antigen. In a particular aspect, the first and second antigen-binding domains both bind to the same epitope on the same antigen. In another particular aspect, each of the first and second antigen-binding domains binds to a different epitope on the same antigen. In a particular aspect, the antigen-binding molecule of the present disclosure is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets one specific antigen. In one embodiment of the above aspect, each of the first and second antigen-binding domains binds to a different antigen. In another aspect of the foregoing, the antigen-binding molecule of the Disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). In this Specification, a clamping antigen-binding molecule means an antigen-binding molecule that specifically binds to an antigen-antigen complex formed from an antigen A and an antigen-binding molecule that binds to that antigen A, thereby increasing the binding activity of the antigen-binding molecule to that antigen A (or stabilizing the antigen-antigen complex formed from the antigen A and the antigen-binding molecule that binds to that antigen A). For example, a CD3 clamping antibody can specifically bind to an antigen-antibody complex formed from CD3 and an antibody with reduced binding ability to CD3 (a CD3-attenuated antibody), thereby increasing the binding activity of the CD3-attenuated antibody to CD3 (or stabilizing the antigen-antibody complex formed from CD3 and the CD3-attenuated antibody). In certain aspects, the first and / or second antigen-binding domains in the antigen-binding molecule of the Disclosure may be antigen-binding domains derived from a clamping antigen-binding molecule (clamping antigen-binding domains). In one embodiment of the above aspect, the first and second antigen-binding domains both have the same amino acid sequence. In another embodiment, each of the first and second antigen-binding domains has a different amino acid sequence.

[0153] In one embodiment of the above aspect, at least one of the two antigens to which the first and second antigen-binding domains bind is a soluble protein or a membrane protein.

[0154] <Function of antigen-binding molecules> In one aspect of the foregoing, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one less bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (for example, a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0155] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has an activity that controls the interaction between two antigen molecules. Although not bound by any particular theory, this interaction-controlling activity is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In certain aspects, the antigen-binding molecule of the present disclosure can enhance or reduce the interaction between two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0156] In a particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are a ligand and its receptor, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the activation of the receptor by the ligand. In another particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are an enzyme and its substrate, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the catalytic reaction of the enzyme with respect to the substrate.

[0157] In another specific embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the Disclosure has the activity to facilitate interaction between a cell expressing a first antigen and a cell expressing a second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen are cytotoxic cells and their target cells, respectively, and the antigen-binding molecule of the Disclosure facilitates damage to the target cells by the cytotoxic cells. Cytotoxic cells are, for example, T cells, NK cells, monocytes, or macrophages.

[0158] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has an activity that controls the activation of two antigen molecules that are activated by association with each other. Although not bound by any particular theory, this activity that controls the activation is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can enhance or reduce the activation of two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). For example, the antigen molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0159] In one aspect of the aforementioned aspect, the antigen-binding molecule of the Disclosure is resistant to protease cleavage. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the Disclosure only in that it has one fewer bond between its two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular aspect, the antigen-binding molecule of the Disclosure exhibits an increased proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment compared to a control antigen-binding molecule. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits a decreased proportion of specific fragments (e.g., Fab monomers) generated after protease treatment compared to a control antigen-binding molecule.

[0160] In one embodiment of the aforementioned aspect, when the antigen-binding molecule of the present disclosure is treated with a protease, a dimer of the antigen-binding domain or a fragment thereof (e.g., a crosslinked Fab dimer) is cleaved. In a particular embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the present disclosure in that it has one fewer bond between two antigen-binding domains is treated with the protease, a monomer of the antigen-binding domain or a fragment thereof is cleaved. In a further embodiment, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). In these embodiments, the protease can cleave the hinge region of the antigen-binding molecule.

[0161] <Pharmaceutical composition> In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule and a pharmaceutically acceptable carrier.

[0162] <Uses of antigen-binding molecules> In one aspect, the present disclosure provides a method for holding two antigen molecules in spatially close proximity, (a) To provide an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more binding sites; (b) Adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. This provides a method that includes this. In certain embodiments, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In further embodiments, the other binding in (b) above is binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The Disclosure also provides a method for holding two antigen molecules in spatial proximity, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for use in holding two antigen molecules in spatial proximity.

[0163] In another aspect, the present disclosure provides a method for controlling the interaction between two antigen molecules, (a) To provide an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more binding sites; (b) Adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. This provides a method that includes this. In certain embodiments, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In further embodiments, the other binding in (b) above is binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The Disclosure also provides a method for controlling the interaction between two antigen molecules, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for use in controlling the interaction between two antigen molecules.

[0164] In another aspect, the present disclosure provides a method for controlling the activity of two antigen molecules that are activated by association with each other, (a) To provide an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more binding sites; (b) Adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c)(b) The antigen-binding molecule prepared in (c)(b) is brought into contact with the two antigen molecules. This provides a method that includes this. In certain embodiments, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In further embodiments, the other binding in (b) above is binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The Disclosure also provides a method for controlling the activity of two antigen molecules that are activated by association with each other, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for use in controlling the activity of two antigen molecules that are activated by association with each other.

[0165] Furthermore, in another aspect, the present disclosure relates to a method for increasing the resistance of an antigen-binding molecule to protease cleavage, (a) To provide an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more binding sites; and (b) Adding another bond to the antigen-binding molecule that links the two antigen-binding domains together. This provides a method that includes this. In a particular embodiment, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between antigen-binding domains originates from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other binding in (b) above is binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0166] The antigen-binding molecules used in these various methods may have the characteristics of antigen-binding molecules described herein.

[0167] <Method for producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having the activity of holding two antigen molecules in spatially close proximity, (a) To provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as a starting point for linking the two antigen-binding domains; (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that another bond is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via two or more binding sites. This provides a method that includes this. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region).

[0168] In another aspect, the present disclosure relates to a method for producing an antigen-binding molecule having activity to control the interaction between two antigen molecules, (a) To provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as a starting point for linking the two antigen-binding domains; (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that another bond is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via two or more binding sites. This provides a method that includes this. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region).

[0169] Furthermore, in another aspect, the present disclosure relates to a method for producing an antigen-binding molecule having activity to control the activation of two antigen molecules that are activated by association with each other, (a) To provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as a starting point for linking the two antigen-binding domains; (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that another bond is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via two or more binding sites. This provides a method that includes this. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region).

[0170] Furthermore, in another aspect, the present disclosure relates to a method for producing an antigen-binding molecule with increased resistance to protease cleavage, (a) To provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as a starting point for linking the two antigen-binding domains; (b) Introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that another bond is added that links the two antigen-binding domains; (c) Introducing the nucleic acid prepared in (b) into host cells; (d) culturing host cells so that the two polypeptides express; and (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via two or more binding sites. This provides a method that includes this. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region). The antigen-binding molecules produced in these various situations may have the characteristics of antigen-binding molecules described herein.

[0171] <Screening method for antigen-binding molecules> In another context, the present disclosure provides a method for identifying a novel set of protein molecules that are activated by association with one another. (a) Provide any two protein molecules; (b) To produce an antigen-binding molecule comprising two antigen-binding domains that bind to each of the two protein molecules, and having the activity to hold the two protein molecules in close proximity, by a manufacturing method of the present disclosure; (c) Contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) Evaluate whether the two protein molecules are activated. This provides a method that includes this. In a particular embodiment, at least one of the protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0172] <Linking of antigen-binding domains> In a non-limiting embodiment, two or more antigen-binding domains contained in the antigen-binding molecule of the Disclosure are linked to each other via one or more bonds. In a preferred embodiment, the antigen-binding domains contained in the antigen-binding molecule of the Disclosure have activity to bind to an antigen on their own. In such embodiment, the antigen-binding molecule of the Disclosure containing two antigen-binding domains can bind to two or more antigen molecules, the antigen-binding molecule of the Disclosure containing three antigen-binding domains can bind to three or more antigen molecules, the antigen-binding molecule of the Disclosure containing four antigen-binding domains can bind to four or more antigen molecules, and the antigen-binding molecule of the Disclosure containing N antigen-binding domains can bind to N or more antigen molecules.

[0173] In certain embodiments, at least one of the bindings between antigen-binding domains contained in the antigen-binding molecule of this disclosure is different from those found in native antibodies (e.g., in the Fab or hinge region of wild-type antibodies). Examples of bindings found between antigen-binding domains in native antibodies (e.g., native IgG antibodies) include disulfide bonds in the hinge region. Bindings between amino acid residues located outside the hinge region may also be bindings between amino acid residues within an antibody fragment (e.g., Fab), and include bindings between heavy chains (HH form), bindings between light chains (LL form), and bindings between heavy chains and light chains (HL or LH form) (see Figure 21). Examples of amino acid residues in the heavy or light chain that serve as the starting point for bindings between antigen-binding domains include amino acid residues at the aforementioned positions within the variable region (VH or VL region) or the constant region (CH1 region, hinge region, or CL region).

[0174] In one non-limiting embodiment, a plurality of amino acid residues located at distant positions on the primary structure of at least one of the two or more antigen-binding domains contained in the antigen-binding molecule of the present disclosure serve as the starting point for binding between the antigen-binding domains. The distance between the plurality of amino acid residues is such that, as a result of the linkage between the antigen-binding domains by binding starting from each amino acid residue, a structure of two or more antigen-binding domains that are sufficiently close together is achieved. The distance between the multiple amino acid residues may be, for example, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 11 or more amino acids, 12 or more amino acids, 13 or more amino acids, 14 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 30 or more amino acids, 35 or more amino acids, 40 or more amino acids, 45 or more amino acids, 50 or more amino acids, 60 or more amino acids, 70 or more amino acids, 80 or more amino acids, 90 or more amino acids, 100 or more amino acids, 110 or more amino acids, 120 or more amino acids, 130 or more amino acids, 140 or more amino acids, 150 or more amino acids, 160 or more amino acids, 170 or more amino acids, 180 or more amino acids, 190 or more amino acids, 200 or more amino acids, 210 or more amino acids, or 220 or more amino acids. Furthermore, the number of bindings between antigen-binding domains, and the number of amino acid residues that initiate such bindings, are such that, as a result of the linkage between antigen-binding domains, a structure of two or more sufficiently close antigen-binding domains is achieved. This number may be, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. In a particular embodiment, as long as the linkage between antigen-binding domains by three or more bindings starting from three or more amino acid residues in each antigen-binding domain results in the formation of structures of two or more sufficiently close antigen-binding domains, the distance between any two amino acid residues selected from the three or more amino acid residues on the primary structure may be seven amino acids or more for at least one pair of amino acid residues, and less than seven amino acids for the remaining pairs of amino acid residues.

[0175] In relation to the antigen-binding domains contained in the antigen-binding molecules of this disclosure, "sufficiently close" means that two or more antigen-binding domains are close enough to achieve the desired function (activity) of the antigen-binding molecules of this disclosure. Examples of such desired functions (activities) include the activity of holding two antigen molecules in spatially close proximity, the activity of controlling the interaction between two antigen molecules, the activity of promoting the activation of a receptor by a ligand, the activity of promoting the catalytic reaction of an enzyme to a substrate, the activity of promoting the interaction between a cell expressing a first antigen and a cell expressing a second antigen, the activity of promoting damage to target cells by cells with cytotoxic activity (e.g., T cells, NK cells, monocytes, macrophages, etc.), the activity of controlling the activation of two antigen molecules that are activated by association with each other, and the resistance of the antigen-binding molecule to protease cleavage.

[0176] In one non-limiting embodiment, the bond between antigen-binding domains in the antigen-binding molecule of the present disclosure may be a covalent bond or a non-covalent bond. Such a covalent bond may be formed by the direct crosslinking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain, for example, a disulfide bond between cysteine ​​residues. The amino acid residues that are directly crosslinked may be located on an antibody fragment such as Fab, or within a hinge region. In another embodiment, a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a crosslinking agent. For example, when crosslinking is performed using an amine-reactive crosslinking agent, crosslinking can be performed via the free amino group of the N-terminal amino acid of the antigen-binding domain, or via the primary amine of the side chain of the lysine residue in the antigen-binding domain.Amine-reactive crosslinking agents include functional groups that form chemical bonds with primary amines (e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, and fluoroesters). Typical examples include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl) suberate), DSP (dithiobis(succinimidyl propionate)), and DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate): 3,3'-dithiobis(sulfosuccinimidylsulfonate), DST (disuccinimidyl tartrate), BSOCOES (bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone), EGS (ethylene glycol bis(succinimidyl succinate)), Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), and DFDNB (1,5-difluoro-2,4-dinitrobenzene) is one example.Other examples of crosslinking agents include carboxylamine-reactive, sulfhydryl-reactive, aldehyde-reactive, and photoreactive crosslinking agents. The non-covalent bond connecting the antigen-binding domain may be an ionic bond, a hydrogen bond, or a hydrophobic bond.

[0177] Whether a particular antigen-binding molecule has a greater number of bindings between its antigen-binding domains compared to a control antigen-binding molecule (for example, an antigen-binding molecule with a structure substantially similar to that of a natural antibody) can be evaluated, for example, by the following method. First, the antigen-binding molecule of interest and the control antigen-binding molecule are treated with a protease that cleaves the antigen-binding domain (for example, papain and Lys-C, which cleave the N-terminal side of the site where hinge regions are cross-linked), and then subjected to non-reductive electrophoresis. Next, an antibody that recognizes a part of the antigen-binding domain (for example, an anti-kappa chain HRP-labeled antibody) is used to detect the fragment present after protease treatment. If only monomers of the antigen-binding domain (e.g., Fab monomers) are detected for the control antigen-binding molecule, and multimers of the antigen-binding domain (e.g., Fab dimers) are detected for the antigen-binding molecule of interest, then the antigen-binding molecule of interest can be evaluated as having a greater number of bindings between its antigen-binding domains compared to the control antigen-binding molecule. The formation of disulfide bonds between cysteines in modified antigen-binding molecu...

Claims

1. A method for producing an antibody preparation, comprising the steps of contacting an antibody solution with a reducing agent, and subsequently re-oxidizing the antibody, wherein the antibody comprises a first antigen-binding domain and a second antigen-binding domain linked to each other by at least one disulfide bond, and the at least one disulfide bond is formed between amino acid residues at EU numbering position 191 in the CH1 region of each of the first antigen-binding domain and the second antigen-binding domain. The method wherein the antibody solution comprises antibodies in two structural isoforms that differ by at least one disulfide bond.

2. The method according to claim 1, wherein the population of antibody structural isoforms having at least one disulfide bond is preferentially enriched or increased.

3. The method according to claim 1 or 2, wherein the antibody is an IgG antibody.

4. The method according to claim 3, wherein the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

5. The method according to any one of claims 1 to 4, wherein the pH of the reducing agent that is contacted with the antibody is 3 to 10.

6. The reducing agents are TCEP, 2-MEA, DTT, cysteine, GSH, and Na. 2 SO 3 The method according to any one of claims 1 to 5, selected from the group consisting of the following.

7. The method according to any one of claims 1 to 6, wherein the contact step is carried out for at least 30 minutes.

8. The method according to any one of claims 1 to 7, wherein the contact step is carried out at a temperature of 20 degrees Celsius to 37 degrees Celsius.

9. The method according to claim 8, wherein the contact step is performed at 23 degrees Celsius, 25 degrees Celsius, or 37 degrees Celsius.

10. The method according to claim 9, wherein the contact step is performed at 23 degrees Celsius.

11. The method according to any one of claims 1 to 10, wherein the concentration of the antibody is from 1 mg / ml to 50 mg / ml.

12. The method according to any one of claims 1 to 11, wherein the antibody is partially purified by affinity chromatography before the step of contacting it with the reducing agent.

13. The method according to any one of claims 1 to 12, further comprising the step of removing the reducing agent by dialysis or chromatography before the step of reoxidizing the antibody.

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