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

Abstract

An object of the present invention is to provide a novel antigen-binding molecule that has the activity of regulating, for example, interactions between antigen molecules. 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 each other via at least one disulfide bond formed between the 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 a preferred form of antibody protein, and a method for removing disulfide heterogeneity of a recombinant antibody protein. TIFF2023512390000215.tif64144

Description

[Technical Field]

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

[0002] Antibodies are proteins that bind specifically to antigens with high affinity. A variety of molecules, from small molecules to proteins, are known to act as antigens. Since the development of monoclonal antibody production techniques, antibody engineering techniques have advanced, making it easier to obtain antibodies that recognize specific molecules. Furthermore, antibody engineering techniques are expanding beyond the modification of proteins themselves to include the addition of new functions, including conjugation with small molecules. For example, cysteine-engineered antibodies, which contain a free cysteine ​​amino acid in the heavy or light chain, are used in medical applications as antibody-drug conjugates (ADCs) (Patent Document 1).

[0003] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Antibodies not only bind to antigens and have agonistic and antagonistic effects, 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). Utilizing these antibody functions, pharmaceuticals for cancer, immune diseases, chronic diseases, infectious diseases, and the like have been developed (Non-Patent Document 1).

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

[0005] However, because such antibodies may not fully exert the expected effects if they remain in their natural IgG form, second-generation antibody drugs have been developed in which the functions of natural IgG antibodies have been artificially enhanced or added, or attenuated or deleted, depending on the application of the antibody. Examples of second-generation antibody drugs include antibodies with enhanced or deleted effector functions (Non-Patent Document 3), antibodies that bind to antigens in a pH-dependent manner (Non-Patent Document 4), and antibodies that bind to two or more types of antigens with a single molecule (antibodies that bind to two types of antigens are generally called "bispecific antibodies") (Non-Patent Document 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 with cancer cells by binding to a protein expressed on the cell membrane of T cells as one antigen and a cancer antigen as the other antigen (Non-Patent Document 7, Non-Patent Document 8, Patent Document 2). Examples of bispecific antibodies 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 are 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 is engineered to form a new antigen-binding site (Fcab) (Non-Patent Document 9), and molecules in which Fab-Fab are connected in tandem (Non-Patent Document 10).

[0007] On the other hand, antibodies with effector functions act on normal cells that express low levels of the target antigen, which can easily cause side effects. Therefore, attempts have been made to make antibody drugs exert their effector functions specifically in target tissues. For example, there are reported antibodies whose binding activity changes upon binding to cellular metabolites (Patent Document 3), antibodies that exhibit antigen-binding ability upon cleavage by proteases (Patent Document 4), and a technology that controls antibody-mediated crosslinking between chimeric antigen receptor T cells (CAR-T cells) and cancer cells by adding a compound (ABT-737) (Non-Patent Document 11).

[0008] Obtaining agonist antibodies against some targets can be difficult, and various methods have been developed, particularly for membrane proteins such as G-protein-coupled receptors (Non-Patent Document 12). Therefore, a simple method for enhancing the agonistic activity of antibodies against such targets is needed. Existing methods include crosslinking anti-DR4 (Death Receptor 4) or anti-DR5 (Death Receptor 5) antibodies (Non-Patent Document 13), multimerizing anti-DR5 (Death Receptor 5) antibody nanobodies (Non-Patent Document 14), converting anti-thrombopoietin receptor antibodies into sc(Fv)2 covalently linked diabodies (Non-Patent Document 15), changing the IgG subclass of anti-CD40 antibodies (Non-Patent Document 16), hexamerizing anti-CD20 antibodies (Non-Patent Document 17), and preparing spherical antibody-like molecules (Patent Document 5). Furthermore, reported techniques using bispecific antibodies include a technique in which two types of appropriate anti-erythropoietin antibodies with different epitopes are combined as a bispecific antibody (Non-Patent Document 18), a technique in which antibodies for guide and effector function are combined as a bispecific antibody (Non-Patent Document 19), and a technique in which multiple types of antibody fragments with different epitopes into which Cys residues have been introduced are combined and conjugated (Non-Patent Document 20, Non-Patent Document 21, Patent Document 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

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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 objective of the present invention is to provide novel antigen-binding molecules (e.g., IgG antibodies) that have the activity of modulating the interaction between two or more antigen molecules, and / or methods for producing or using such antigen-binding molecules. More specifically, the present invention solves the problem that conventional antibodies (e.g., wild-type IgG) lack controlled mobility of the two antigen-binding domains (e.g., two Fab arms) by introducing one or more engineered disulfide bonds between the two antigen-binding domains (two Fabs) of the antibody through the introduction of mutations into the heavy and / or light chains. 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 the antibody, such antibodies can form one or more disulfide bonds between the two antigen-binding domains (two Fabs). [Means for solving the problem]

[0012] The antigen-binding molecules of the present invention comprise 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 that are not within the hinge region. The terms "linked" and "formed" include cases where a disulfide bond has already formed and cases where a disulfide bond has not formed but can be formed later under appropriate conditions.

[0013] In one non-limiting aspect, one or more engineered disulfide bonds between the two Fabs of an IgG antibody allow for 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 a corresponding wild-type IgG antibody that does not have one or more engineered disulfide bonds. In one non-limiting aspect, one or more engineered disulfide bonds between the two Fabs of an IgG antibody improve the agonist activity of the IgG antibody compared to a corresponding wild-type IgG antibody that does not have one or more engineered disulfide bonds. In addition, in another non-limiting aspect, one or more engineered disulfide bonds between the two Fabs of an IgG antibody improve the resistance of the IgG antibody to protease digestion compared to a corresponding wild-type IgG antibody that does not have one or more engineered disulfide bonds.

[0014] While preparing antibodies capable of forming one or more engineered disulfide bonds between their two Fabs, the present inventors further discovered that multiple conformational isoforms of the same antibody (same sequence) but with different disulfide structures, specifically, isoforms with "paired cysteines" and isoforms with "free or unpaired cysteines" (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 with one or more engineered disulfide bonds between their two Fabs. More specifically, the present invention describes methods for increasing the structural uniformity and relative abundance of antibodies in the "paired cysteine" form, i.e., antibodies with one or more engineered disulfide bonds formed between their two Fabs. In other words, the present invention describes methods for reducing the relative abundance of antibodies in the "free or unpaired cysteine" form, i.e., antibodies that do not have an engineered disulfide bond formed between the two Fabs of the antibody.

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

[0016] More specifically, the present invention provides: [1] A method for (i) producing an antibody preparation, (ii) purifying an antibody having a desired conformation, or (iii) improving the homogeneity of an antibody preparation, comprising: 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 wherein the at least one disulfide bond can be formed between amino acid residues that are not within a hinge region. [2] A method for (i) producing an antibody preparation, (ii) purifying an antibody preparation, or (iii) improving the homogeneity of an antibody preparation, comprising: The method comprises isolating a fraction of antibodies having a desired three-dimensional structure through one or more chromatographic steps selected from the group consisting of reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, and electrophoresis; wherein the antibodies having the desired three-dimensional structure are characterized by having at least one disulfide bond formed between amino acid residues that are not within the hinge region. [2A] The method according to [2], wherein one or more chromatography steps are ion exchange chromatography (IEC) and / or hydrophobic interaction chromatography (HIC), or mixed-mode chromatography of IEC and HIC. [3] The method of 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 within the hinge region. [3A] The method of [3], wherein the antibody preparation comprises two structural isoforms that differ only by at least one disulfide bond formed between amino acid residues that are not within the hinge region. [3B] The method of any one of [1] to [3A], wherein the population of antibody structural isoforms having at least one disulfide bond formed between amino acid residues that are not within the hinge region is preferentially enriched or increased. [3C] The method of any one of [1] to [3B], wherein a homogeneous antibody preparation is produced, the antibody having at least one disulfide bond formed between amino acid residues that are not within the hinge region in a molar ratio of at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%. [3D] The method of any one of [1] to [3C], wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a hinge region or does not comprise a hinge region. [3E] The method according to any one of [1] to [3D], wherein the amino acid residue not within the hinge region is an introduced or engineered 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 of 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 of 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 of 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 of [5], wherein the at least one disulfide bond is formed between the antigen-binding domains at any one of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, and 201 to 214 (EU numbering) in the CH1 region. [5.2] The at least one disulfide bond is located at any one of 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 (EU numbering) in the CH1 region. The method of [5], wherein the amino acid sequence is formed between the antigen-binding domains at any one of 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 of [5], wherein the at least one disulfide bond is formed between the antigen-binding domains at any one of positions 134, 135, 136, 137, 191, 192, 193, 194, 195, and 196 (EU numbering) in the CH1 region. [5.4] The method of [5], wherein the at least one disulfide bond is formed between the antigen-binding domains at any one of positions 135, 136, and 191 (EU numbering) in the CH1 region. [5.5] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues 119, 120, 121, 122, and 123 (EU numbering). [5.6] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues at positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140 (EU numbering). [5.7] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues 148, 149, and 150 (EU numbering). [5.8] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues at positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167 (EU numbering). [5.9] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues 174, 175, 176, 177, and 178 (EU numbering). [5.10] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering). [5.11] The method of [5], wherein the at least one disulfide bond is formed between amino acid residues in the first antigen-binding domain and the second antigen-binding domain selected from the group consisting of amino acid residues at positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214 (EU numbering). [5.12] The method of [5], wherein the difference in amino acid residue positions between the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. [5.13] The method of [5], wherein at least one disulfide bond linking the two antigen-binding domains is formed by linking the amino acid residue at position 135 (EU numbering) in the CH1 region of the first antigen-binding domain with the amino acid residue at any one of positions 132 to 138 (EU numbering) in the CH1 region of the second antigen-binding domain. [5.14] The method of [5], wherein at least one disulfide bond linking the two antigen-binding domains is formed by linking the amino acid residue at position 136 (EU numbering) in the CH1 region of the first antigen-binding domain with the amino acid residue at any one of positions 133 to 139 (EU numbering) in the CH1 region of the second antigen-binding domain. [5.15] The method of [5], wherein at least one disulfide bond linking the two antigen-binding domains is formed by linking the amino acid residue at position 191 (EU numbering) in the CH1 region of the first antigen-binding domain with the amino acid residue at any one of positions 188 to 194 (EU numbering) in the CH1 region of the second antigen-binding domain. [5.16] The method according to [5], wherein one disulfide bond is formed between the two antigen-binding domains at position 135 (EU numbering) in the CH1 region. [5.17] The method according to [5], wherein one disulfide bond is formed between the two antigen-binding domains at position 136 (EU numbering) in the CH1 region. [5.18] The method according to [5], wherein one disulfide bond is formed between the two antigen-binding domains at position 191 (EU numbering) 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 any one of [5] to [5A], wherein one disulfide bond is formed between the amino acid residue at position 191 (EU numbering) in the CH1 region of each of the first antigen-binding domain and the second antigen-binding domain; [6A] The method of [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 positions 131 to 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 residue at position 132 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; (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 residue at position 134 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; (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 residue at position 138 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. [6B] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more oppositely charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [6C] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more negatively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [6D] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more negatively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 193 to 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). including one, two or more of: the other antigen-binding domain of the first and second antigen-binding domains has the following amino acid residues (according to EU numbering) in each CH1 region: (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 following: [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). and the other of the first and second antigen-binding domains has the following amino acid residues (according to EU numbering) in each CH1 region: (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 of [6] or [6A], comprising one or more of: [6F-2] The method of [6] or [6A], wherein each of the first and second antigen-binding domains comprises any of the combinations of specific charge mutations (according to EU numbering) in each CH1 region listed in Table 7, Table 82, or Table 85. [6G] The method of [6] or [6A], wherein either one of the first or second antigen-binding domains contains one, two, or more hydrophobic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more hydrophobic amino acid residues at positions 193 to 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 of [6] or [6A], wherein either one of the first or second antigen-binding domains comprises one "knob" amino acid residue at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more "hole" amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [6J] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more "hole" amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one "knob" amino acid residue at positions 193 to 195 (EU numbering) in each CH1 region. [6K] The method of [6I] or [6J], wherein the "knob" amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "hole" amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (T), or serine (S). [6L] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more aromatic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [6M] The method of [6] or [6A], wherein either one of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more aromatic amino acid residues at positions 193 to 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 of [6] or [6A], wherein each of the first and second antigen-binding domains comprises any of the combinations of specific hydrophobic amino acid mutations (according to EU numbering) in each CH1 region listed in Table 10. [7] The method of 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 of [7], wherein the amino acid residues forming at least one disulfide bond between the two antigen-binding domains are located at any one of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213, according to the Kabat numbering system, in the CL region. [7.2] The method of [7], wherein the amino acid residue that forms at least one disulfide bond between the two antigen-binding domains is located at a position in the CL region 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 (Kabat numbering). [7.3] The method according to [7], wherein the amino acid residue through which at least one disulfide bond is formed between the two antigen-binding domains is located at position 126 (Kabat numbering) in the CL region. [7.4] The method of [7], wherein at least one disulfide bond 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. [7.5] The method of [7], wherein the amino acid residues forming at least one disulfide bond between the two antigen-binding domains are located at positions independently selected from the group consisting of positions 108, 109, 110, 111, and 112 (Kabat numbering). [7.6] The method of [7], wherein the amino acid residues through which at least one disulfide bond is formed between the two antigen-binding domains are located at positions independently selected from the group consisting of positions 151, 152, 153, 154, 155, and 156 (Kabat numbering). [7.7] The method of [7], wherein the amino acid residues forming at least one disulfide bond between the two antigen-binding domains are located at positions independently selected from the group consisting of positions 184, 185, 186, 187, 188, 189, and 190 (Kabat numbering). [7.8] The method of [7], wherein the amino acid residues forming at least one disulfide bond between the two antigen-binding domains are located at positions independently selected from the group consisting of positions 200, 201, 202, and 203 according to the Kabat numbering system. [7.9] The method of [7], wherein the amino acid residues through which at least one disulfide bond is formed between the two antigen-binding domains are located at positions independently selected from the group consisting of positions 208, 209, 210, 211, 212, and 213 (Kabat numbering). [7.10] The method according to any one of [7] to [7.9], wherein the difference in the position of the amino acid residue where 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 connecting the two antigen-binding domains is formed by linking the amino acid residue at position 126 (Kabat numbering) in the CL regions of the two antigen-binding domains. [8] The method of 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 of [8], wherein the amino acid residues in the CH1 region are selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering), and the amino acid residues in the CL region are selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 (Kabat numbering). [8.2] The method of [8], wherein at least one disulfide bond linking the two antigen-binding domains is formed by linking the amino acid residue at position 191 (EU numbering) in the CH1 region of the first antigen-binding domain with the amino acid residue at position 126 (Kabat numbering) 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 of any one of [1] to [4], wherein the at least one disulfide bond is formed between the variable regions of the first antigen-binding domain and the second antigen-binding domain. [9.1] The method of [9], wherein the amino acid residues that form at least one disulfide bond between the antigen-binding domains are present in the VH region. [9.2] The method of [9], wherein the amino acid residues forming at least one disulfide bond between the antigen-binding domains are located at a position in the VH region selected from the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b (Kabat numbering) in the VH region. [9.3] The method of [9], wherein an amino acid residue that forms at least one disulfide bond between the antigen-binding domains is present in the VL region. [9.4] The method of [9], wherein the amino acid residue that forms at least one disulfide bond between the antigen-binding domains is located at a position selected from the group consisting of positions 21, 27, 58, 77, 100, 105, and 107, according to the Kabat numbering system, in the VL region (subclass κ). [9.5] The method of [9], wherein the amino acid residues forming at least one disulfide bond between the antigen-binding domains are located at a position selected from the group consisting of positions 6, 19, 33, and 34, according to the Kabat numbering system, in the VL region (subclass λ). [9A] The method of [4], wherein the amino acid residues that form at least one disulfide bond between the two antigen-binding domains are present in the VHH region. [9B] The method of [9A], wherein the amino acid residue that forms at least one disulfide bond between the antigen-binding domains is located at a position selected from the group consisting of 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 according to Kabat numbering.

[10] The 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 binding of the two antigen-binding domains to two antigens that are in spatial proximity to each other; (b) the at least one disulfide bond holds the first antigen-binding domain and the second antigen-binding domain in closer spatial proximity to each other than in the same corresponding antibody lacking 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 relative to a corresponding identical antibody lacking the at least one disulfide bond; (d) the at least one disulfide bond increases the resistance of the antibody to protease cleavage relative to a corresponding identical antibody lacking 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 a corresponding identical antibody lacking the at least one disulfide bond; (f) the method produces an antibody preparation that is more homogeneous than the same antibody preparation that has not been treated by the method; (g) the method produces an antibody preparation whose biological activity is increased compared to the same antibody not treated by the method; (h) the method produces an antibody that has enhanced ability to hold two antigen molecules in close spatial proximity compared to the same antibody not treated by the method; (i) the method produces an antibody that has enhanced stability compared to the same antibody not treated by the method; and (j) The method preferentially enriches antibodies having at least one disulfide bond formed outside the hinge region, and the preferentially enriched form has a pharmaceutically desirable property selected from any of (a) to (i) above, compared to a preparation not treated by the method.

[11] The method of 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 of

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

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

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

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

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

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

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

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

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

[13] , wherein the first and second antigen-binding domains each have a different amino acid sequence. [14F] The method of any one of [1] to [14E], wherein at least one of the two antigens bound by the first and second antigen-binding domains is a soluble protein. [14G] The method of any one of [1] to [14E], wherein at least one of the two antigens bound by the first and second antigen-binding domains is a membrane protein. [14H] The method according to any one of [1] to [14G], which has the activity of regulating the interaction between two antigen molecules. [14I] The method according to [14H], which can enhance or weaken 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 a ligand and its receptor, respectively, and the antibody has the activity of promoting activation of the receptor by the ligand. [14K] The method of any one of [14H] to [14I], wherein the two antigen molecules are an enzyme and its substrate, respectively, and the antigen-binding molecule has the activity of promoting the catalytic reaction between the enzyme and the substrate. [14L] The method according to any one of [14H] to [14I], wherein the two antigen molecules are both proteins present on the cell surface, and the antibody has the activity of promoting interaction between cells expressing the first antigen and cells expressing the second antigen. [14M] Any of the methods described in [14L], wherein the cells expressing the first antigen are cells with cytotoxic activity, the cells expressing the second antigen are their target cells, and the antibody promotes damage to the target cells by the cells with cytotoxic activity. [14N] The method according to [14M], wherein the cells having cytotoxic activity are T cells, NK cells, monocytes, or macrophages. [14O] The method of [14N], wherein the antibody having at least one disulfide bond enhances or weakens the activation of two antigen molecules compared to the same corresponding antibody that does not have the 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-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[15] the method of any one of

[14] to [14P], wherein the first antigen-binding domain and the second antigen-binding domain are each capable of binding to CD3;

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

[15] , wherein the reducing reagent contacted with the antibody has a pH of about 3 to about 10. [16A] The method according to

[16] , wherein the reducing reagent contacted with the antibody has a pH of about 6, 7, or 8. [16B] The method according to

[16] , wherein the reducing reagent contacted with the antibody has a pH of about 7. [16C] The method according to

[16] , wherein the reducing reagent contacted with the antibody has a pH of about 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 from about 0.01 mM to about 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, or 100 mM, preferably about 0.01 mM to 25 mM.

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

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

[19] , wherein the contacting step is carried out for about 2 to about 48 hours. [20B] The method of any one of [1] to

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

[21] The method of any one of [1] to [20B], wherein the contacting 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 of any one of [1] to

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

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

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

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

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

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

[23] , further comprising isolating a fraction of the contacted antibodies having a desired conformation. [24A] The method according to

[24] , wherein the isolation technique is selected from the group consisting of reverse 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 isolation method 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 the reducing agent, preferably by dialysis, more preferably by chromatography.

[25] An IgG antibody preparation prepared by the method of any one of [1] to [24B], which has a homogeneous population of IgG antibodies having at least one disulfide bond outside the hinge region.

[26] An IgG antibody preparation prepared by the method of any one of [1] to

[25] , comprising IgG antibodies having at least one disulfide bond outside the hinge region in a molar ratio of at least 50%, 60%, 70%, 80%, or 90%, preferably at least 95%.

[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 the antibody 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 bonds. [2] the antigen-binding molecule of [1], wherein at least one of the bonds linking the two antigen-binding domains is a covalent bond; [3] the antigen-binding molecule of [2], wherein a covalent bond is formed by direct cross-linking between an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain; [4] the antigen-binding molecule of [3], wherein the cross-linked amino acid residue is cysteine; [5] The antigen-binding molecule of [4], wherein the formed covalent bond is a disulfide bond. [6] the antigen-binding molecule of [2], wherein a covalent bond is formed by cross-linking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a cross-linking agent; [7] The antigen-binding molecule of [6], wherein the crosslinking agent is an amine-reactive crosslinking agent. [8] the antigen-binding molecule of [7], wherein the cross-linked amino acid residue is lysine; [9] the antigen-binding molecule of [1], wherein at least one of the bonds linking the two antigen-binding domains is a non-covalent bond;

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

[11] The antigen-binding molecule of

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

[12] the antigen-binding molecule of

[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] the antigen-binding molecule of any one of [1] to

[12] , wherein at least one of the amino acid residues serving as the origin of binding between the antigen-binding domains is an artificially introduced mutated amino acid residue;

[14] the antigen-binding molecule of

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

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

[14] , wherein at least one of the first and second antigen-binding domains has antigen-binding activity by itself;

[16] the antigen-binding molecule of any one of [1] to

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

[17] the antigen-binding molecule of 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 in the first antigen-binding domain and the second antigen-binding domain.

[18] the antigen-binding molecule of 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 in the first antigen-binding domain and the second antigen-binding domain.

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

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

[20] The antigen-binding molecule of

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

[21] the antigen-binding molecule of

[19] or

[20] , wherein at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the antibody fragment;

[22] the antigen-binding molecule of

[21] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located in a constant region;

[23] the antigen-binding molecule of

[22] , wherein the constant region is derived from a human;

[24] the antigen-binding molecule of

[22] or

[23] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within the CH1 region;

[25] the antigen-binding molecule of

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

[26] the antigen-binding molecule of

[24] or

[25] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219 (EU numbering) in the CH1 region;

[27] The amino acid residues that serve as the origin of binding between antigen-binding domains are at 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 (EU numbering) in the CH1 region. 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.

[28] the antigen-binding molecule of

[27] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at position 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 (EU numbering) in the CH1 region;

[29] the antigen-binding molecule of

[28] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located at position 135, 136, or 191 (EU numbering) in the CH1 region;

[30] the antigen-binding molecule of 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 of

[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 positions 119, 120, 121, 122, and 123 (EU numbering);

[32] the antigen-binding molecule of

[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 positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140 (EU numbering);

[33] the antigen-binding molecule of

[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 positions 148, 149, and 150 (EU numbering).

[34] the antigen-binding molecule of

[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 positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167 (EU numbering);

[35] the antigen-binding molecule of

[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 positions 174, 175, 176, 177, and 178 (EU numbering);

[36] the antigen-binding molecule of

[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 positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering);

[37] the antigen-binding molecule of

[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 positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214 (EU numbering);

[38] the antigen-binding molecule of

[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 positions 218 and 219 (EU numbering).

[39] the antigen-binding molecule of 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 within 3 amino acids;

[40] the antigen-binding molecule of

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

[41] the antigen-binding molecule of

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

[42] the antigen-binding molecule of

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

[43] the antigen-binding molecule of

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

[44] the antigen-binding molecule of

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

[45] the antigen-binding molecule of

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

[42] , wherein one disulfide bond is formed between the amino acid residue at position 191 (EU numbering) in each CH1 region of the first antigen-binding domain and the second antigen-binding domain. [45B] The antigen-binding molecule of [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 antigen-binding domain and the second antigen-binding domain: (a) between amino acid residues at any of positions 131 to 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 residue at position 132 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; (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 residue at position 134 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; (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 residue at position 138 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. [45C] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more oppositely charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45D] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more negatively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45E] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more negatively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 193 to 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). including one, two or more of: the other of the first and second antigen-binding domains has the following amino acid residues (according to EU numbering) in each CH1 region: (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 antigen-binding molecule of [45A] or [45B], comprising one, two or more of: [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). and the other of the first and second antigen-binding domains has the following amino acid residues (according to EU numbering) in each CH1 region: (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 antigen-binding molecule of [45A] or [45B], comprising one or more of: [45G-2] The antigen-binding molecule of [45A] or [45B], wherein each of the first and second antigen-binding domains contains any of the combinations of specific charge mutations (according to EU numbering) in each CH1 region listed in Table 7, Table 82, or Table 85. [45H] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more hydrophobic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more hydrophobic amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45I-1] The antigen-binding molecule of [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 of [45A] or [45B], wherein each of the first and second antigen-binding domains comprises any of the combinations of specific hydrophobic amino acid mutations (according to EU numbering) in each CH1 region listed in Table 10. [45J] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains comprises one "knob" amino acid residue at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more "hole" amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45K] The antigen-binding molecule of [45A] or [45B], wherein either one of the first or second antigen-binding domains contains one, two, or more "hole" amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one "knob" amino acid residue at positions 193 to 195 (EU numbering) in each CH1 region. [45L] The antigen-binding molecule of [45J] or [45K], wherein the "knob" amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "hole" amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (T), or serine (S). [45M] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more aromatic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45N] The antigen-binding molecule of [45A] or [45B], wherein either one of the first and second antigen-binding domains contains one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains contains one, two, or more aromatic amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. [45O] The antigen-binding molecule of [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 of

[22] or

[23] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within the CL domain;

[47] the antigen-binding molecule of

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

[48] ​​the antigen-binding molecule of

[46] or

[47] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213, Kabat numbering, in the CL region;

[49] the antigen-binding molecule of

[48] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located 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, Kabat numbering, in the CL region;

[50] the antigen-binding molecule of

[49] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at position 126 according to the Kabat numbering system in the CL region;

[51] the antigen-binding molecule of 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 of

[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 positions 108, 109, 110, 111, and 112 according to the Kabat numbering system;

[53] the antigen-binding molecule of

[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 positions 121, 122, 123, 124, 125, 126, 127, and 128 according to the Kabat numbering system;

[54] the antigen-binding molecule of

[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 positions 151, 152, 153, 154, 155, and 156 according to the Kabat numbering system;

[55] the antigen-binding molecule of

[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 positions 184, 185, 186, 187, 188, 189, and 190 according to the Kabat numbering system;

[56] the antigen-binding molecule of

[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 positions 195 and 196 according to the Kabat numbering system;

[57] the antigen-binding molecule of

[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 positions 200, 201, 202, and 203 according to the Kabat numbering system.

[58] the antigen-binding molecule of

[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 positions 208, 209, 210, 211, 212, and 213 according to the Kabat numbering system;

[59] the antigen-binding molecule of 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 within 3 amino acids;

[60] the antigen-binding molecule of

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

[61] the antigen-binding molecule of 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 of

[61] , wherein the amino acid residues in the CH1 domain are selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering), and the amino acid residues in the CL domain are selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 (Kabat numbering);

[63] the antigen-binding molecule of

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

[64] the antigen-binding molecule of

[21] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within a variable region;

[65] the antigen-binding molecule of

[64] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within the VH region;

[66] the antigen-binding molecule of

[65] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one selected from the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b, according to the Kabat numbering, in the VH region;

[67] the antigen-binding molecule of

[64] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located in the VL region;

[68] the antigen-binding molecule of

[67] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one selected from the group consisting of positions 21, 27, 58, 77, 100, 105, and 107, according to the Kabat numbering system, in the VL region (subclass κ).

[69] the antigen-binding molecule of

[67] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one selected from the group consisting of positions 6, 19, 33, and 34, Kabat numbering, in the VL region (subclass λ).

[70] the antigen-binding molecule of

[64] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within the VHH domain;

[71] the antigen-binding molecule of

[70] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one selected from the group consisting of 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, according to Kabat numbering, in the VHH region;

[72] The antigen-binding molecule of 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 of

[72] , wherein the non-antibody protein is one of a pair of a ligand and a receptor that specifically bind to each other.

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

[73] , wherein the antigen-binding domain comprises a hinge region;

[75] the antigen-binding molecule of

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

[76] the antigen-binding molecule of

[75] , wherein the cysteine ​​residue is located at position 226 and / or 229 (EU numbering) in the hinge region;

[77] the antigen-binding molecule of

[74] or

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

[78] the antigen-binding molecule of

[77] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is located at any one selected from the group consisting of positions 216, 218, and 219 (EU numbering) in the hinge region.

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

[78] , wherein the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more bonds;

[80] the antigen-binding molecule of

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

[81] the antigen-binding molecule of

[80] , wherein the amino acid residue that serves as the origin of binding between the antigen-binding domains is located within a hinge region;

[82] the antigen-binding molecule of

[81] , wherein the amino acid residue serving as the origin of binding between the antigen-binding domains is a cysteine ​​residue in a hinge region;

[83] The antigen-binding molecule of 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 between cysteine ​​residues present in the hinge region.

[84] the antigen-binding molecule of

[83] , wherein the cysteine ​​residue is located at position 226 and / or 229 (EU numbering) in the hinge region;

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

[79] to

[84] , wherein at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the antibody fragment and at least one of the amino acid residues is present in the hinge region.

[86] the antigen-binding molecule of

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

[87] the antigen-binding molecule of any one of [1] to

[86] , wherein the antigen-binding domain comprises an Fc region;

[88] The antigen-binding molecule of

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

[89] the antigen-binding molecule of

[88] , wherein the amino acid mutations that promote multimerization comprise 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 of

[88] or

[89] , wherein the multimerization is hexamerization.

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

[87] to

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

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

[92] the antigen-binding molecule of any one of [1] to

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

[93] the antigen-binding molecule of

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

[94] The antigen-binding molecule of

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

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

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

[96] the antigen-binding molecule of

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

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

[93] to

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

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

[91] , wherein at least one of the two antigens bound by the first and second antigen-binding domains is a soluble protein;

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

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

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

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

[101] The antigen-binding molecule of

[100] , which can enhance or attenuate 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 of

[100] only in that it has one fewer bond between the two antigen-binding domains.

[102] The antigen-binding molecule of

[100] or

[101] , wherein the two antigen molecules are a ligand and its receptor, respectively, and the antigen-binding molecule has the activity of promoting activation of the receptor by the ligand.

[103] the antigen-binding molecule of

[100] or

[101] , wherein the two antigen molecules are an enzyme and its substrate, respectively, and the antigen-binding molecule has the activity of promoting the catalytic reaction of the enzyme with the substrate;

[104] The antigen-binding molecule of

[100] or

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

[105] The antigen-binding molecule of

[104] , wherein the cells expressing the first antigen are cells with cytotoxic activity, the cells expressing the second antigen are their target cells, and the antigen-binding molecule promotes damage to the target cells by the cells with cytotoxic activity.

[106] the antigen-binding molecule of

[105] , wherein the cell having cytotoxic activity is a T cell, a NK cell, a monocyte, or a macrophage;

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

[99] , which has the activity of regulating the activation of two antigen molecules that are activated by their association with each other.

[108] The antigen-binding molecule of

[107] , which enhances or attenuates 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 of

[107] only in that it has one less bond between the two antigen-binding domains.

[109] The antigen-binding molecule of

[107] or

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

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

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

[111] The antigen-binding molecule of

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

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

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

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

[113] The antigen-binding molecule of

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

[112] only in that the control antigen-binding molecule has one fewer bond between the two antigen-binding domains.

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

[99] , which is resistant to protease cleavage.

[115] The antigen-binding molecule of

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

[114] only in that it has one less bond between the two antigen-binding domains.

[116] the antigen-binding molecule of

[115] , which has an increased proportion of full-length molecules remaining after protease treatment, compared to the control antigen-binding molecule;

[117] the antigen-binding molecule of

[115] or

[116] , which has a reduced proportion of specific fragments generated after protease treatment compared to the control antigen-binding molecule;

[118] the antigen-binding molecule of any one of [1] to

[99] , which, when treated with a protease, excises a dimer of the antigen-binding domain or a fragment thereof;

[119] The antigen-binding molecule of

[118] , wherein when a control antigen-binding molecule is treated with the protease, a monomer of the antigen-binding domain or a fragment thereof is cleaved out, and the control antigen-binding molecule differs from the antigen-binding molecule of

[118] only in that the control antigen-binding molecule has one less bond between the two antigen-binding domains.

[120] The antigen-binding molecule of

[118] or

[119] , wherein the hinge region is cleaved by a protease.

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

[101] to

[106] ,

[108] to

[109] ,

[111] ,

[113] ,

[115] to

[117] , and

[119] to

[120] , wherein the one missing bond is formed starting from a mutant amino acid residue.

[122] the antigen-binding molecule of

[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 the antigen-binding molecule of 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 an interaction between two antigen molecules, comprising: (a) providing an antigen-binding molecule comprising 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) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules.

[125] A method for regulating the activity of two antigen molecules that are activated by their association with each other, comprising: (a) providing an antigen-binding molecule comprising 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) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules.

[126] A method for holding two antigen molecules in close spatial proximity, comprising: (a) providing an antigen-binding molecule comprising 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) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules.

[127] A method for positioning two antigen-binding domains in close spatial proximity and / or reducing the mobility of two antigen-binding domains, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains to each other;

[128] A method for increasing the resistance of an antigen-binding molecule to protease cleavage, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains to each other;

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

[129] A method for producing an antigen-binding molecule having an activity of regulating the interaction between two antigen molecules, comprising: (a) providing 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; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing a host cell to express the two polypeptides; and (e) Obtaining an antigen-binding molecule that is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more bonds.

[130] A method for producing an antigen-binding molecule that has the activity of regulating the activation of two antigen molecules that are activated by their association with each other, the method comprising: (a) providing 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; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing a host cell to express the two polypeptides; and (e) Obtaining an antigen-binding molecule that is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more bonds.

[131] A method for producing an antigen-binding molecule having the activity of holding two antigen molecules in close spatial proximity, comprising: (a) providing 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; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing a host cell to express the two polypeptides; and (e) Obtaining an antigen-binding molecule that is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more bonds.

[132] A method for producing an antigen-binding molecule in which two antigen-binding domains are located in close spatial proximity and / or the mobility of the two antigen-binding domains is reduced, comprising: (a) providing 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; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing a host cell to express the two polypeptides; and (e) Obtaining an antigen-binding molecule that is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more bonds.

[133] A method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) providing 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; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing a host cell to express the two polypeptides; and (e) Obtaining an antigen-binding molecule that is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more bonds.

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

[134] A method for identifying a novel set of protein molecules that are activated by associating with each other, comprising: (a) providing any two protein molecules; (b) producing an antigen-binding molecule comprising two antigen-binding domains that bind to the two protein molecules, respectively, by the method of any one of

[129] to

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

[135] The method according to

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

[0023] [Figure 1] 1 shows a non-reducing SDS-PAGE gel image for analyzing OKT3 and its variants with cysteine ​​substitutions (see Example 1). The two dashed lines indicate an upper band and a lower band. The lower band can be considered to correspond to an antibody with one or more engineered disulfide bonds formed between the CH1 regions. [Figure 2] Figure 2 shows a non-reducing SDS-PAGE gel image for analyzing OKT3 variants with cysteine ​​substitutions and OKT3-KiH (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 3] Figure 3 shows a non-reducing SDS-PAGE gel image for analyzing OKT3-KiH variants with cysteine ​​substitutions (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 4] Figure 4 shows a non-reducing SDS-PAGE gel image for analyzing OKT3-KiH variants with cysteine ​​substitutions (see Example 1). The two dashed lines indicate the upper and lower bands. [Figure 5]Figure 5 shows an image of a non-reducing SDS-PAGE gel (left panel) listing the 2-MEA concentration of each sample; and a graph (right panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 4). 20 mg / mL of antibody was reacted by mixing with various concentrations of 2-MEA. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM 2-MEA). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 6] Figure 6 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the 2-MEA concentration of each sample; and a graph (lower panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 4). 20 mg / mL of antibody was reacted by mixing with various concentrations of 2-MEA. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM 2-MEA). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 7] Figure 7 shows an image of a non-reducing SDS-PAGE gel (left panel) listing the 2-MEA concentration of each sample; and a graph (right panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 4). 1 mg / mL of antibody was reacted by mixing with various concentrations of 2-MEA. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM 2-MEA). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 8]Figure 8 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the 2-MEA concentration of each sample; and a graph (lower panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 4). 1 mg / mL of antibody was reacted by mixing with various concentrations of 2-MEA. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM 2-MEA). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 9] Figure 9 shows an image of a non-reducing SDS-PAGE gel (left panel) listing the TCEP concentration of each sample; and a graph (right panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) 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 (cross-linking rate or % cross-linking) for the control (0 mM TCEP). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 10] Figure 10 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the TCEP concentration of each sample; and a graph (lower panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 5). 20 mg / mL of antibody was reacted by mixing with each concentration of TCEP. ND means that no band was detected. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM TCEP). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 11]Figure 11 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the TCEP concentration of each sample; and a graph (lower panel) showing the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for each sample (see Example 5). 1 mg / mL of antibody was reacted by mixing with various concentrations of TCEP. ND means that no band was detected. The leftmost bar and dashed line represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking) for the control (0 mM TCEP). The numbers within the bars represent the ratio of the lower band to the upper band (cross-linking rate or % cross-linking). [Figure 12] Figure 12 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the reagent concentrations for each sample; and a graph 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 various concentrations 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 represent the ratio of the lower band to the upper band (crosslinking rate or % crosslinking). [Figure 13] Figure 13 shows an image of a non-reducing SDS-PAGE gel (upper panel) listing the reagent concentrations for each sample; and a graph (lower panel) showing the ratio of the lower band to the upper band (crosslinking rate or % crosslinking) for samples reacted with GSH (left) or NaSO (right) (see Example 6). 20 mg / mL of antibody was reacted by mixing with various concentrations of GSH or NaSO. 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 represent the ratio of the lower band to the upper band (crosslinking rate or % crosslinking). [Figure 14]Figure 14 shows an image of a non-reducing SDS-PAGE gel (see Example 7). 20 mg / mL of antibody was reacted by mixing with 2-MEA or TCEP at 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 an image of a non-reducing SDS-PAGE gel (see Example 7). 20 mg / mL of antibody was reacted by mixing with 2-MEA or TCEP at 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 (hatched) 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] FIG. 17 shows a chromatogram of cation exchange chromatography performed on an OKT3.S191C antibody sample as described in Example 8-1. [Figure 18]18 shows a gel image of a non-reducing 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] FIG. 19 shows a chromatogram of cation exchange chromatography performed on an OKT3.S191C0110 antibody sample as described in Example 8-2. [Figure 20] Figure 20 shows a gel image of a non-reducing SDS-PAGE analysis of OKT3.S191C0110 antibody sample 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 examples of modified antibodies in which Fabs are cross-linked, as described in Reference Example 1. This figure schematically shows the structural differences between a wild-type antibody (WT), a modified antibody in which the CH1 regions of antibody H chains are cross-linked (HH type), a modified antibody in which the CL regions of antibody L chains are cross-linked (LL type), and a modified antibody in which the CH1 region of the antibody H chain is cross-linked with the CL region of the antibody L chain (HL type or LH type). [Figure 22] Figure 22 shows the results of measuring the CD3-mediated agonistic activity of a wild-type anti-CD3ε antibody molecule (CD3-G4s) and modified antibody molecules (CD3-G4sLL, CD3-G4sHH) produced by linking its Fab-Fab via an additional disulfide bond, as described in Reference Example 4-3. [Figure 23] Figure 23 shows the results of measuring the CD3-mediated agonist activity of the wild-type anti-CD3ε antibody molecule (OKT3-G1s) and modified antibody molecules (OKT3-G1sLL, OKT3-G1sHH) produced by linking its Fab-Fab via an additional disulfide bond, as described in Reference Example 4-3. [Figure 24] Figure 24 shows the results of measuring the CD3- and / or CD28-mediated agonistic activity of a wild-type anti-CD3ε antibody molecule (CD3-G1s), an anti-CD28 antibody molecule (CD28-G1s), an anti-CD3ε x 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-Fabs of the bispecific antibody via an additional disulfide bond, as described in Reference Example 4-3. [Figure 25] Figure 25 shows the results of measuring the CD3- and / or CD28-mediated agonistic activity of the wild-type anti-CD3ε antibody molecule (OKT3-G1s), the anti-CD28 antibody molecule (CD28-G1s), the anti-CD3ε x 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 via an additional disulfide bond, as described in Reference Example 4-3. [Figure 26] 26 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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] 27 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions into the heavy-chain constant region of the 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] 28 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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] 29 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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]30 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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] 31 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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] 32 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions into the heavy-chain constant region of the anti-IL6R antibody, as described in Reference Example 5-2 (Figure 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]33 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAH.xxx-G1T4) produced by introducing cysteine ​​substitutions into the heavy-chain variable region of the anti-IL6R antibody, and a modified antibody (MRAH-G1T4.xxx) produced by introducing cysteine ​​substitutions 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] 34 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) prepared by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) prepared by introducing cysteine ​​substitutions into the light-chain constant region of an 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] 35 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an 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]36 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an 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] 37 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an 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] 38 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an 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]39 shows the results of protease treatment of an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) prepared by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) prepared by introducing cysteine ​​substitutions into the light-chain constant region of an 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 an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an anti-IL6R antibody, as described in Reference Example 6-2 (7 / 10). Each protease-treated antibody 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 an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an anti-IL6R antibody, as described in Reference Example 6-2 (8 / 10). Each protease-treated antibody 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 an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an anti-IL6R antibody (9 / 10), as described in Reference Example 6-2. Each protease-treated antibody 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 an anti-IL6R antibody (MRA), a modified antibody (MRAL.xxx-k0) produced by introducing cysteine ​​substitutions into the light-chain variable region of an anti-IL6R antibody, and a modified antibody (MRAL-k0.xxx) produced by introducing cysteine ​​substitutions into the light-chain constant region of an anti-IL6R antibody (10 / 10), as described in Reference Example 6-2. 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 an anti-IL6R antibody (MRA) and a modified antibody (MRAL-k0.K126C) produced by introducing cysteine ​​substitutions 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 an anti-kappa chain antibody or an anti-human Fc antibody. [Figure 45] Figure 45 shows the molecular weights of each band obtained by treating an antibody sample with protease and their predicted structures, as described in Reference Example 7-2. Below each molecular structure, whether the molecule can react with an anti-kappa chain antibody or an anti-Fc antibody (whether a band is detected in the electrophoresis of Figure 44) is also shown. [Figure 46]Figure 46 shows the results of measuring the CD3-mediated agonistic activity of an anti-CD3 antibody molecule (OKT3), modified antibody molecules (H_T135C, H_S136C, H_S191C, L_K126C) produced by linking its Fab-Fab with an additional disulfide bond, 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 the CD3-mediated agonistic activity of an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (OKT3_KiH) prepared 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) prepared by linking the Fab-Fabs of the OKT3 antibody molecule with an additional disulfide bond, and an anti-KLH antibody (IC17) (negative control), as described in Reference Example 14-4. [Figure 48]Figure 48 shows the results of experiments using an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (H_S191C) prepared by linking its Fab-Fab with an additional disulfide bond, a modified antibody molecule (OKT3_KiH) prepared 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) prepared by linking its Fab-Fab with an additional disulfide bond, and a fragment of OKT3_KiH, as described in Reference Example 15-4. FIG. 1 shows the results of measuring the CD3-mediated agonist activity of modified antibody molecules (0004 / / 0004, 0004 / / 0006) prepared by introducing positively charged amino acid substitutions into one heavy chain constant region and negatively charged amino acid substitutions into the other heavy chain constant region, modified antibody molecules (0004 / / OKT3, OKT3 / / 0004, OKT3 / / 0006) prepared by introducing positively or negatively charged amino acid substitutions into one heavy chain constant region of OKT3_KiH, and an anti-KLH antibody molecule (IC17) (negative control). [Figure 49] Figure 49 shows the results of measuring the CD3-mediated agonist activity of an anti-CD3 antibody molecule (OKT3), modified antibody molecules (dh1, dh2, dh3) prepared by removing the disulfide bond in its hinge region, modified antibody molecules (H_S191C_dh1, H_S191C_dh2, H_S191C_dh3) prepared by further linking their Fab-Fabs with an additional disulfide bond, and an anti-KLH antibody molecule (IC17) (negative control), as described in Reference Example 16-4. [Figure 50]Figure 50 shows the results of measuring the CD3-mediated agonist activity of an anti-CD3 monospecific antibody molecule (OKT3-G1s), a modified antibody molecule (OKT3-G1sHH) prepared by linking its Fab-Fabs with an additional disulfide bond, a modified antibody molecule (CD3-G1sLL) prepared by linking the Fab-Fabs of the anti-CD3 monospecific antibody (CD3-G1s) with an additional disulfide bond, an anti-CD3 biparatopic antibody molecule (CD3 / / OKT3-G1s), modified antibody molecules (CD3 / / OKT3-G1sHH, CD3 / / OKT3-G1sLH) prepared by linking its Fab-Fabs with an additional disulfide bond, and a combination of CD3-G1sLL and OKT3-G1s (CD3-G1sLL+OKT3-G1s), as described in Reference Example 20. [Figure 51A] Figure 51A shows the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3×anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fabs via an additional disulfide bond, as described in Reference Example 22-1. Figure 51A shows the agonistic 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-Fabs via an additional disulfide bond. [Figure 51B]Figure 51B shows the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3×anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fabs via an additional disulfide bond, as described in Reference Example 22-1. Figure 51B shows the agonistic 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-Fabs via an additional disulfide bond. [Figure 51C] Figure 51C shows the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3×anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fabs via an additional disulfide bond, as described in Reference Example 22-1. Figure 51C shows the agonistic 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 prepared by linking its Fab-Fabs via an additional disulfide bond (CD3 / / 949-G1silentLH, CD3 / / 949-G1silentHH, CD3 / / 949-G1silentLL, CD3 / / 949-G1silentHL). [Figure 51D]Figure 51D shows the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3×anti-PD1 bispecific antibody and modified antibody molecules prepared by linking its Fab-Fabs via an additional disulfide bond, as described in Reference Example 22-1. Figure 51D shows the agonistic 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-Fabs via an additional disulfide bond. [Figure 52] Figure 52 shows the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3 x anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), as well as modified antibody molecules (OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentLH, OKT3 / / 949-G1silentLL) produced by linking the Fab-Fabs via an additional disulfide bond, as described in Reference Example 22-2. [Figure 53A]Figure 53A shows the results of evaluating the T cell-dependent cancer cell proliferation inhibitory effect when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding-attenuated CD3 bispecific antibody were used in combination, as described in Reference Example 23-1. When the CD28 / CD3 clamping bispecific antibody and the GPC3 / binding-attenuated CD3 bispecific antibody were used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells), the GPC3 / binding-attenuated CD3 bispecific antibody brought the target cells and effector cells into close proximity, and the CD28 / CD3 clamping bispecific antibody activated the effector cells. Figure 53A shows the inhibitory effect on cancer cell proliferation when the GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) was used as the antibody for targeting T cells to cancer cells, and the GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), KLH / CD3 clamping bispecific antibody molecule (KLH / clamp CD3), CD28 / CD3 clamping bispecific antibody molecule (CD28 / clamp CD3), or a modified antibody molecule (CD28 / clamp CD3_HH) produced by linking the Fab-Fabs of these bispecific antibody molecules via an additional disulfide bond was used as the antibody for activating T cells. [Figure 53B]Figure 53B, like 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-attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-1. Figure 53B shows the inhibitory effect on cancer cell proliferation when an engineered antibody molecule (GPC3 / attCE115_LL) constructed by linking the Fab-Fabs of a GPC3 / binding-attenuated CD3 bispecific antibody with an additional disulfide bond was used as the antibody for targeting T cells to cancer cells, and when an engineered 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), or an engineered antibody molecule (CD28 / clamp CD3_HH) constructed by linking the Fab-Fabs of the bispecific antibody molecule with an additional disulfide bond was used as the antibody 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 / binding-attenuated CD3 bispecific antibody are used in combination, as described in Reference Example 23-2. When the CD28 / CD3 clamping bispecific antibody and the 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), the GPC3 / binding-attenuated CD3 bispecific antibody brings the target cells and effector cells into close proximity, and the CD28 / CD3 clamping bispecific antibody activates the effector cells. Figure 54A shows the amount of IL-6 production in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells) when a 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 a CD28 / CD3 clamping bispecific antibody via an additional disulfide bond were used alone or in combination. [Figure 54B] Figure 54B, like 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 54B shows the amount of IL-6 produced in the presence of effector cells (T cells) alone, when a GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) prepared by linking the Fab-Fab of a CD28 / CD3 clamping bispecific antibody via an additional disulfide bond are used, either alone or in combination. [Figure 54C] Figure 54C, like 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 of a GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) produced by linking the Fab-Fab of a CD28 / CD3 clamping bispecific antibody via an additional disulfide bond, when used alone 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 showing the mechanism of action of T cell-dependent cancer cell proliferation inhibition 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 55A shows the mechanism of action of cancer cell proliferation inhibition 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 showing the mechanism of action of T cell-dependent cancer cell growth inhibition 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 cancer cell growth inhibition when a modified antibody molecule 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 / binding-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 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 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-attenuated 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 modified to introduce an additional disulfide bond between the Fab-Fab of a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding-attenuated CD3 bispecific antibody are used in combination in the presence of effector cells (T cells) alone. [Figure 57A]Figure 57A shows the results of measuring the agonist activity of a CD8 / CD28 bispecific antibody molecule (CD8 / CD28-P587) and modified antibody molecules (CD8 / CD28-P587(HH), CD8 / CD28-P587(LL), CD8 / CD28-P587(HL), and CD8 / CD28-P587(LH)) created by linking its Fab-Fab domains via an additional disulfide bond, as described in Reference Example 24. An anti-KLH antibody molecule (KLH-P587) was used as a negative control. Results obtained using peripheral blood mononuclear cells (PBMCs) from two different donors are shown (top: donor A, bottom: donor B). Figure 57A shows the percentage of divided regulatory T (Treg) cells contained in PBMCs. [Figure 57B] Figure 57B shows the results of measuring the agonist activity of a 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)) created by linking the Fab-Fab of the antibody via an additional disulfide bond, as described in Reference Example 24. Figure 57B shows the percentage of divided CD8α-positive T cells in PBMCs. [Figure 58] FIG. 58 shows a chromatogram of cation exchange chromatography (CIEX) performed on an antibody sample of an OKT3 variant having a charged amino acid substitution, as described in Example 9-3. [Figure 59] FIG. 59 shows a chromatogram of cation exchange chromatography (CIEX) performed on an antibody sample of an OKT3 variant having a charged amino acid substitution, as described in Example 2-2 and Example 9-3. [Figure 60]60 shows a scatter plot of the ratios of the lower band to the upper band (non-reducing SDS-PAGE gel images) 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 for the MRA variant samples shown in Table 87, and the X axis represents the ratio of the lower band to the upper band for the OKT3 variant samples shown in Table 87. [Figure 61A] FIG. 61A shows a chromatogram of cation exchange chromatography (CIEX) performed on an antibody sample of an OKT3 variant having a charged amino acid substitution, as described in Example 10-3. [Figure 61B] FIG. 61B shows a chromatogram of cation exchange chromatography (CIEX) performed on an antibody sample of an MRA variant with a charged amino acid substitution, as described in Example 10-3. [Figure 62A] Figure 62A is a schematic diagram showing the effect of additional amino acid mutations on enhancing Fab cross-linking of engineered disulfide bonds. (Left) The G1T4.S191C variant, which has a cysteine ​​substitution, for example, at S191C (EU numbering) in CH1, contains a mixture of cross-linking and non-cross-linking antibodies. (Center) The G1T4.S191C variant, which contains the additional amino acid mutation X (X can be either a charged amino acid, a hydrophobic amino acid, or a knob-hole amino acid), shows a higher percentage of cross-linking antibodies. (Right) Amino acid positions (EU numbering) at the CH1-CH1 interface where the additional amino acid mutation X (X can be either a charged amino acid, a hydrophobic amino acid, or a knob-hole amino acid) can promote cross-linking of engineered disulfide bonds. [Figure 62B] Figure 62B is a schematic diagram showing the effect of additional mutations on the separation of cross-linked and non-cross-linked Fabs by chromatographic methods such as CIEX. DETAILED DESCRIPTION OF THE INVENTION

[0024] I. Definition As used herein, 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 one embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment or derivative thereof.

[0025] As used herein, the term "antigen-binding domain" refers to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When an antigen has a large molecular weight, the antigen-binding domain can bind only to a specific part of the antigen. This specific part is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain comprises 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 comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In a specific embodiment, the antigen-binding domain comprises a hinge region.

[0026] As used herein, the term "specifically binds" means that one of the molecules involved in specific binding binds without showing any significant binding to molecules other than its one or more binding partner molecules. This expression is also used when an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. When the epitopes bound by an antigen-binding domain are contained in multiple different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens containing the epitopes.

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

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

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

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

[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 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0032] In one embodiment of the present invention, the constant region is preferably an antibody constant region, more preferably an IgG1-, IgG2-, IgG3-, or IgG4-type antibody constant region, and even more preferably a human IgG1-, IgG2-, IgG3-, or IgG4-type antibody constant region. In another embodiment of the present invention, the constant region is preferably a heavy chain constant region, more preferably an IgG1-, IgG2-, IgG3-, or IgG4-type heavy chain constant region, and even more preferably a human IgG1-, IgG2-, IgG3-, or IgG4-type heavy chain constant region. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these sequences can be used in the present invention. The amino acid-altered constant regions of the present invention may also contain other amino acid mutations or modifications, as long as they contain the amino acid mutations of the present invention.

[0033] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example, from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system. In naturally occurring IgG antibodies, the cysteine ​​residue at EU numbering position 220 in the hinge region is known to form a disulfide bond with the cysteine ​​residue at EU numbering position 214 in the antibody light chain. Furthermore, it is known that the cysteine ​​residues at EU numbering positions 226 and 229 in the hinge region of two antibody heavy chains form disulfide bonds. The "hinge region" is generally defined as spanning positions 216 to 238 (EU numbering) or positions 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper hinge, the middle 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), Central hinge: 226th to 230th (EU numbering) or 239th to 243rd (Kabat numbering), Lower hinge: 231-238 (EU numbering) or 244-251 (Kabat numbering). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions (see, for example, Brekke et al., 1995, Immunol (Table 1 of Today 16: 85-90)). Hinge regions herein include wild-type hinge regions as well as variants in which amino acid residues in the wild-type hinge region have been changed by substitution, addition, or deletion. The term "disulfide bonds formed between amino acids not in the hinge region" (or "disulfide bonds formed between amino acids other than the hinge region") refers to disulfide bonds formed, connected, or linked through amino acids located in any region of an antibody other than the "hinge region" defined above. For example, such disulfide bonds are formed, connected, or linked through amino acids located at any position in an antibody other than the hinge region (e.g., from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system). In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located in 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 positions 119-123, 131-140, 148-150, 155-167, 174-178, 188-197, and 201-214 (EU numbering) in the CH1 region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at 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 (EU numbering) in the CH1 region. , 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, 214. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering) in the CH1 region.In one preferred embodiment, such a disulfide bond is formed, connected, or linked through the amino acid located at EU numbering position 191 in the CH1 region.

[0034] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

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

[0036] "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 binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

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

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

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

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

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

[0042] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0043] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

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

[0045] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

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

[0047] "Contacting" means providing or exposing in a solution. The antibody, protein, or polypeptide can be contacted with a reducing reagent and can also be bound to a solid support (e.g., an affinity column or a chromatography matrix). Preferably, the solution is buffered. To maximize the yield of the antibody / protein having the desired conformation, the pH of the solution is selected to maintain the stability of the antibody / protein and to optimize disulfide exchange. In the practice of the present invention, the pH of the solution is preferably not strongly acidic. Thus, 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 for a particular embodiment of the present invention can be easily determined experimentally by one 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 comprised of a compound 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 in which the cells producing the recombinant protein are grown. In further embodiments, the reducing agent may be added to the LC mobile phase during the LC separation step to separate the recombinant protein. In certain embodiments, the protein is immobilized on the stationary phase of the LC column, and the reducing agent is part of the mobile phase. In certain embodiments, intact IgG antibodies may be eluted as a heterogeneous mixture, as 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 contemplated that this more uniform peak of interest may 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 a desired conformation (e.g., an antibody in a "paired cysteine" form, with one or more engineered disulfide bonds formed between the two Fabs of the antibody, e.g., between amino acid residues not in the hinge region). The optimal absolute concentration and molar ratio of the reducing agent depend on the concentration of total IgG and, in some circumstances, the specific IgG subclass. When used to prepare IgG1 molecules, it also depends on the number and accessibility of unpaired cysteines in the protein. Generally, the concentration of free thiols 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 reducing agent concentration is 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, or 100 mM. In some preferred embodiments, 0.05 mM to 1 mM of 2-MEA can be used. In some preferred embodiments, 0.01 mM to 25 mM TCEP can be used.

[0051] Contacting the recombinant protein preparation with the reducing agent is carried out for a time sufficient to increase the relative proportion of the desired conformation. Any relative increase in proportion is desirable, including, for example, conversion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, and even 80% or 90% of the protein having the undesired conformation to protein having the desired conformation. The contacting can be carried out by providing the reducing agent to the fermentation medium in which the protein is being produced. Alternatively, the contacting is carried out during partial purification of the protein from the cell culture in which it is produced. In yet other embodiments, the contacting is carried out after the protein has been eluted from a chromatography column but before any further processing. Essentially, the contacting can 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) can be carried out prior to the contacting.

[0052] The contacting may be performed by an antibody attached to the stationary phase of a chromatography column, with the reducing agent being part of the mobile phase; in this case, the contacting may be performed as part of a chromatographic purification procedure. Representative 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 reviewed in (Li et al., 2004).

[0053] When the contacting step is performed on a partially or highly purified preparation of recombinant protein, the contacting step can be performed for a short period of time, such as from about 1 hour to about 4 hours, and for a long period of time, such as from about 6 hours to about 4 days. Contacting steps of about 2 to about 48 hours or about 16 hours have been found to work well. The contacting step can also be performed between other steps, such as on a solid phase, or between filtration or any other steps in purification.

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

[0055] Additionally, it is contemplated that the method may be performed at high pressure. High hydrostatic pressure (1000-2000 bar) in combination with low, non-denaturing concentrations of guanidine hydrochloride below 1 M has previously been used to disaggregate (solubilize) and refold several denatured proteins produced as inclusion bodies by Escherichia coli (E-coli), including human growth hormone and lysozyme, as well as b-lactamase (St John et al., Proc Natl Acad Sci USA, 96:13029-13033 (1999)). b-lactamase was refolded with high yields of active protein, even without the addition of GdmHCl. In another study (Seefeldt et al., Protein Sci, 13:2639-2650 (2004)), the refolding yield of the mammalian cell-produced protein bikunin obtained by high-pressure-modulated refolding at 2000 bas was 70% by RP-HPLC, 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 disaggregation. The interaction of high pressure with proteins is similar to the interaction of proteins with chaotropic agents. Therefore, it is contemplated that the method of the present invention uses high pressure 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] The recombinant antibody / protein preparation can be contacted with the reducing agent in various amounts, as needed. For example, the methods of the present invention have been successfully performed at analytical laboratory scale (1-50 mL), preparative scale (50 mL-10 L), and manufacturing scale (10 L or more). The methods of the present invention can be performed reproducibly on both small and large scales. Thus, the antibody concentration can be in industrial quantities (in terms of gram quantities) (e.g., industrial quantities for a particular IgG) or milligram quantities. In certain embodiments, the concentration of the 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. Recombinant IgG1 molecules at these concentrations are specifically contemplated.

[0057] In certain embodiments, proteins produced using a medium containing a reducing agent are further treated in another treatment step utilizing a chaotropic denaturant, such as sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride (GuHCl). A significant amount of chaotropic agent is required to observe appreciable unfolding. In some embodiments, the treatment step uses 0.1 M to 2 M of a chaotrope, which produces an effect equivalent to the use of 0.1 M to 2 M of guanidine hydrochloride. In certain embodiments, oxidative refolding is achieved in the presence of approximately 1.0 M of guanidine hydrochloride, or an amount of another chaotropic agent that produces the same or similar amount of refolding as 1 M of guanidine hydrochloride. In some embodiments, the method uses about 1.5 M to 0.5 M of a chaotrope. The amount of chaotropic agent used is based on the structural stability of the protein in the presence of the chaotrope. The chaotrope should be present enough to disrupt the local tertiary and / or quaternary structure of the protein's domain interactions, but less than that required to completely unfold the secondary structure of the molecule and / or individual domains. To determine the point at which a protein begins to unfold by equilibrium denaturation, one skilled in the art can titrate the chaotrope into a solution containing the protein and monitor the structure by techniques such as circular dichroism or fluorescence. There are other parameters that can be used in place of chaotropes to unfold or slightly disrupt the structure of a protein. Temperature and pressure are two basic parameters previously used to alter protein structure and may be used in place of chaotropic agents during contact with a redox agent. The inventors contemplate that any parameter shown to denature or disrupt protein structure can be utilized by one skilled in the art in place of a 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 inactivated, 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, removal of the reducing agent may be performed by dialysis, buffer exchange, or any of the chromatographic methods described herein.

[0059] The term "preferentially enriched (or increased)" refers to an increase in the relative abundance of a desired form, or 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 antibody structural isoforms, e.g., antibodies having at least one disulfide bond formed between amino acid residues other than the hinge region. In one embodiment, the at least one disulfide bond is formed between the amino acid residue at EU numbering position 191 in the CH1 region of each of the first and second antigen-binding domains. In certain embodiments, the methods produce homogeneous antibody preparations having at least 50%, 60%, 70%, 80%, 90%, and preferably at least 95% molar ratio of uniform antibodies having at least one disulfide bond formed other than the hinge region.

[0060] A "homogeneous" population of antibodies refers to an antibody population that primarily comprises a single form of antibody, e.g., at least 50%, 60%, 70%, 80%, or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100%, of the antibodies in a solution or composition are in a properly folded conformation. Similarly, a "homogeneous" population of antibodies having at least one disulfide bond formed outside the hinge region refers to a population of such antibodies that primarily comprises a single properly folded conformation, e.g., at least 50%, 60%, 70%, 80%, or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100% molar ratio of such antibodies having at least one disulfide bond formed outside the hinge region. In a preferred embodiment, the "homogeneous" population of antibodies comprises at least one disulfide bond formed between the amino acid residue at EU numbering position 191 in each CH1 region of the first antigen-binding domain and the second antigen-binding domain (i.e., the "paired cysteines" at EU numbering position 191 in the CH1 region). In a preferred embodiment, the methods of the present invention produce a homogeneous antibody population or 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 conformations in a mixture can be performed using any of a variety of analytical and / or qualitative techniques. If two conformations are differentially resolved through a separation technique, such as chromatography, electrophoresis, filtration, or other purification technique, the relative proportions of the conformations in the mixture can be determined using such a purification technique. For example, at least two distinct conformations of a recombinant IgG can be separated using hydrophobic interaction chromatography. Furthermore, far-ultraviolet circular dichroism has been used to estimate the secondary structural makeup of proteins (Perczel et al., 1991, Protein Engrg. 4:669-679), and such a technique can determine whether alternative conformations of a protein exist. Yet another technique used to determine conformation is fluorescence spectroscopy, which can be used to identify complementary differences in tertiary structure that can be assigned to tryptophan and tyrosine fluorescence. Other techniques that can be used to determine conformational differences and thus the relative proportions of conformations are online SEC to measure aggregation state, differential scanning calorimetry to measure melting transitions (Tm) and component enthalpies, and chaotropic unfolding. Yet another technique that can be used to determine conformational differences and thus the relative proportions of conformations is LC / MS detection to determine protein heterogeneity.

[0062] Alternatively, if differences in activity exist between antibody / protein conformations, determining the relative proportions of conformations in the mixture can be accomplished by means of activity assays (e.g., ligand binding, enzymatic activity, biological activity, etc.). Protein biological activity can also be used. Alternatively, binding assays can be used, in which activity is expressed as activity units / mg protein.

[0063] In some embodiments, described in detail herein below, the present invention uses IEC chromatography to determine antibody / protein heterogeneity. In such cases, the antibody is purified to "homogeneity" or is considered to be "homogeneous," meaning that no polypeptide peaks or fractions corresponding to other polypeptides are detectable upon analysis by IEC chromatography. In certain embodiments, the antibody is purified to "homogeneity" or is considered to be "homogeneous," such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the relevant art will recognize that multiple bands corresponding to polypeptides can be visualized by SDS-PAGE due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptides of the present invention are purified to substantial homogeneity, as indicated by a single polypeptide band upon 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] Herein, examples of conditions for SDS-PAGE analysis are as follows: Sample Buffer Solution (x4) without 2-mercaptoethanol can be used to prepare electrophoresis samples. The samples can be treated for 10 minutes under conditions of an analyte concentration of 50 or 100 micrograms / mL and 70°C, and then subjected to non-reducing SDS-PAGE. In non-reducing SDS-PAGE, electrophoresis can 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 captured, and the bands can be quantified using an imaging device. In the gel image, multiple bands, for example, two bands, i.e., an "upper band" and a "lower band," can be observed for antibody variant samples. In this case, the molecular weight of the upper band can correspond to that of the parent antibody (before modification). Structural changes, such as cross-linking via disulfide bonds in Fab, can be caused by cysteine ​​substitution and can result in changes in electrophoretic mobility. In this case, the lower band may be considered to correspond to an antibody having one or more engineered disulfide bonds formed between the CH1 regions. Antibody variant samples with additional cysteine ​​substitutions may exhibit a higher ratio of lower band to upper band compared to control samples. The additional cysteine ​​substitutions may enhance / promote disulfide bond cross-linking of Fab; increase the percentage or structural uniformity of antibody preparations with engineered disulfide bonds formed at the variant positions; or reduce the percentage of antibody preparations without engineered disulfide bonds formed at the variant positions. As used herein, the term "ratio of lower band to 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) As used herein, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain consisting of a pair of an antibody light chain variable region (VL (light chain variable region)) and an antibody heavy chain variable region (VH (heavy chain variable region)). In 1988, Skerra and Pluckthun discovered that homogeneous, active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH and VL were associated in a manner that allowed them to bind to antigens.

[0066] scFv, single chain antibodies, and sc(Fv)2 As used herein, the terms "scFv," "single-chain antibody," or "sc(Fv)2" all refer to antibody fragments that contain, within a single polypeptide chain, variable regions from both the heavy and light chains but lack constant regions. Generally, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables them to form the desired structure that will allow antigen binding. Single-chain antibodies are discussed in detail by Plückthun in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994). See also International Patent Application Publication No. WO 1988 / 001649; U.S. Pat. Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0067] An scFv is an antigen-binding domain in which the VH and VL constituting the Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

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

[0069] As used herein, examples of the form of the antigen-binding domain that constitutes sc(Fv)2 include antibodies characterized in that two VH units and two VL units are arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). 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 order configurations are also possible. [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' A "Fab" is composed of one light chain and one heavy chain CH1 region and variable region. The heavy chain of a wild-type Fab molecule cannot form disulfide bonds with another heavy chain molecule. The term "Fab" encompasses not only wild-type Fab molecules, but also Fab variants in which amino acid residues in the wild-type Fab have been altered by substitution, addition, or deletion. In certain embodiments, mutated amino acid residues (e.g., substituted, added, or inserted cysteine ​​or lysine residues) contained in the Fab variants can form disulfide bonds with another heavy chain molecule or portion thereof (e.g., a Fab molecule).

[0071] An scFab is an antigen-binding domain in which one light chain and one heavy chain CH1 region and variable region constituting a Fab are linked by a peptide linker, which can maintain the light chain and the heavy chain CH1 region and variable region 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, digesting the immunoglobulins (monoclonal antibodies) near the disulfide bond between the hinge regions of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of the two heavy chains, producing two homologous antibody fragments in which an light chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked by a disulfide bond at the C-terminal region to an heavy chain fragment containing a VH (heavy chain variable region) and a CHγ1 (the γ1 region of the heavy chain constant region). Each of these two homologous antibody fragments is called Fab'.

[0073] "F(ab')2" is composed of two light chains and two heavy chains containing constant regions, i.e., portions of the CH1 and CH2 domains, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably prepared by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment by adsorption onto a protein A column. Such a protease is not particularly limited, as long as it can cleave a full-length antibody to produce F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0074] Single Domain Antibodies As used herein, the term "single-domain antibody" is not particularly limited in its structure, as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing of VH and VL, whereas single-domain antibodies are known to be able to exhibit antigen-binding activity solely through their own domain structure, without pairing with any other domain. Single-domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer. Examples of single domain antibodies include, but are not limited to, camelid VHHs, shark VHHs, and the like. NAR and antibody fragments comprising all or a portion of the VH domain or all or a portion of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments comprising all or a portion of the VH / VL domains of an antibody, include, but are not limited to, single-domain antibodies artificially produced starting from a human antibody VH or VL, such as those described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a 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 genes capable of producing single-domain antibodies have been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which genes capable of producing single-domain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO 2015 / 143414 and U.S. Patent Publication No. US 2011 / 0123527 A1. Humanized single-chain antibodies can also be obtained by replacing the framework sequences of single-domain antibodies obtained from animals with human germline sequences or sequences similar thereto. Humanized single-domain antibodies (e.g., humanized VHHs) are one embodiment of the single-domain antibodies of the present invention. Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. 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)).

[0075] "Binding activity" refers to the strength of the total noncovalent 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, binding activity is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of these binding forces. The binding activity of a molecule X to its partner Y can generally be expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." 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 an "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 antibody or an "antagonist" antigen-binding molecule or 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 phrase "substantially reduced" or "substantially different" refers to a difference between two values ​​(usually one for a molecule and one for a reference / comparator molecule) that is sufficiently large that one of skill in the art would consider the difference between the two values ​​to be statistically significant in terms of the biological characteristic measured by the values ​​(e.g., KD value).

[0079] As used herein, the terms "substantially similar" or "substantially the same" refer to a similarity between two numerical values ​​(e.g., between one relating to an antibody of the invention and one relating to a reference / comparator antibody) that is sufficiently high that one of skill in the art would consider the difference between the two numerical values ​​to have little or no biological and / or statistical significance in terms of the biological characteristic measured by the numerical values ​​(e.g., KD values).

[0080] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

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

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

[0083] II. Antigen-binding molecules In one aspect, the present disclosure is based in part on the discovery that 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, exhibits enhanced or attenuated activities compared to a control antigen-binding molecule comprising antigen-binding domains that are not linked or are linked via fewer bonds. In a specific embodiment, an antigen-binding molecule is provided that has the activity of holding two or more antigen molecules in close spatial proximity. The antigen-binding molecule of the present disclosure is useful, for example, in that it can control the activation of two antigen molecules that are activated by their association with each other. In another specific embodiment, an antigen-binding molecule is provided that is resistant to protease digestion due to the linkage between the antigen-binding domains.

[0084] A. Exemplary Antigen-Binding Molecules <Structure of antigen-binding molecules> 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 embodiment of the above aspect, at least one of the one or more bonds connecting the two antigen-binding domains is a covalent bond. In a specific embodiment, the covalent bond is formed by directly cross-linking 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 cross-linking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a cross-linking agent, e.g., an amine-reactive cross-linking agent, and the cross-linked amino acid residue is, e.g., lysine.

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

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

[0088] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains has antigen-binding activity by itself (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 by themselves.

[0089] In one embodiment of the above aspect, the first and second antigen-binding domains are both antigen-binding domains of the same type. As described below, examples of proteins that constitute antigen-binding domains include polypeptides derived from antibodies or non-antibody proteins, and fragments thereof (e.g., Fab, Fab', scFab, Fv, scFv, single-domain antibodies, etc.). When the proteins that constitute the first and second antigen-binding domains have the same structure from the viewpoint of molecular morphology, these antigen-binding domains are considered to be of the same type.

[0090] In one embodiment of the above aspect, at least one bond linking 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, respectively.

[0091] The positions of amino acid residues in the antigen-binding domain can be indicated according to the Kabat numbering or EU numbering system (also referred to 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, when amino acid residues that form the bond origin between the first and second antigen-binding domains are present at the same corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by the same numbers according to the Kabat numbering or EU numbering system. Alternatively, when amino acid residues that form the bond origin between the first and second antigen-binding domains are present at different but 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 comprises an antibody fragment that binds to a specific antigen. In certain embodiments, the antibody fragment is a Fab, Fab', scFab, Fv, scFv, or single-domain antibody. In certain embodiments, at least one of the amino acid residues that form the origin of binding between the antigen-binding domains is present within the antibody fragment.

[0093] In one embodiment of the above aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the constant region. In a specific embodiment, the amino acid residue is present in the CH1 region, for example, at any one of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219 (EU numbering) in the CH1 region. In certain embodiments, the amino acid residues are located at 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, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 33 The amino acid residue is located at any position selected from the group consisting of: 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 certain embodiments, the amino acid residue is located at position 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 (EU numbering) in the CH1 region. In certain embodiments, the amino acid residue is located at position 135, 136, or 191 (EU numbering) in the CH1 region. In one embodiment of the above aspect, the constant region is of human origin. In a specific embodiment, the subclass of the heavy chain constant region is any of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a specific embodiment, the subclass of the CH1 region is any of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

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

[0095] In one embodiment of the above aspect, the difference in the position of the amino acid residue that serves as the binding origin 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 binding origin in the CH1 region of the first antigen-binding domain and the position of the amino acid residue that serves as the binding origin in the CH1 region of the second antigen-binding domain, respectively, the difference (i.e., the distance) is within 3 amino acids, based on EU numbering. In a specific embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 135 (EU numbering) in the CH1 region of the first antigen-binding domain with any amino acid residue at positions 132 to 138 (EU numbering) in the CH1 region of the second antigen-binding domain. In a specific embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 136 (EU numbering) in the CH1 region of the first antigen-binding domain with any amino acid residue at positions 133 to 139 (EU numbering) in the CH1 region of the second antigen-binding domain. In a specific embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at position 191 (EU numbering) in the CH1 region of the first antigen-binding domain with any amino acid residue at positions 188 to 194 (EU numbering) in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at position 135 (EU numbering) in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at position 136 (EU numbering) in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 191 (EU numbering) in the CH1 regions of the two antigen-binding domains.

[0096] In one embodiment of the aforementioned aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is located in the CL region, for example, at any one of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213, according to the Kabat numbering, in the CL region. In certain embodiments, the amino acid residue is located 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 (Kabat numbering) in the CL region. In certain embodiments, the amino acid residue is located at position 126 (Kabat numbering) in the CL region. In one embodiment of the above aspect, the constant region is of human origin. In a particular embodiment, the subclass of the CL region is kappa or lambda.

[0097] In one embodiment of the foregoing aspects, at least one bond connecting the first and second 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. In certain embodiments, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of positions 108, 109, 110, 111, and 112 (Kabat numbering). In certain embodiments, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 (Kabat numbering). In certain embodiments, 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 positions 151, 152, 153, 154, 155, and 156 (Kabat numbering). In certain embodiments, 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 positions 184, 185, 186, 187, 188, 189, and 190 (Kabat numbering). In certain embodiments, 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 positions 195 and 196 (Kabat numbering). In certain embodiments, 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 positions 200, 201, 202, and 203 (Kabat numbering). 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 positions 208, 209, 210, 211, 212, and 213 according to the Kabat numbering system.

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

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

[0100] In one embodiment of the above aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is located in a variable region. In a specific embodiment, the amino acid residue is located in the VH region, for example, at any position selected from the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b according to the Kabat numbering in the VH region. In a specific embodiment, the amino acid residue is located in the VL region, for example, at any position selected from the group consisting of positions 21, 27, 58, 77, 100, 105, and 107 according to the Kabat numbering in the VL region (subclass κ) and positions 6, 19, 33, and 34 according to the Kabat numbering in the VL region (subclass λ). In certain embodiments, the amino acid residue is present in the VHH region, for example, at any position selected from the group consisting of 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 according to the Kabat numbering system of the VHH region.

[0101] In one embodiment of the above aspect, 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. In a specific embodiment, the non-antibody protein is either a pair of a ligand and a receptor that specifically bind to each other. Such receptors include, for example, receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic 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 comprise a hinge region. In a specific embodiment, at least one of the cysteine ​​residues present in the wild-type hinge region is substituted with another amino acid residue. Such a cysteine ​​residue is, for example, present at EU numbering positions 226 and / or 229 in the wild-type hinge region. In a specific embodiment, at least one of the amino acid residues serving as the origin of binding between the antigen-binding domains is present in the hinge region, for example, at any one selected from the group consisting of EU numbering positions 216, 218, and 219 in the hinge region.

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

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

[0105] In certain embodiments, at least one of the amino acid residues that form the bond between the antigen-binding domains is present in the antibody fragment, and at least one is present in the hinge region. In an exemplary embodiment, the antigen-binding molecule of the present disclosure is F(ab')2, in which both the first and second antigen-binding domains comprise Fab and hinge regions.

[0106] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure further comprises an Fc region, for example, a full-length antibody. In certain embodiments, one or more amino acid mutations that promote multimerization of the Fc region have been introduced into the Fc region of the antigen-binding molecule of the present disclosure. 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 certain embodiments, the multimerization is hexamerization.

[0107] <Antigens to which antigen-binding molecules bind> In one embodiment of the above aspect, the first and second antigen-binding domains both bind to the same antigen. In a specific embodiment, the first and second antigen-binding domains bind to the same epitope on the same antigen. In another specific embodiment, the first and second antigen-binding domains each bind to a different epitope on the same antigen. In a specific embodiment, 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 above aspect, each of the first and second antigen-binding domains binds to a different antigen. In another embodiment of the above aspect, the antigen-binding molecule of the present disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). As used herein, a clamping antigen-binding molecule refers to an antigen-binding molecule that specifically binds to an antigen-antigen-binding molecule complex formed between an antigen A and an antigen-binding molecule that binds to the antigen A, thereby increasing the binding activity of the antigen-binding molecule that binds to the antigen A (or stabilizing the antigen-antigen-binding molecule complex formed between the antigen A and the antigen-binding molecule that binds to the antigen A). For example, a CD3-clamping antibody specifically binds to an antigen-antibody complex formed between CD3 and an antibody with reduced binding ability to CD3 (a CD3 antibody with reduced binding), thereby increasing the CD3-binding activity of the CD3 antibody with reduced binding ability (or stabilizing the antigen-antibody complex formed between CD3 and the CD3 antibody with reduced binding ability). In a specific embodiment, the first and / or second antigen-binding domain in the antigen-binding molecule of the present disclosure can be an antigen-binding domain derived from a clamping antigen-binding molecule (clamping antigen-binding domain). In one embodiment of the above aspects, the first and second antigen-binding domains both have the same amino acid sequence, hi another embodiment, the first and second antigen-binding domains each have a different amino acid sequence.

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

[0109] <Function of antigen-binding molecules> In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region). In another embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of regulating the interaction between two antigen molecules. Without being bound by a particular theory, it is believed that the activity of regulating the interaction results from the antigen-binding molecule of the present disclosure holding two antigen molecules in close spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can enhance or attenuate the interaction between two antigen molecules compared to a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). In a specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure are a ligand and its receptor, respectively, and the antigen-binding molecule of the present disclosure has the activity of promoting activation of the receptor by the ligand. In another specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure are an enzyme and its substrate, respectively, and the antigen-binding molecule of the present disclosure has the activity of promoting the catalytic reaction of the enzyme on the substrate. In yet another specific embodiment, the two antigen molecules bound by the antigen-binding molecule of the present disclosure are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the present disclosure has the activity of promoting 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, respectively, a cell with cytotoxic activity and its target cell, and the antigen-binding molecule of the present disclosure promotes damage to the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, a T cell, a NK cell, a monocyte, or a macrophage.

[0110] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of regulating the activation of two antigen molecules that are activated by their association with each other. Without being bound by a particular theory, it is believed that the activity of regulating activation is achieved as a result of the antigen-binding molecule of the present disclosure holding two antigen molecules in close spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can enhance or attenuate the activation of two antigen molecules compared to a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond between the antigen-binding domains is derived from 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). For example, such antigen molecules are selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0111] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has two antigen-binding domains that are located in closer spatial proximity and / or have reduced mobility of the two antigen-binding domains. In a specific embodiment, the antigen-binding molecule of the present disclosure has two antigen-binding domains that are located closer to each other and / or have reduced mobility of the two antigen-binding domains compared to a control antigen-binding molecule, and the control antigen-binding molecule differs 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 embodiment, the one fewer bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond 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 that is not present in the wild-type Fab or hinge region).

[0112] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure is resistant to protease cleavage. In a specific embodiment, the antigen-binding molecule of the present disclosure has increased resistance to protease cleavage compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present disclosure only in that it has one less bond between the two antigen-binding domains. In a further embodiment, the one less bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains and that is not present in wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in wild-type Fab or hinge region). In a specific embodiment, the antigen-binding molecule of the present disclosure has 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 specific embodiment, the antigen-binding molecule of the present disclosure has 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 above aspect, when an antigen-binding molecule of the present disclosure is treated with a protease, dimers of antigen-binding domains or fragments thereof (e.g., cross-linked Fab dimers) are excised. In a specific embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the present disclosure only in that it has one less bond between the two antigen-binding domains is treated with the protease, monomers of the antigen-binding domains or fragments thereof are excised. In a further embodiment, the one less bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond between the antigen-binding domains is derived from 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 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 the present disclosure only in that the number of bonds between the two antigen-binding domains is reduced by one, and the reduced bond is formed via a mutant amino acid residue, such as an artificially introduced cysteine ​​residue.

[0115] <Pharmaceutical Composition> In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure 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 close spatial proximity, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; (b) adding to the antigen-binding molecule at least one bond that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bonds, and in this case, some or all of the one or more bonds are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for maintaining two antigen molecules in close spatial proximity, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of this disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of this disclosure for use in maintaining two antigen molecules in close spatial proximity.

[0117] In another aspect, the present disclosure provides a method for controlling an interaction between two antigen molecules, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; (b) adding to the antigen-binding molecule at least one bond that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bonds, and in this case, some or all of the one or more bonds are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, in at least one bond in (b) above, the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for controlling the interaction between two antigen molecules, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of this disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of this disclosure for use in controlling the interaction between two antigen molecules.

[0118] In yet another aspect, the present disclosure provides a method for regulating the activity of two antigen molecules that are activated by association with each other, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; (b) adding to the antigen-binding molecule at least one bond that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bonds, and in this case, some or all of the one or more bonds are bonds in which the amino acid residue serving as the bond origin between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, in at least one bond in (b) above, the amino acid residue serving as the bond origin between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for regulating the activity of two antigen molecules that are activated by their association with each other, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of this disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of this disclosure for use in regulating the activity of two antigen molecules that are activated by their association with each other.

[0119] In yet another aspect, the present disclosure provides a method for positioning two antigen-binding domains in close spatial proximity and / or reducing the flexibility of two antigen-binding domains, the method comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains to each other; In a specific embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bonds, and in this case, some or all of the one or more bonds are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, in at least one bond in (b) above, the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region).

[0120] In yet another aspect, the present disclosure provides a method for increasing resistance to protease cleavage of an antigen-binding molecule, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains to each other; In a specific embodiment, the two antigen-binding domains in the antigen-binding molecule of (a) above may be linked to each other via one or more bonds, and in this case, some or all of the one or more bonds are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, in at least one bond in (b) above, the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region).

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

[0122] <Method of producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of holding two antigen molecules in close spatial proximity, comprising: (a) providing 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; (b) introducing mutations into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 one or more bonds. and preferably further comprising the step of contacting the antibody preparation with a reducing agent.

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

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

[0125] In certain embodiments, the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and NaSO. In some preferred embodiments, the reducing agent is TCEP. In certain embodiments, the concentration of the reducing agent is about 0.01 mM to about 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 0.01 mM to 25 mM TCEP.

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

[0127] In certain embodiments, the contacting 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) prior to the contacting. 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 certain embodiments, the contacting step preferentially enriches or enriches the population of antibody structural isoforms having at least one disulfide bond formed between amino acid residues that are not in a hinge region. In certain embodiments, the contacting step produces a homogeneous antibody preparation having at least 50%, 60%, 70%, 80%, 90%, and preferably at least 95% molar ratio of said antibodies having at least one disulfide bond formed between amino acid residues that are not in a hinge region.

[0129] In certain embodiments, the contacting step produces an antibody preparation that is more homogeneous than the same antibody preparation that has not been treated by the method. In certain embodiments, the contacting step produces an antibody preparation that has an increase in its biological activity compared to the same antibody not treated by the method. In certain embodiments, the contacting step produces an antibody that has enhanced ability to hold two antigen molecules in close spatial proximity relative to the same antibody not treated by the method. In certain embodiments, the contacting step produces an antibody that has enhanced stability compared to the same antibody not treated by the method.

[0130] In certain embodiments, the contacting step preferentially enriches antibodies having at least one disulfide bond formed outside of the hinge region, and the preferentially enriched form has a pharmaceutically desirable property selected from any of the following (a)-(e), relative to a preparation not treated by the contacting 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 binding of the two antigen-binding domains to binding to two antigens that are in spatial proximity to each other; (b) the at least one disulfide bond holds the first antigen-binding domain and the second antigen-binding domain in closer spatial proximity to each other than in the same corresponding antibody lacking 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 relative to a corresponding identical antibody lacking the at least one disulfide bond; (d) the at least one disulfide bond increases the resistance of the antibody to protease cleavage relative to a corresponding identical antibody lacking 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 a corresponding identical antibody lacking the at least one disulfide bond. In a specific embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the origin of a bond for linking the two antigen-binding domains, and in this case, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains are amino acid residues present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond derived from a mutant amino acid residue that is not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

[0131] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of regulating an interaction between two antigen molecules, comprising: (a) providing 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; (b) introducing mutations into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 one or more bonds. The present invention provides a method comprising: In a specific embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the origin of a bond for linking the two antigen-binding domains, and in this case, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains are amino acid residues present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond derived from a mutant amino acid residue that is not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

[0132] In yet another aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity that controls the activation of two antigen molecules that are activated by associating with each other, comprising: (a) providing 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; (b) introducing mutations into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 one or more bonds. The present invention provides a method comprising: In a specific embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the origin of a bond for linking the two antigen-binding domains, and in this case, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains are amino acid residues present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond derived from a mutant amino acid residue that is not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

[0133] In yet another aspect, the present disclosure provides a method for producing an antigen-binding molecule in which two antigen-binding domains are located in close spatial proximity and / or the flexibility of the two antigen-binding domains is reduced, comprising: (a) providing 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; (b) introducing mutations into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 one or more bonds. The present invention provides a method comprising: In a specific embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the origin of a bond for linking the two antigen-binding domains, and in this case, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains are amino acid residues present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond derived from a mutant amino acid residue that is not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

[0134] In yet another aspect, the present disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) providing 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; (b) introducing mutations into the nucleic acid encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 one or more bonds. The present invention provides a method comprising: In a specific embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the origin of a bond for linking the two antigen-binding domains, and in this case, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains are amino acid residues present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond derived from a mutant amino acid residue that is not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

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

[0136] <Method for screening antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel set of protein molecules that are activated by associating with one another, the method comprising: (a) providing any two protein molecules; (b) producing an antigen-binding molecule comprising two antigen-binding domains that bind to the two protein molecules, respectively, by the production method of the present disclosure; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) assessing whether the two protein molecules are activated; The present invention provides a method comprising: In certain embodiments, at least one of the two protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic 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 molecules> 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 alone (i.e., one antigen-binding domain alone has antigen-binding activity). In a specific embodiment, both the first and second antigen-binding domains have antigen-binding activity alone.

[0138] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains comprises an antibody fragment that binds to a specific antigen. In a specific embodiment, the first and / or second antigen-binding domain comprises a hinge region. The amino acid residues that serve as the starting point for binding between the antigen-binding domains are present in the first and second antigen-binding domains, respectively, and the bond between the antigen-binding domains is formed by linking these amino acid residues. In a specific embodiment, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is present in the antibody fragment. In a specific embodiment, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is present in the hinge region. In a specific embodiment, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is present in the antibody fragment, and at least one of said amino acid residues is present in the hinge region.

[0139] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains has multiple amino acid residues that serve as binding origins between the antigen-binding domains, located at positions that are 7 amino acids or more apart from each other on the primary structure. This means that between any two amino acid residues of the multiple amino acid residues, there are 6 or more amino acid residues that are not the relevant amino acid residues. In certain embodiments, the combination of multiple amino acid residues that serve as binding origins between the antigen-binding domains may include a pair of amino acid residues that are located less than 7 amino acids apart on the primary structure. In certain embodiments, when the first and second antigen-binding domains are linked to each other via three or more bonds, three or more amino acid residues, including a pair of amino acid residues that are 7 amino acids or more apart from each other on the primary structure, can serve as binding origins between the antigen-binding domains. In certain embodiments, amino acid residues present at the same position in the first antigen-binding domain and the second antigen-binding domain link to each other to form a bond, while in certain embodiments, amino acid residues present at different positions in the first antigen-binding domain and the second antigen-binding domain link to each other to form a bond.

[0140] The positions of amino acid residues in the antigen-binding domain can be indicated according to the Kabat numbering or EU numbering system (also referred to 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, when amino acid residues that form the bond origin between the first and second antigen-binding domains are present at the same corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by the same numbers according to the Kabat numbering or EU numbering system. Alternatively, when amino acid residues that form the bond origin between the first and second antigen-binding domains are present at different but 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 embodiment of the above aspect, at least one of the two or more bonds connecting the antigen-binding domains is a covalent bond. In a specific embodiment, the covalent bond is formed by directly cross-linking 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 may be, for example, a cysteine, and the covalent bond formed may be, for example, a disulfide bond. At least one of the cross-linked cysteine ​​residues may be present in the hinge region. In another specific embodiment, a covalent bond is formed by cross-linking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a cross-linking agent, e.g., an amine-reactive cross-linking agent, and the cross-linked amino acid residue is, e.g., lysine.

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

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

[0144] In one embodiment of the above aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the constant region. In a specific embodiment, the amino acid residue is present in the CH1 region, for example, at a position selected from the group consisting of 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 (EU numbering) in the CH1 region. In an exemplary embodiment, the amino acid residue is located at position 191 (EU numbering) in the CH1 region, and a bond is formed between the amino acid residues at position 191 (EU numbering) in the CH1 regions of the two antigen-binding domains. In some embodiments of the above aspects, one disulfide bond is formed between the amino acid residue at position 191 (EU numbering) in each CH1 region of the first antigen-binding domain and the second antigen-binding domain. In some embodiments of the above aspects, 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 positions 131 to 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 residue at position 132 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; (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 residue at position 134 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; (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 residue at position 138 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. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one, two or more charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two or more oppositely charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more negatively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one, two, or more negatively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more positively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises the following amino acid residues (according to EU numbering) in each CH1 region: (a) the amino acid residue at position 136 being glutamic acid (E) or aspartic acid (D); (b) the amino acid residue at position 137, which is glutamic acid (E) or aspartic acid (D); (c) the amino acid residue at position 138 being glutamic acid (E) or aspartic acid (D); including one, two or more of: The other antigen-binding domain of the first and second antigen-binding domains has the following amino acid residues (according to EU numbering) in each CH1 region: (d) the amino acid residue at position 193 being lysine (K), arginine (R), or histidine (H); (e) the amino acid residue at position 194 being lysine (K), arginine (R), or histidine (H); and (f) the amino acid residue at position 195 being lysine (K), arginine (R), or histidine (H); Contains one, two or more of: In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises the following amino acid residues (according to EU numbering) in each CH1 region: (a) the amino acid residue at position 136 being lysine (K), arginine (R), or histidine (H); (b) the amino acid residue at position 137 being lysine (K), arginine (R), or histidine (H); (c) the amino acid residue at position 138 being lysine (K), arginine (R), or histidine (H); and The other antigen-binding domain of the first and second antigen-binding domains has 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 being glutamic acid (E) or aspartic acid (D); and (f) the amino acid residue at position 195 being glutamic acid (E) or aspartic acid (D); Contains one or more of: In some embodiments of the above aspects, one of the first and second antigen-binding domains comprises one, two, or more hydrophobic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more hydrophobic amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, the hydrophobic amino acid residue is alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp).

[0145] In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one "knob" amino acid residue at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two or more "hole" amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one, two or more "hole" amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one "knob" amino acid residue at positions 193 to 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 "hole" 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 above aspects, either one of the first and second antigen-binding domains comprises one, two, or more aromatic amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more positively charged amino acid residues at positions 193 to 195 (EU numbering) in each CH1 region. In some embodiments of the above aspects, either one of the first and second antigen-binding domains comprises one, two, or more positively charged amino acid residues at positions 136 to 138 (EU numbering) in each CH1 region; and the other of the first and second antigen-binding domains comprises one, two, or more aromatic amino acid residues at positions 193 to 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 certain embodiments, at least one of the amino acid residues that form the origin of the bond between the antigen-binding domains is located in the hinge region, e.g., at a position selected from the group consisting of EU numbering positions 216, 218, and 219 in the hinge region. In certain embodiments, at least one of the amino acid residues that serve as the origin of the bond between the antigen-binding domains is located in 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 in the CL region. In an exemplary embodiment, the amino acid residue is located at EU numbering position 126 in the CL region, and the amino acid residue at EU numbering position 126 in the CL regions of the two antigen-binding domains is linked to form a bond. In certain embodiments, an amino acid residue in the CH1 region of the first antigen-binding domain links to an amino acid residue in the CL region of the second antigen-binding domain to form a bond. In an exemplary embodiment, an amino acid residue at position 191 (EU numbering) in the CH1 region of the first antigen-binding domain links to an amino acid residue at position 126 (EU numbering) in the CL region of the second antigen-binding domain to form a bond.

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

[0148] In one embodiment of the above aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is located in a variable region. In a specific embodiment, the amino acid residue is located in the VH region, e.g., at a position selected from the group consisting of positions 8, 16, 28, 74, and 82b according to the Kabat numbering in the VH region. In a specific embodiment, the amino acid residue is located in the VL region, e.g., at a position selected from the group consisting of positions 100, 105, and 107 according to the Kabat numbering in the VL region.

[0149] In one embodiment of the above aspect, both the first and second antigen-binding domains comprise a Fab and hinge region. In certain embodiments, at least one of the amino acid residues that form the bond between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region, such as a cysteine ​​residue in the hinge region, such as the cysteine ​​residues at positions 226 and 229 (EU numbering). In another specific embodiment, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is a mutated 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. Such a mutated amino acid residue can be introduced into the wild-type Fab or hinge region by techniques such as amino acid substitution. In each of the CH1 region, hinge region, CL region, VH region, and VL region, amino acid residues that can serve as the starting point for binding between the antigen-binding domains are disclosed herein, and for example, a cysteine ​​residue 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 binding between antigen-binding domains (e.g., cysteine ​​residues) may be substituted with other amino acid residues or deleted, such as those at positions 220, 226, and 229 (EU numbering) in the hinge region and 214 in the CL region. In certain embodiments, the antigen-binding molecule of the present disclosure is a F(ab')2, in which the first and second antigen-binding domains both comprise a Fab and hinge region.

[0150] In one embodiment of the above aspect, 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. In a specific embodiment, the non-antibody protein is either a pair of a ligand and a receptor that specifically bind to each other. Examples of the receptor include receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0151] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure further comprises an Fc region, for example, a full-length antibody. In certain embodiments, one or more amino acid mutations that promote multimerization of the Fc region have been introduced into the Fc region of the antigen-binding molecule of the present disclosure. 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 certain embodiments, the multimerization is hexamerization.

[0152] <Antigens to which antigen-binding molecules bind> In one embodiment of the above aspect, the first and second antigen-binding domains both bind to the same antigen. In a specific embodiment, the first and second antigen-binding domains both bind to the same epitope on the same antigen. In another specific embodiment, the first and second antigen-binding domains each bind to a different epitope on the same antigen. In a specific embodiment, 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 aspects, each of the first and second antigen-binding domains binds to a different antigen. In another embodiment of the above aspect, the antigen-binding molecule of the present disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). As used herein, a clamping antigen-binding molecule refers to an antigen-binding molecule that specifically binds to an antigen-antigen-binding molecule complex formed between an antigen A and an antigen-binding molecule that binds to the antigen A, thereby increasing the binding activity of the antigen-binding molecule that binds to the antigen A (or stabilizing the antigen-antigen-binding molecule complex formed between the antigen A and the antigen-binding molecule that binds to the antigen A). For example, a CD3-clamping antibody specifically binds to an antigen-antibody complex formed between CD3 and an antibody with reduced binding ability to CD3 (a CD3 antibody with reduced binding), thereby increasing the CD3-binding activity of the CD3 antibody with reduced binding ability (or stabilizing the antigen-antibody complex formed between CD3 and the CD3 antibody with reduced binding ability). In a specific embodiment, the first and / or second antigen-binding domain in the antigen-binding molecule of the present disclosure can be an antigen-binding domain derived from a clamping antigen-binding molecule (clamping antigen-binding domain). In one embodiment of the above aspects, the first and second antigen-binding domains both have the same amino acid sequence, hi another embodiment, the first and second antigen-binding domains each have a different amino acid sequence.

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

[0154] <Function of antigen-binding molecules> In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains (e.g., a cysteine ​​residue that is not present in a wild-type Fab or hinge region).

[0155] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of regulating the interaction between two antigen molecules. Without being bound by a particular theory, it is believed that the activity of regulating the interaction is achieved as a result of the antigen-binding molecule of the present disclosure holding two antigen molecules in close spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can enhance or attenuate the interaction between two antigen molecules compared to a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond between the antigen-binding domains is derived from 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).

[0156] In a specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure are a ligand and its receptor, respectively, and the antigen-binding molecule of the present disclosure has the activity of promoting activation of the receptor by the ligand. In another specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure are an enzyme and its substrate, respectively, and the antigen-binding molecule of the present disclosure has the activity of promoting the catalytic reaction of the enzyme on the substrate.

[0157] In yet another specific embodiment, the two antigen molecules bound by the antigen-binding molecule of the present disclosure are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the present disclosure has the activity of promoting 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, respectively, a cell with cytotoxic activity and its target cell, and the antigen-binding molecule of the present disclosure promotes damage to the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, a T cell, a NK cell, a monocyte, or a macrophage.

[0158] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of regulating the activation of two antigen molecules that are activated by their association with each other. Without being bound by a particular theory, it is believed that the activity of regulating activation is achieved as a result of the antigen-binding molecule of the present disclosure holding two antigen molecules in close spatial proximity. In a specific embodiment, the antigen-binding molecule of the present disclosure can enhance or attenuate the activation of two antigen molecules compared to a control antigen-binding molecule, which differs 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 embodiment, the one fewer bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond between the antigen-binding domains is derived from 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). For example, the antigen molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0159] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure is resistant to protease cleavage. In a specific embodiment, the antigen-binding molecule of the present disclosure has increased resistance to protease cleavage compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present disclosure only in that it has one less bond between the two antigen-binding domains. In a further embodiment, the one less bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains and that is not present in wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in wild-type Fab or hinge region). In a specific embodiment, the antigen-binding molecule of the present disclosure has 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 specific embodiment, the antigen-binding molecule of the present disclosure has 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 above aspect, when an antigen-binding molecule of the present disclosure is treated with a protease, dimers of antigen-binding domains or fragments thereof (e.g., cross-linked Fab dimers) are excised. In a specific embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the present disclosure only in that it has one less bond between the two antigen-binding domains is treated with the protease, monomers of the antigen-binding domains or fragments thereof are excised. In a further embodiment, the one less bond can be selected from bonds in which the amino acid residue serving as the starting point of the bond between the antigen-binding domains is derived from 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 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 of the present disclosure 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 close spatial proximity, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more bonds; (b) adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other bond in (b) above is a bond in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for holding two antigen molecules in close spatial proximity, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of the present disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of the present disclosure for use in holding two antigen molecules in close spatial proximity.

[0163] In another aspect, the present disclosure provides a method for controlling an interaction between two antigen molecules, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more bonds; (b) adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other bond in (b) above is a bond in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for controlling the interaction between two antigen molecules, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of the present disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of the present disclosure for use in controlling the interaction between two antigen molecules.

[0164] In yet another aspect, the present disclosure provides a method for regulating the activity of two antigen molecules that are activated by association with each other, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more bonds; (b) adding another bond to the antigen-binding molecule that links the two antigen-binding domains together; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other bond in (b) above is a bond in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region). The present disclosure also provides a method for regulating the activity of two antigen molecules that are activated by their association with each other, comprising contacting the two antigen molecules with an antigen-binding molecule or pharmaceutical composition of the present disclosure. The present disclosure further provides an antigen-binding molecule or pharmaceutical composition of the present disclosure for use in regulating the activity of two antigen molecules that are activated by their association with each other.

[0165] Furthermore, in another aspect, the present disclosure provides a method for increasing resistance to protease cleavage of an antigen-binding molecule, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more bonds; and (b) adding another bond to the antigen-binding molecule that links the two antigen-binding domains to each other; The present invention provides a method comprising: In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from an amino acid residue present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other bond in (b) above is a bond in which the amino acid residue serving as the origin of the bond between the antigen-binding domains is derived from a mutant amino acid residue not present in a wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region).

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

[0167] <Method of producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of holding two antigen molecules in close spatial proximity, comprising: (a) providing 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 comprises one or more amino acid residues that serve as a bond origin for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add another bond connecting the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 bonds. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more amino acid residues that serve as the origin of the bond between the 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 bond in (b) above is a bond derived from a mutant amino acid residue that does not exist in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that does not exist in the wild-type Fab or hinge region).

[0168] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of regulating an interaction between two antigen molecules, comprising: (a) providing 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 comprises one or more amino acid residues that serve as a bond origin for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add another bond connecting the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 bonds. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more amino acid residues that serve as the origin of the bond between the 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 bond in (b) above is a bond derived from a mutant amino acid residue that does not exist in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that does not exist in the wild-type Fab or hinge region).

[0169] Furthermore, in another aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of regulating the activation of two antigen molecules that are activated by associating with each other, comprising: (a) providing 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 comprises one or more amino acid residues that serve as a bond origin for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add another bond connecting the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 bonds. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more amino acid residues that serve as the origin of the bond between the 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 bond in (b) above is a bond derived from a mutant amino acid residue that does not exist in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that does not exist in the wild-type Fab or hinge region).

[0170] Furthermore, in another aspect, the present disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) providing 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 comprises one or more amino acid residues that serve as a bond origin for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so as to add another bond connecting the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cells to express the two polypeptides; and (e) Obtaining 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 bonds. The present invention provides a method comprising: In a specific embodiment, some or all of the one or more amino acid residues that serve as the origin of the bond between the 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 bond in (b) above is a bond derived from a mutant amino acid residue that does not exist in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that does not exist in the wild-type Fab or hinge region). The antigen-binding molecules produced in these various aspects may have the characteristics of the antigen-binding molecules described herein.

[0171] <Method for screening antigen-binding molecules> In another aspect, the present disclosure provides a method for identifying a novel set of protein molecules that are activated by associating with one another, the method comprising: (a) providing any two protein molecules; (b) producing, by the production method of the present disclosure, an antigen-binding molecule comprising two antigen-binding domains that bind to the two protein molecules, respectively, and that has the activity of holding the two protein molecules in close proximity; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) assessing whether the two protein molecules are activated; The present invention provides a method comprising: In certain embodiments, at least one of the protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic 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 comprised in an antigen-binding molecule of the present disclosure are linked to each other via one or more bonds. In a preferred embodiment, each antigen-binding domain comprised in an antigen-binding molecule of the present disclosure has antigen-binding activity alone. In this embodiment, an antigen-binding molecule of the present disclosure comprising two antigen-binding domains can bind to two or more antigen molecules, an antigen-binding molecule of the present disclosure comprising three antigen-binding domains can bind to three or more antigen molecules, an antigen-binding molecule of the present disclosure comprising four antigen-binding domains can bind to four or more antigen molecules, and an antigen-binding molecule of the present disclosure comprising N antigen-binding domains can bind to N or more antigen molecules.

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

[0174] In one non-limiting embodiment, multiple amino acid residues located at positions distant from each other in the primary structure of at least one of the two or more antigen-binding domains comprised in an antigen-binding molecule of the present disclosure serve as the starting points for binding between the antigen-binding domains. The distance between the multiple amino acid residues is such that the two or more antigen-binding domains are structured in sufficient proximity as a result of linkage between the antigen-binding domains via bonds originating from each amino acid residue. 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 bonds between the antigen-binding domains and the number of amino acid residues that serve as the starting points for the bonds are such that a structure of two or more antigen-binding domains in sufficient proximity is achieved as a result of the linkage between the antigen-binding domains through the bonds. 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 specific embodiment, as long as a structure of two or more antigen-binding domains that are sufficiently close together is achieved as a result of linking the antigen-binding domains by three or more bonds starting from three or more amino acid residues in each antigen-binding domain, the distance between any two amino acid residues selected from the three or more amino acid residues in the primary structure may be seven or more amino acids in at least one pair of amino acid residues, and may be less than seven amino acids in the remaining pairs of amino acid residues.

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

[0176] In a non-limiting embodiment, the bond between the antigen-binding domains contained 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 direct cross-linking between an amino acid residue in a first antigen-binding domain and an amino acid residue in a second antigen-binding domain, for example, a disulfide bond between cysteine ​​residues. The directly cross-linked amino acid residue may be present on an antibody fragment such as Fab, or may be present in the 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 using 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 the primary amine of the side chain of a lysine residue in the antigen-binding domain.Amine-reactive crosslinkers include functional groups that form chemical bonds with primary amines (e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluoroesters). Representative examples include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl) suberate), DSP (dithiobis(succinimidyl propionate)), DTSSP (3,3'-dithiobis(sulfosuccinimidyl) propionate):3,3'-dithiobis(sulfosuccinimidyl sulfonate)), 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: 1,5-difluoro-2,4-dinitrobenzene).Other examples of crosslinkers include carboxyl-amine reactive, sulfhydryl reactive, aldehyde reactive, and photoreactive crosslinkers. The non-covalent bond linking the antigen-binding domains may be an ionic bond, a hydrogen bond, or a hydrophobic bond.

[0177] Whether the number of bonds between antigen-binding domains is higher than that of a control antigen-binding molecule (e.g., an antigen-binding molecule having a structure substantially similar to that of a native antibody) can be assessed, 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 excises the antigen-binding domain (e.g., proteases such as papain and Lys-C that cleave the N-terminal side of the site where the hinge regions are cross-linked), followed by non-reducing electrophoresis. Next, an antibody that recognizes a portion of the antigen-binding domain (e.g., an anti-kappa chain HRP-labeled antibody) is used to detect fragments present after protease treatment. If only antigen-binding domain monomers (e.g., Fab monomers) are detected for the control antigen-binding molecule, but antigen-binding domain multimers (e.g., Fab dimers) are detected for the antigen-binding molecule of interest, the antigen-binding molecule of interest can be assessed as having a higher number of bonds between antigen-binding domains than the control antigen-binding molecule. The formation of disulfide bonds between cysteines in modified antigen-binding molecules prepared by introducing cysteines into control antigen-binding molecules can be evaluated, for example, by the following method. First, the antigen-binding molecule of interest is incubated with chymotrypsin in 20 mM phosphate buffer (pH 7.0), and the mass of the peptide predicted to be produced from the amino acid sequence of each antibody is detected by LC / MS. If a component corresponding to the theoretical mass of the peptide produced when the newly introduced cysteines form a disulfide bond is detected, it can be determined that the introduced cysteines have formed a disulfide bond. Furthermore, if a disulfide bond-reducing agent (e.g., tris(2-carboxyethyl)phosphine) is added to a sample containing the antigen-binding molecule and then the sample is analyzed, and the component is no longer detected, this further supports the accuracy of the above evaluation.

[0178] <Resistance to protease cleavage> In a non-limiting embodiment, the antigen-binding molecule of the present disclosure is resistant to protease cleavage. In a specific embodiment, the antigen-binding molecule of the present disclosure has increased resistance to protease cleavage compared to a control antigen-binding molecule (e.g., an antigen-binding molecule having a structure substantially similar to a native antibody structure) that has one or more fewer bonds between the antigen-binding domains than the antigen-binding molecule. In a further embodiment, the one fewer bond can be selected from bonds derived from a mutant amino acid residue that serves as the starting point for the bond between the antigen-binding domains and that is not present in wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in wild-type Fab or hinge region). An antigen-binding molecule can be evaluated as having increased resistance to protease cleavage (improved protease resistance) when, for example, the proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment is increased or the proportion of specific fragments (e.g., Fab monomers) generated after protease treatment is decreased compared to a control antigen-binding molecule. In certain embodiments, the proportion of full-length molecules remaining after protease treatment, based on the total number of antigen-binding molecules, can be, for example, 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 7.5% or more, 10% or more, 12.5% ​​or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more. In another specific embodiment, the proportion of antigen-binding domain (e.g., Fab) monomers generated after protease treatment can be, for example, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, based on the total antigen-binding molecules. In another specific embodiment, the proportion of dimers of antigen-binding domains (e.g., Fab) generated after protease treatment, relative to the total number of antigen-binding mol...

Claims

1. A method for producing an antibody preparation, comprising the steps of contacting an antibody solution with a reducing agent and, following said contacting, reoxidizing the antibody, wherein said antibody comprises a first antigen-binding domain and a second antigen-binding domain that are linked to each other via at least one disulfide bond, said at least one disulfide bond being 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; The method, wherein the antibody solution comprises two structural isoforms of the antibody that differ only in the at least one disulfide bond.

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

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

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

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

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

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

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

9. 9. The method of claim 8, wherein the contacting step is carried out at 23 degrees Celsius, 25 degrees Celsius, or 37 degrees Celsius.

10. 10. The method of claim 9, wherein the contacting step is carried out at 23 degrees Celsius.

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

13. 13. The method of any one of claims 1 to 12, further comprising removing the reducing agent by dialysis or chromatographic methods prior to reoxidizing the antibody.

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