Antigen-binding molecules comprising two linked antigen-binding domains

Abstract

To provide antigen-binding molecules having an activity to control the interaction between two or more antigen molecules, or to provide a method for producing or using such antigen-binding molecules.SOLUTION: There is provided an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, the antigen-binding domain comprising a hinge region, the first and second antigen-binding domains comprising an antibody fragment that binds to a specific antigen, the first antigen-binding domain and the second antigen-binding domain being linked to each other via two or more bonds that link amino acid residues at specific positions, at least one of the bonds being a covalent bond, and at least one of the amino acid residues that serve as the origin of the bond between the antigen-binding domains being present in the CH1 region and / or the CL region of the antibody fragment, having increased resistance to protease cleavage compared to a control antigen-binding molecule, and having one fewer bond between the two antigen-binding domains.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to an antigen-binding molecule comprising a first and a second antigen-binding domain linked to each other, a method for producing the antigen-binding molecule, a method for using the antigen-binding molecule, and a pharmaceutical composition comprising the antigen-binding molecule. The present disclosure also relates to a method for increasing the resistance of an antigen-binding molecule to protease cleavage. [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). Meanwhile, antibody engineering technology has not only contributed to the development of antibody engineering as a tool for protein detection, analysis, and purification, but also contributed to the development of protein engineering in general, by using antibody molecules themselves as model proteins to improve the functions of proteins other than antibodies.

[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, natural IgG antibodies may not fully exert the expected effects, and therefore second-generation antibody drugs have been developed in which the functions of natural IgG antibodies are artificially enhanced or added, or attenuated or deleted, to enhance, add, or attenuate or delete the functions according to the intended use 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 antigens with a single molecule (antibodies that bind to two types of antigens are generally referred to as "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 by crosslinking cytotoxic T cells with cancer cells, using 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 used as 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 that utilize effector functions also act on normal cells that express low levels of the target antigen, making them susceptible to side effects. Therefore, attempts have been made to make antibody drugs exert their effector functions specifically on target tissues. For example, there are reported antibodies whose binding ability 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 covalent 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 creating 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 [Non-licensed document 2] Clinical and Experimental Immunology (2009) 157, 9-19 [Non-licensed document 3] Current Pharmaceutical Biotechnology (2016) 17, 1298-1314

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0011] Attempts to enhance or attenuate the agonistic activity and effector function of the above-mentioned antibody drugs are still in development, and further attempts are expected. The present invention was made in light of this situation, and aims to provide novel antigen-binding molecules that have the activity to regulate the interaction between two or more antigen molecules, or methods for producing or using such antigen-binding molecules. The present invention is useful for screening and developing antibody drugs, and is also thought to be applicable to various other protein engineering techniques. [Means for solving the problem]

[0012] In a non-limiting embodiment, the present inventors introduced amino acid mutations into the antigen-binding domains of an antigen-binding molecule (e.g., an antibody) that contains two antigen-binding domains (e.g., Fab portions) and has agonistic activity, thereby creating a molecule in which the antigen-binding domains are linked together, and found that this greatly improved agonistic activity. Furthermore, in a non-limiting embodiment, the present inventors discovered an antigen-binding molecule that has acquired resistance to protease digestion due to the linkage between the antigen-binding domains.

[0013] The present disclosure is based on these findings and specifically includes the embodiments exemplified below. [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 type of amino acid residue to be crosslinked 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 type of amino acid residue to be crosslinked 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 the same type of antigen-binding domains;

[17] the antigen-binding molecule of any 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 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 any one of Fab, Fab', scFab, Fv, scFv, and 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 present 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 any one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and 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, or 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 that serves 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 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 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 of the 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 of the 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 of the 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;

[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 region;

[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 that serves 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 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 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 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 region;

[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 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 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 that serves 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 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 the hinge region;

[82] the antigen-binding molecule of

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

[83] the antigen-binding molecule of any 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 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 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 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 amino acid mutations at at least one site selected from the group consisting of positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447 (EU numbering);

[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.

[0014]

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

[91] , wherein the first and second antigen-binding domains bind to the same type of 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 the first and second antigen-binding domains bind to different epitopes on the antigen;

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

[91] , wherein the first and second antigen-binding domains bind to different types of antigens;

[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 of

[93] to

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

[98] the antigen-binding molecule of any 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.

[0015]

[100] The antigen-binding molecule of any 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 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 have the activity of promoting interaction between cells expressing the first antigen and cells expressing the 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 damage to the target cells by the cells with cytotoxic activity is promoted.

[106] the antigen-binding molecule of

[105] , wherein the cells having cytotoxic activity are T cells, NK cells, monocytes, or macrophages;

[107] the antigen-binding molecule of any 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 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 the control antigen-binding molecule has one fewer bond between the two antigen-binding domains.

[112] The antigen-binding molecule of any 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 less bond between the two antigen-binding domains.

[114] The antigen-binding molecule of any 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 the control antigen-binding molecule 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 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 fewer 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 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;

[0016]

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

[122] and a pharmaceutically acceptable carrier.

[0017]

[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 controlling 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 the 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 to the antigen-binding molecule at least one bond that links the two antigen-binding domains to each other.

[0018]

[129] A method for producing an antigen-binding molecule having an activity that regulates 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 the host cell so as 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.

[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 the host cell so as 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.

[131] A method for producing an antigen-binding molecule that has 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 the host cell so as 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.

[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, 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 the host cell so as 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.

[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 the host cell so as 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.

[0019]

[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 of

[129] to

[133] ; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) determining 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]

[0020] [Figure 1] Figure 1 shows examples of modified antibodies in which Fabs are cross-linked, as described in Example 1. This figure schematically illustrates 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, LH type). [Figure 2] Figure 2 shows the results of measuring the CD3-mediated agonistic activity of a natural anti-CD3ε antibody molecule (CD3-G4s) and modified antibody molecules (CD3-G4sLL, CD3-G4sHH) in which the Fab-Fab domains of the natural anti-CD3ε antibody molecule are linked via an additional disulfide bond, as described in Example 4-3. [Figure 3] Figure 3 shows the results of measuring the CD3-mediated agonistic activity of a natural anti-CD3ε antibody molecule (OKT3-G1s) and modified antibody molecules (OKT3-G1sLL, OKT3-G1sHH) in which the Fab-Fab domains are linked via an additional disulfide bond, as described in Example 4-3. [Figure 4]Figure 4 shows the results of measuring the CD3- and / or CD28-mediated agonistic activity of a natural 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 in which the Fab-Fabs are linked via an additional disulfide bond (CD3 / / CD28-G1sLL, CD3 / / CD28-G1sHH, CD3 / / CD28-G1sLH, CD3 / / CD28-G1sHL), as described in Example 4-3. [Figure 5] Figure 5 shows the results of measuring the CD3- and / or CD28-mediated agonistic activity of a natural anti-CD3ε antibody molecule (OKT3-G1s), an anti-CD28 antibody molecule (CD28-G1s), an anti-CD3ε x anti-CD28 bispecific antibody (OKT3 / / CD28-G1s), and modified antibody molecules in which the Fab-Fabs are linked via an additional disulfide bond (OKT3 / / CD28-G1sHH, OKT3 / / CD28-G1sHL), as described in Example 4-3. [Figure 6] 6 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (1 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 7] 7 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (2 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 8]8 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (3 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 9] 9 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (4 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 10] 10 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (5 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 11] 11 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (6 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 12]12 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (7 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 13] 13 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitutions in its heavy chain variable region, and a modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitutions in its heavy chain constant region, as described in Example 5-2 (8 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 14] 14 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in the light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in the light-chain constant region, as described in Example 6-2 (1 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 15] 15 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in the light chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in the light chain constant region, as described in Example 6-2 (2 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 16]16 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (3 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 17] 17 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (4 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 18] 18 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (5 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 19] 19 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (6 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 20]20 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (7 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 21] 21 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (8 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 22] 22 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in the light chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in the light chain constant region, as described in Example 6-2 (9 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 23] 23 shows the results of protease treatment of an anti-IL6R antibody (MRA), its modified antibody (MRAL.xxx-k0) with cysteine ​​substitutions in its light-chain variable region, and a modified antibody (MRAL-k0.xxx) with cysteine ​​substitutions in its light-chain constant region, as described in Example 6-2 (10 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody. [Figure 24]24 shows the results of protease treatment of an anti-IL6R antibody (MRA) and its modified antibody (MRAL-k0.K126C) with cysteine ​​substitutions in the light-chain constant region, as described in Example 7-2. Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and bands were detected using an anti-kappa chain antibody or an anti-human Fc antibody. [Figure 25] Figure 25 shows the molecular weights of each band obtained by treating an antibody sample with protease and their predicted structures, as described in 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 24) is also shown. [Figure 26] Figure 26 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) in which the Fab-Fab domains of the OKT3 antibody molecule are linked by an additional disulfide bond, and an anti-KLH antibody molecule (IC17) (negative control), as described in Example 13-4. [Figure 27] Figure 27 shows the results of measuring the CD3-mediated agonist activity of an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (OKT3_KiH) in which a Knobs-into-Holes (KiH) modification that promotes heterodimerization has been introduced into the heavy chain constant region of OKT3, modified antibody molecules in which the Fab-Fabs of the OKT3 antibody molecule have been linked by an additional disulfide bond (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), and an anti-KLH antibody molecule (IC17) (negative control), as described in Example 14-4. [Figure 28]Figure 28 shows the results of experiments using an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (H_S191C) in which the Fab-Fab pairs of the antibody molecule are linked via an additional disulfide bond, a modified antibody molecule (OKT3_KiH) in which a Knobs-into-Holes (KiH) modification that promotes heterodimerization has been introduced into the heavy chain constant region of OKT3, a modified antibody molecule (H_S191C_KiH) in which the Fab-Fab pairs of the antibody molecule are linked via an additional disulfide bond, and one of the heavy chain constant regions of OKT3_KiH. FIG. 1 shows the results of measuring the CD3-mediated agonist activity of modified antibody molecules (0004 / / 0004, 0004 / / 0006) in which positively charged amino acid substitutions have been introduced 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) in which positively or negatively charged amino acid substitutions have been introduced into one heavy chain constant region of OKT3_KiH, and an anti-KLH antibody molecule (IC17) (negative control). [Figure 29] Figure 29 shows the results of measuring the CD3-mediated agonist activity of an anti-CD3 antibody molecule (OKT3), modified antibody molecules (dh1, dh2, dh3) in which the disulfide bond in the hinge region has been removed, modified antibody molecules (H_S191C_dh1, H_S191C_dh2, H_S191C_dh3) in which the Fab-Fabs of these antibodies have been linked by an additional disulfide bond, and an anti-KLH antibody molecule (IC17) (negative control), as described in Example 16-4. [Figure 30]Figure 30 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) in which the Fab-Fabs of the anti-CD3 monospecific antibody (CD3-G1s) are linked by an additional disulfide bond, a modified antibody molecule (CD3-G1sLL) in which the Fab-Fabs of the anti-CD3 monospecific antibody (CD3-G1s) are linked by an additional disulfide bond, an anti-CD3 biparatopic antibody molecule (CD3 / / OKT3-G1s), modified antibody molecules (CD3 / / OKT3-G1sHH, CD3 / / OKT3-G1sLH) in which the Fab-Fabs of the anti-CD3 monospecific antibody (CD3 / / OKT3-G1s) are linked by an additional disulfide bond, and a combination of CD3-G1sLL and OKT3-G1s (CD3-G1sLL+OKT3-G1s), as described in Example 20. [Figure 31A] Figures 31A to 31D show the results of measuring the CD3- and / or PD1-mediated agonistic activity of an anti-CD3×anti-PD1 bispecific antibody and modified antibody molecules in which the Fab-Fabs are linked via an additional disulfide bond, as described in Example 22-1. (A) 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 in which the Fab-Fabs are linked via an additional disulfide bond (OKT3 / / 117-G1silentHH, OKT3 / / 117-G1silentHL, OKT3 / / 117-G1silentLL). [Figure 31B] (B) 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) in which the Fab-Fab pairs are linked via an additional disulfide bond is shown. [Figure 31C](C) 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), as well as modified antibody molecules (CD3 / / 949-G1silentLH, CD3 / / 949-G1silentHH, CD3 / / 949-G1silentLL, CD3 / / 949-G1silentHL) in which the Fab-Fab pairs are linked by an additional disulfide bond. [Figure 31D] (D) 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), as well as modified antibody molecules (OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentLL) in which the Fab-Fab pairs are linked via an additional disulfide bond. [Figure 32] Figure 32 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) in which the Fab-Fabs of the bispecific antibody molecule are linked via an additional disulfide bond, as described in Example 22-2. [Figure 33A]Figures 33A and 33B show 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 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. (A) The inhibitory effect on cancer cell proliferation is shown when the GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) is used as an 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), and a modified antibody molecule (CD28 / clamp CD3_HH) in which the Fab-Fab units of these bispecific antibodies are linked by an additional disulfide bond are used as antibodies for activating T cells. [Figure 33B] (B) The inhibitory effect on cancer cell proliferation is shown when the following antibodies are used to target T cells to cancer cells: a modified antibody molecule (GPC3 / attCE115_LL) in which the Fab-Fab of a GPC3 / binding-attenuated CD3 bispecific antibody is linked by an additional disulfide bond; and a GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), a KLH / CD3 clamping bispecific antibody molecule (KLH / clamp CD3), a CD28 / CD3 clamping bispecific antibody molecule (CD28 / clamp CD3), and a modified antibody molecule in which the Fab-Fab of the same is linked by an additional disulfide bond (CD28 / clamp CD3_HH). [Figure 34A]Figures 34A-C show 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 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. (A) IL-6 production levels 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) in which the Fab-Fab of a CD28 / CD3 clamping bispecific antibody is linked via an additional disulfide bond was used alone or in combination. [Figure 34B] (B) IL-6 production levels in the presence of effector cells (T cells) alone when the GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and a modified antibody molecule (CD28 / clamp CD3_HH) in which the Fab-Fab of a CD28 / CD3 clamping bispecific antibody is linked via an additional disulfide bond (Figure 1B) were used alone or in combination. [Figure 34C] (C) The inhibitory effect of the GPC3 / binding-attenuated CD3 bispecific antibody molecule (GPC3 / attCE115) and the modified antibody molecule (CD28 / clamp CD3_HH) in which the Fab-Fab of the CD28 / CD3 clamping bispecific antibody is linked by 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 35A]Figures 35A and 35B are schematic diagrams 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 Example 23-1 (ε in the figure represents CD3ε). (A) The mechanism of action of cancer cell growth 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 35B] (B) The mechanism of action of cancer cell proliferation inhibition in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells) 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. [Figure 36A] Figures 36A and 36B are schematic diagrams 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 Example 23-2 (ε in the figure represents CD3ε). (A) The diagram 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 36B] (B) Mechanism of cytokine production in the presence of effector cells (T cells) alone when a modified antibody molecule, modified to introduce an additional disulfide bond between the Fab-Fab of a CD28 / CD3 clamping bispecific antibody, is used in combination with a GPC3 / binding-attenuated CD3 bispecific antibody. [Figure 37A]Figures 37A and 37B show 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)) linked by an additional disulfide bond between the Fabs, as described in Example 24. An anti-KLH antibody molecule (KLH-P587) was used as a negative control. Results are shown for peripheral blood mononuclear cells (PBMCs) from two different donors (top: donor A, bottom: donor B). (A) The percentage of divided regulatory T (Treg) cells in PBMCs is shown. [Figure 37B] (B) The percentage of divided CD8α-positive T cells in PBMCs. DETAILED DESCRIPTION OF THE INVENTION

[0021] 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.

[0022] 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," or "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.

[0023] As used herein, the term "specifically binds" refers to a state in which one of the specifically binding molecules binds without showing any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies to cases in which an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0024] 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%.

[0025] 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.

[0026] 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 variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0027] "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 (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0028] 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.

[0029] 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.

[0030] In one embodiment of the present invention, the constant region is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 antibody constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 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 heavy chain constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 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 antibodies, 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 may be used in the present invention. Note that 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.

[0031] The term "hinge region" refers to the antibody heavy chain polypeptide portion that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example, from approximately position 216 to approximately position 230 according to the EU numbering system, or from approximately position 226 to approximately 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. As used herein, the term "hinge region" encompasses not only the wild-type but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type.

[0032] 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.

[0033] "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.

[0034] "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.

[0035] 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.)).

[0036] 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).

[0037] 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.

[0038] "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.

[0039] 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.

[0040] 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.

[0041] 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."

[0042] 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.

[0043] 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.

[0044] "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.

[0045] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the minimum 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 and active Fvs 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). The Fvs prepared from the periplasmic fraction contained an association of VH and VL in a manner that allowed them to bind to antigens.

[0046] scFv, single chain antibody, or sc(Fv)2 As used herein, the terms "scFv," "single-chain antibody," or "sc(Fv)2" refer to an antibody fragment that contains, in a single polypeptide chain, variable regions from both the heavy and light chains but lacks 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 may enable 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 and U.S. Pat. Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may also be bispecific and / or humanized.

[0047] 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.

[0048] 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. Suitable examples include bispecific sc(Fv)2s that recognize two different epitopes present in the same antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2s can be produced by methods known to those skilled in the art. For example, they can be produced by linking scFvs with a linker such as a peptide linker.

[0049] As used herein, the antigen-binding domain constituting sc(Fv)2 may be configured as an antibody in which two VHs and two VLs 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 VHs and two VLs 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]

[0050] Fab, F(ab')2, or Fab' "Fab" is composed of one light chain and the CH1 region and variable region of one heavy chain. The heavy chain of a wild-type Fab molecule cannot form a disulfide bond with another heavy chain molecule. The present specification encompasses not only wild-type Fabs, but also modified Fabs in which amino acid residues have been substituted, added, or deleted from the wild-type. In certain embodiments, the mutated amino acid residues (e.g., substituted, added, or inserted cysteine ​​or lysine residues) contained in the modified Fabs can form disulfide bonds with another heavy chain molecule or portion thereof (e.g., a Fab molecule).

[0051] 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.

[0052] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating an immunoglobulin (monoclonal antibody) with a protease (protease) such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, treating IgG with papain cleaves the antibody upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which an light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and an heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (the γ1 region of the heavy chain constant region) are linked by a disulfide bond at their C-terminal regions. These two homologous antibody fragments are each referred to as Fab'.

[0053] "F(ab')2" comprises two light chains and two heavy chains comprising constant regions, i.e., portions of the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably obtained 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. There are no particular limitations on the protease, as long as it can digest a full-length antibody so as to produce F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH. Examples of such a protease include pepsin and ficin.

[0054] Single domain antibodies (also called monodomain antibodies) As used herein, the term "single-domain antibody" is not limited by its structure, as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, such as 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 Examples of single-domain antibodies include antigen-binding molecules that inherently lack light chains, such as those described in U.S. Pat. No. 6,248,516 B1, or 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 human antibody VL, such as those described in U.S. Pat. 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-domain antibodies can also be obtained by substituting human germline sequences or sequences similar thereto for the framework sequences of single-domain antibodies obtained from animals. Humanized single-domain antibodies (e.g., humanized VHHs) are also an 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)), or 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)).

[0055] "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 the molecule's binding partner (e.g., an antigen). Here, binding activity is not strictly limited to 1:1 interactions 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.

[0056] 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.

[0057] 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.

[0058] 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).

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 various 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.

[0064] 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.

[0065] 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 type of the cross-linked amino acid residue is, for example, cysteine, and the covalent bond formed 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, such as an amine-reactive cross-linking agent, and the type of amino acid residue cross-linked is, for example, lysine.

[0066] 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).

[0067] The bond between the antigen-binding domains (the bond connecting the two antigen-binding domains) is formed by linking amino acid residues that serve as the origin of binding in each of the first and second antigen-binding domains. 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 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 binding between the antigen-binding domains are disclosed herein in the CH1 region, CL region, and hinge region as constant regions, and the VH region, VL region, and VHH region as variable regions, and cysteine ​​residues can be introduced into these sites, for example.

[0068] In one embodiment of the above aspect, 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.

[0069] 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 perspective of molecular shape, they are considered to be of the same type.

[0070] 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.

[0071] The positions of amino acid residues on the antigen-binding domains 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. On the other hand, 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.

[0072] 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.

[0073] 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.

[0074] 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 a specific embodiment, 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 a specific embodiment, 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, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 148, 149, and 150. In certain embodiments, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. In certain embodiments, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178. 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 EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197. 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 EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.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.

[0075] 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, the difference is within 3 amino acids, respectively, 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 and second 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 to 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 to 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 connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues 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.

[0076] 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.

[0077] In one embodiment of the above aspect, 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 a specific embodiment, 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 a specific embodiment, 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, 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, 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, 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, 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.

[0078] 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 CL region of the first antigen-binding domain and 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 is within 3 amino acids, respectively, according to EU 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 126 (Kabat numbering) in the CL regions of the two antigen-binding domains.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 the bond 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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 are 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 site selected from the group consisting of EU numbering 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.

[0087] <Types of antigens that antigen-binding molecules bind to> In one embodiment of the above aspect, the first and second antigen-binding domains both bind to the same type of antigen. In a specific embodiment, the first and second antigen-binding domains bind to the same epitope on the same type of antigen. In another specific embodiment, the first and second antigen-binding domains bind to different epitopes on the same type of 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 type of antigen. In another embodiment of the above aspect, the first and second antigen-binding domains bind to different types of antigens. 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 have the same amino acid sequence, while in another embodiment, the first and second antigen-binding domains have different amino acid sequences.

[0088] 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.

[0089] <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 (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains. 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 (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains. 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. The cell expressing the first antigen and the cell expressing the second antigen are, for example, a cell with cytotoxic activity and a target cell thereof, respectively, and the antigen-binding molecule of the present disclosure promotes the destruction of the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, any of T cells, NK cells, monocytes, and macrophages.

[0090] 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 any 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 derived from a mutant amino acid residue (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains. The antigen molecule is, for example, 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.

[0091] 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 flexibility 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 flexibility 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 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 the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0092] 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 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 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.

[0093] 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 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 these embodiments, the protease can cleave the hinge region of the antigen-binding molecule.

[0094] 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.

[0095] <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.

[0096] <Uses of antigen-binding molecules> In one aspect, the present disclosure provides a method for holding two antigen molecules in close spatial proximity, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains of the antigen-binding molecule in (a) 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 that serves 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 present in a hinge region). In a further embodiment, the at least one bond in (b) is a bond in which the amino acid residue that serves as the origin of the bond between the antigen-binding domains is 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). 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 holding two antigen molecules in close spatial proximity.

[0097] In another aspect, the present disclosure provides a method for controlling the interaction between two antigen molecules, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains of the antigen-binding molecule in (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 that serves 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 present 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 that serves as the origin of the bond between the antigen-binding domains is 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). 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 controlling the interaction between two antigen molecules.

[0098] In another aspect, the present disclosure provides a method for regulating the activity of two antigen molecules that are activated by their association with each other, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, the two antigen-binding domains of the antigen-binding molecule in (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 residues that serve as the origin of the bond between the antigen-binding domains are derived from amino acid residues present in a wild-type Fab or hinge region (e.g., cysteine ​​residues 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 (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the origin of the bond between the antigen-binding domains. The present disclosure also provides a method for regulating the activity of two antigenic molecules that are activated by their association with each other, comprising contacting the two antigenic 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 regulating the activity of two antigenic molecules that are activated by their association with each other.

[0099] In another aspect, the present disclosure provides a method for positioning two antigen-binding domains in close spatial proximity and / or reducing the flexibility of the two antigen-binding domains, the method comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding to the antigen-binding molecule at least one bond that links the two antigen-binding domains to each other. In a specific embodiment, the two antigen-binding domains of the antigen-binding molecule in (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 that serves 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 present 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 that serves as the origin of the bond between the antigen-binding domains is 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).

[0100] In yet another aspect, the present disclosure provides a method for increasing the resistance of an antigen-binding molecule to protease cleavage, the method comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding to the antigen-binding molecule at least one bond linking the two antigen-binding domains to each other. In a specific embodiment, the two antigen-binding domains of the antigen-binding molecule in (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).

[0101] The antigen-binding molecules used in these various methods may have the characteristics of the antigen-binding molecules described herein.

[0102] <Method of producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having the activity of holding two antigen molecules in close spatial proximity, 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 that at least one bond linking the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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. 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., cysteine ​​residues 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).

[0103] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule having an activity of regulating the interaction between two antigen molecules, 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 that at least one bond linking the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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. 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., cysteine ​​residues 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).

[0104] In yet another aspect, the present disclosure provides 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 that at least one bond linking the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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. 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., cysteine ​​residues 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).

[0105] 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 mobility of the two antigen-binding domains is reduced, 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 that at least one bond linking the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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. 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., cysteine ​​residues 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).

[0106] In yet another aspect, the present disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, 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 that at least one bond linking the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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. 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., cysteine ​​residues 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).

[0107] The antigen-binding molecules produced in these various aspects may have the characteristics of the antigen-binding molecules described herein.

[0108] <Method for screening antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel pair of protein molecules that are activated by associating with each other, 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; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) determining whether the two protein molecules are activated. 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.

[0109] 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.

[0110] 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 bond between the antigen-binding domains is formed by linking amino acid residues present in each of the first and second antigen-binding domains, which serve as the bond origin. In a specific embodiment, at least one of the amino acid residues that serve as the bond origin 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 bond origin 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 bond origin between the antigen-binding domains is present in the antibody fragment, and at least one is present in the hinge region.

[0111] 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 located at the same positions in the first and second antigen-binding domains are linked to each other to form a bond, while in certain embodiments, amino acid residues located at different positions in the first and second antigen-binding domains are linked to each other to form a bond.

[0112] The positions of amino acid residues on the antigen-binding domains 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. On the other hand, 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.

[0113] In one embodiment of the above aspect, at least one of the two or more bonds linking 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 type of cross-linked amino acid residue is, for example, cysteine, and the covalent bond formed is, 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, such as an amine-reactive cross-linking agent, and the type of amino acid residue cross-linked is, for example, lysine.

[0114] 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.

[0115] In one embodiment of the above aspect, the antibody fragment is any of a Fab, a Fab', an scFab, an Fv, an scFv, and a single domain antibody.

[0116] 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 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 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 hinge region, for example, at any one selected from the group consisting of positions 216, 218, and 219 (EU numbering) 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 any 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 a bond is formed by linking the amino acid residue at EU numbering position 126 in the CL regions of the two antigen-binding domains. 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.

[0117] 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.

[0118] 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 positions 8, 16, 28, 74, and 82b (Kabat numbering) of the VH region. In a specific embodiment, the amino acid residue is located in the VL region, e.g., at positions 100, 105, and 107 (Kabat numbering) of the VL region.

[0119] 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. The amino acid residues that can serve as the starting point for binding between the antigen-binding domains in each of the CH1 region, hinge region, CL region, VH region, and VL region 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 an F(ab')2, in which both the first and second antigen-binding domains comprise an Fab and hinge region.

[0120] 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.

[0121] 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 are 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 site selected from the group consisting of EU numbering 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.

[0122] <Types of antigens that antigen-binding molecules bind to> In one embodiment of the above aspect, the first and second antigen-binding domains both bind to the same type of antigen. In a specific embodiment, the first and second antigen-binding domains bind to the same epitope on the same type of antigen. In another specific embodiment, the first and second antigen-binding domains bind to different epitopes on the same type of 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 type of antigen. In another embodiment of the above aspect, the first and second antigen-binding domains bind to different types of antigens. 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 have the same amino acid sequence, while in another embodiment, the first and second antigen-binding domains have different amino acid sequences.

[0123] 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.

[0124] <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 (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains.

[0125] 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 (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains.

[0126] 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.

[0127] 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. The cell expressing the first antigen and the cell expressing the second antigen are, for example, a cell with cytotoxic activity and a target cell thereof, respectively, and the antigen-binding molecule of the present disclosure promotes the destruction of the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, any of T cells, NK cells, monocytes, and macrophages.

[0128] 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 any 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 derived from a mutant amino acid residue (e.g., a cysteine ​​residue not present in a wild-type Fab or hinge region) that serves as the starting amino acid residue for the bond between the antigen-binding domains. The antigen molecule is, for example, 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.

[0129] 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 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 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.

[0130] 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 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 these embodiments, the protease can cleave the hinge region of the antigen-binding molecule.

[0131] <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.

[0132] <Uses of antigen-binding molecules> In one aspect, the present disclosure provides a method for holding two antigen molecules in close spatial proximity, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the originating amino acid residue 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 originating amino acid residue of the other bond in (b) above 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 holding two antigen molecules in close spatial proximity.

[0133] In another aspect, the present disclosure provides a method for controlling the interaction between two antigen molecules, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the originating amino acid residue 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 originating amino acid residue of the bond between the antigen-binding domains in (b) above 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 controlling the interaction between two antigen molecules.

[0134] In another aspect, the present disclosure provides a method for regulating the activity of two antigen molecules that are activated by their association with each other, the method 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 to each other; and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules. In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the originating amino acid residue 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 present in the hinge region). In a further embodiment, the originating amino acid residue of the bond between the antigen-binding domains in (b) above 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 antigenic molecules that are activated by their association with each other, comprising contacting the two antigenic 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 regulating the activity of two antigenic molecules that are activated by their association with each other.

[0135] In yet another aspect, the present disclosure provides a method for increasing the resistance of an antigen-binding molecule to protease cleavage, the method 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 to the antigen-binding molecule another bond linking the two antigen-binding domains to each other. In a specific embodiment, some or all of the one or more bonds in (a) above are bonds in which the originating amino acid residue 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 originating amino acid residue of the bond between the antigen-binding domains in (b) above 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).

[0136] The antigen-binding molecules used in these various methods may have the characteristics of the antigen-binding molecules described herein.

[0137] <Method of producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule that has the activity of holding two antigen molecules in close spatial proximity, 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, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the origin of a bond 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 linking the two antigen-binding domains; (c) introducing the nucleic acid prepared in (b) into a host cell; (d) culturing the host cell so as 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 two or more bonds. 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).

[0138] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule that has the activity of regulating the interaction between two antigen molecules, 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, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the origin of a bond connecting 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 prepared in (b) into a host cell; (d) culturing the 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 two or more bonds. In a specific embodiment, some or all of the one or more amino acid residues that serve as the origin of a bond between the antigen-binding domains in (a) above are amino acid residues present in a 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 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) that serves as the origin of the bond between the antigen-binding domains.

[0139] In yet another aspect, the present disclosure provides 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, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the origin of a bond 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 linking the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so as 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 two or more bonds. 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).

[0140] In yet another aspect, the present disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, 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, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the origin of a bond connecting 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 prepared in (b) into a host cell; (d) culturing the 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 two or more bonds. 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 a 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 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) that serves as the origin of the bond between the antigen-binding domains. The antigen-binding molecules produced in these various aspects may have the characteristics of the antigen-binding molecules described herein.

[0141] <Method for screening antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel pair of protein molecules that are activated by associating with each other, the method comprising: (a) providing any two protein molecules; (b) producing, by the production method of the present disclosure, antigen-binding molecules that comprise two antigen-binding domains that bind to the two protein molecules, respectively, and have the activity of holding the two protein molecules in close proximity; (c) contacting the antigen-binding molecules produced in (b) with the two protein molecules; and (d) determining whether the two protein molecules are activated. 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.

[0142] <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.

[0143] 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 1). 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).

[0144] In a 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 origin of binding between the antigen-binding domains. The distance between the multiple amino acid residues is such that, as a result of linkage between the antigen-binding domains via bonds originating from each amino acid residue, a structure of two or more antigen-binding domains that are sufficiently close to each other is achieved. The multiple amino acid residues may be separated by, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 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 origin of the bonds are such that a structure of two or more antigen-binding domains that are sufficiently close together is achieved as a result of the linkage between the antigen-binding domains through the bonds; for example, the number may be 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 originating 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.

[0145] 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 destruction 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.

[0146] 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., isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, fluoroester, etc.), and representative examples include disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl tartrate (DST), bis(2-(succinimidooxycarbonyloxy) ethyl)sulfone (BSOCOES), ethylene glycol bis(succinimidyl Examples of crosslinkers include ethylene glycol bis(sulfosuccinimidyl succinate) (Sulfo-EGS), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), and 1,5-difluoro-2,4-dinitrobenzene (DFDNB). 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.

[0147] Whether or not there is increased inter-antigen-binding domain binding compared to 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., a protease that cleaves N-terminally from the site where the hinge regions are cross-linked, such as papain or Lys-C), 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 in the control antigen-binding molecule, whereas antigen-binding domain multimers (e.g., Fab dimers) are detected in the antigen-binding molecule of interest, the antigen-binding molecule of interest can be assessed as having increased inter-antigen-binding domain binding compared to the control antigen-binding molecule. The formation of disulfide bonds between cysteines in modified antigen-binding molecules in which cysteines have been introduced 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 sample containing the antigen-binding molecule is analyzed after adding an agent that reduces disulfide bonds (e.g., tris(2-carboxyethyl)phosphine) and the component is no longer detected, this further supports the accuracy of the above evaluation.

[0148] <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 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 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. Examples of proteases include, but are not limited to, Lys-C, plasmin, human neutrophil elastase (HNE), papain, and the like.

[0149] In a further aspect, the antigen-binding molecule according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1 to 7 below.

[0150] 1. Affinity of antigen-binding molecules In certain embodiments, the antigen-binding molecules provided herein have an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13M, e.g. 10 -9 M~10 -13 It has a dissociation constant (KD) of 1 M.

[0151] 2. Antibody fragment In certain embodiments, the antigen-binding molecules provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described herein. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); in addition, see WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0152] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0153] 3. Chimeric and humanized antibodies In certain embodiments, the antigen-binding molecule provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include their antigen-binding fragments.

[0154] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0155] 4. Human antibodies In certain embodiments, the antigen-binding molecules provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0156] 5. Library-derived antigen-binding molecules Antigen-binding molecules of the present invention may be isolated by screening combinatorial libraries for antigen-binding molecules with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antigen-binding molecules with the desired binding properties. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0157] 6. Multispecific antigen binding molecules In certain embodiments, the antigen-binding molecules provided herein are multispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules). Multispecific antigen-binding molecules are monoclonal antigen-binding molecules that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for a specific antigen (e.g., CD3), and the other is for any other antigen (e.g., CD28 or a cancer antigen). In certain embodiments, a bispecific antigen-binding molecule may bind to two different epitopes on a single antigen. Bispecific antigen-binding molecules can be prepared as full-length antibodies or antibody fragments.

[0158] Techniques for producing multispecific antigen-binding molecules include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antigen-binding molecules can be constructed by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); creating bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1993)). (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0159] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0160] 7. Antigen-binding molecule variants In certain embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecule. Amino acid sequence variants of the antigen-binding molecule may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding molecule or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the antigen-binding molecule, and / or insertions into the amino acid sequence of the antigen-binding molecule, and / or substitutions of residues in the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0161] a) Substitution, insertion, and deletion mutants In certain embodiments, antigen-binding molecule variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in the table below under the heading of "preferred substitutions." More substantial changes are provided in the table under the heading of "exemplary substitutions," and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antigen-binding molecule of interest, and the products may be screened for desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0162] TIFF0007811967000001.tif160170

[0163] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0164] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Typically, the resulting variants selected for further study will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain the specific biological properties of the parent antigen-binding molecule. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0165] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve the affinity of antigen-binding molecules. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigens, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antigen-binding molecule variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0166] In certain embodiments, substitutions, insertions, or deletions can be made within one or more HVRs, as long as such modifications do not substantially reduce the ability of the antigen-binding molecule to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs.Such modifications can be, for example, outside the antigen contact residues of HVRs.In certain embodiments of the above-mentioned mutant VH and VL sequences, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

[0167] A useful method for identifying residues or regions of an antigen-binding molecule that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the interaction between the antigen-binding molecule and the antigen is affected. Further substitutions can be introduced into amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, the crystal structure of the complex of the antigen and the antigen-binding molecule can be analyzed to identify contact points between the antigen-binding molecule and the antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or can be excluded from the substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0168] Amino acid sequence insertions include fusions ranging in length from one residue to 100 or more residues at the amino and / or carboxyl termini, as well as insertions of single or multiple amino acid residues within the sequence. An example of a terminal insertion is an antigen-binding molecule with an N-terminal methionyl residue. Other insertional variants of antigen-binding molecules include those fused to the N- or C-terminus of the antigen-binding molecule with an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antigen-binding molecule.

[0169] b) Glycosylation variants In certain embodiments, the antigen-binding molecules provided herein are modified to increase or decrease the degree of glycosylation of the antigen-binding molecules. Addition or deletion of glycosylation sites to an antigen-binding molecule can be conveniently achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0170] When an antigen-binding molecule contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antigen-binding molecules of the present invention may be performed to create antigen-binding molecule variants with specific improved properties.

[0171] In one embodiment, antigen-binding molecule variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antigen-binding molecules can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variation among multiple antigen-binding molecules, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antigen-binding molecule variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Including Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antigen-binding molecules include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0172] Further provided are antigen-binding molecule variants having bisected oligosaccharides, for example, biantennary oligosaccharides added to the Fc region of the antigen-binding molecule are bisected by GlcNAc. Such antigen-binding molecule variants may have reduced fucosylation and / or improved ADCC function. Examples of such antigen-binding molecule variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antigen-binding molecule variants having at least one galactose residue in the oligosaccharide added to the Fc region are also provided. Such antigen-binding molecule variants may have improved CDC function. Such antigen-binding molecule variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0173] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antigen-binding molecule provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0174] In certain embodiments, antigen-binding molecule variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications in which their in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. To confirm reduced / lack of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antigen-binding molecule lacks FcγR binding (and thus likely lacks ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antigen-binding molecule is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0175] Antigen-binding molecules with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0176] Certain antigen-binding molecule variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0177] In certain embodiments, the antigen-binding molecule variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0178] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0179] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0180] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0181] d) Cysteine-engineered antigen-binding molecule variants In certain embodiments, it may be desirable to create cysteine-engineered antigen-binding molecules (e.g., "thioMAbs") in which one or more residues of the antigen-binding molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antigen-binding molecule. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antigen-binding molecule, which may be used to conjugate the antigen-binding molecule to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0182] e) Antigen-binding molecule derivative In certain embodiments, the antigen-binding molecules provided herein may be further modified to contain additional nonprotein moieties known in the art and readily available. Moieties suitable for derivatization of antigen-binding molecules include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3 dioxolane, poly-1,3,6 trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antigen-binding molecule can vary, and if one or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the specific properties or functions of the antigen-binding molecule to be improved, whether the antigen-binding molecule derivative will be used in therapy under specified conditions, etc.

[0183] In the context of the antigen-binding molecules of the present disclosure, desired properties (activities) are not particularly limited, and examples include binding activity, neutralizing activity, cytotoxic activity, agonistic activity, antagonistic activity, enzymatic activity, etc. Agonistic activity is the activity of inducing a change in some physiological activity by, for example, transmitting a signal intracellularly upon binding of an antibody to an antigen such as a receptor. Examples of physiological activities include, but are not limited to, proliferation activity, survival activity, differentiation activity, transcription activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation / dephosphorylation activity, redox activity, metastasis activity, nucleolytic activity, dehydration activity, cell death-inducing activity, and apoptosis-inducing activity.

[0184] In another embodiment, a conjugate of an antigen-binding molecule and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, including but not limited to, a wavelength that heats the non-protein moiety to a temperature that is not harmful to normal cells but that kills cells in close proximity to the antigen-binding molecule-non-protein moiety.

[0185] B. Recombinant Methods and Constructs For example, as described in U.S. Patent No. 4,816,567, antigen-binding molecules can be produced using recombinant methods or constructs. In one embodiment, an isolated nucleic acid encoding an antigen-binding molecule of the present disclosure (a polypeptide comprising an antigen-binding domain described herein) is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antigen-binding molecule (e.g., the light chain and / or the heavy chain of the antigen-binding molecule). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and an amino acid sequence comprising the VH of the antigen-binding molecule, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding molecule. In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cell) or lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell)). In one embodiment, there is provided a method for producing an antigen-binding molecule of the present disclosure, comprising culturing a host cell comprising a nucleic acid encoding the antigen-binding molecule as described above under conditions suitable for expression of the antigen-binding molecule, and optionally recovering the antigen-binding molecule from the host cell (or host cell culture medium).

[0186] For recombinant production of the antigen-binding molecules of the present disclosure, nucleic acids encoding the antigen-binding molecules (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antigen-binding molecules).

[0187] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells as described herein. For example, antigen-binding molecules may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (Also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, antigen-binding molecules may be isolated in a soluble fraction from bacterial cell paste and further purified.

[0188] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antigen-binding molecules with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antigen-binding molecule-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0189] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antigen-binding molecules. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly transformation of Spodoptera frugiperda cells.

[0190] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antigen-binding molecules in transgenic plants).

[0191] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). (described in

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

[0192] C. Assay The antigen-binding molecules provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0193] 1. Binding and other assays In one aspect, the antigen-binding molecules of the present disclosure are tested for their antigen-binding activity by known methods, such as ELISA, Western blot, etc.

[0194] 2.Activity measurement method In one aspect, a method for identifying an antigen-binding molecule having biological activity is provided. Biological activity may include, for example, the activity of holding two antigen molecules in close spatial proximity, the activity of controlling 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 destruction of target cells by cells with cytotoxic activity (e.g., T cells, NK cells, monocytes, macrophages, etc.), the activity of controlling the activation of two antigen molecules activated by their association with each other, and resistance to protease cleavage. Also provided are antigen-binding molecules that have such biological activity in vivo and / or in vitro.

[0195] Furthermore, antigen-binding molecules of the present disclosure can exhibit various biological activities depending on the type of antigen molecule to which they bind. Examples of such antigen-binding molecules include antigen-binding molecules that bind to the T cell receptor (TCR) complex (e.g., CD3) and have the activity of inducing T cell activation (agonistic activity); antigen-binding molecules that bind to the TNF receptor superfamily (e.g., OX40 and 4-1BB) or other costimulatory molecules (e.g., CD28 and ICOS) and have the activity of promoting said activation (agonistic activity); and the like. In certain embodiments, the biological activity exhibited through binding to such antigen molecules is enhanced or attenuated by linking two or more antigen-binding domains contained in the antigen-binding molecule of the present disclosure. Without being limited by theory, in certain embodiments, such enhancement or attenuation may be achieved by regulating the interaction between two or more antigen molecules (e.g., promoting the association of two or more antigen molecules) through binding to an antigen-binding molecule of the present disclosure.

[0196] In certain embodiments, antigen-binding molecules of the present disclosure are tested for such biological activity. Whether two antigen molecules are in close spatial proximity can be evaluated using techniques such as crystal structure analysis of a complex consisting of an antigen and an antigen-binding molecule, electron microscopy, or structural analysis by electron tomography. Whether two antigen-binding domains are in close spatial proximity or whether the mobility of the two antigen-binding domains is reduced can also be evaluated using techniques similar to those described above. For a method for analyzing the three-dimensional structure of IgG molecules using electron tomography, see Zhang et al., Sci. Rep. 5:9803 (2015), among others. Electron tomography can display the frequency of possible structures of the molecule being measured in a histogram, allowing structural changes such as reduced domain mobility to be evaluated using a distribution. For example, when the relationship between the possible values ​​of structural parameters such as the distance and angle between two domains and their frequency of occurrence is represented in a histogram, a decrease in the distribution range can be determined to indicate a decrease in the mobility of the two domains. The activity exhibited by the interaction between two antigen molecules can be evaluated using an appropriate activity measurement system selected from known systems depending on the type of antigen molecule in question. The effect on protease cleavage can be evaluated using methods known to those skilled in the art or the methods described in the Examples below.

[0197] D. Pharmaceutical Formulations (Pharmaceutical Compositions) Pharmaceutical formulations of the antigen-binding molecules described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antigen-binding molecules having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of their use (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0198] Exemplary lyophilized antigen-binding molecule formulations are described in U.S. Patent No. 6,267,958. Aqueous antigen-binding molecule formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0199] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Such active ingredients are present in suitable combinations and in amounts that are effective for the purpose intended.

[0200] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0201] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antigen-binding molecules, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0202] Preparations to be used for in vivo administration are generally sterile, and sterility is readily accomplished, for example, by filtration through sterile filtration membranes. [Example]

[0203] The following are examples of antigen-binding molecules and methods of the present disclosure. In light of the general description above, it will be understood that various other embodiments may be practiced.

[0204] [Example 1] Concept of Fab cross-linked antibody Agonist antibodies have superior properties compared to natural ligands and their fusion proteins in terms of stability, pharmacokinetics, manufacturing methods, etc., and their development as pharmaceuticals is progressing. However, it is generally difficult to obtain agonist antibodies with stronger activity than simple binding antibodies or neutralizing antibodies, and methods to solve this problem are needed. The properties required for an agonist antibody are thought to depend on the type of ligand. It has been reported that the activation of agonist antibodies against the TNF receptor superfamily, represented by Death receptor (DR), OX40, 4-1BB, and CD40, is dependent on the multimerization of the antibody and ligand. Techniques to enhance this effect include the use of natural ligands, cross-linking with anti-Fc antibodies, cross-linking via FcγRs, multimerization of antibody binding domains, and multimerization via antibody Fc. Furthermore, regardless of multimerization, adjusting the distance between antigen-binding sites using antibody Fab structures or scFvs can also enhance agonist activity depending on the type of antigen. Another approach has been reported in which bispecific antibodies, which can bind to different epitopes within the same antigen, are used as agonist antibodies against cytokine receptors. Furthermore, a method has been reported in which two types of Fab are cross-linked using chemical conjugation to improve agonist activity. In addition to the above, there is a need for methods to improve the activity of agonist antibodies, but no simple method for achieving this has been reported. Therefore, the inventors developed a method for cross-linking Fabs with minimal mutation introduction, demonstrated that agonist activity was actually enhanced, and completed this invention. An example embodiment is shown in Figure 1.

[0205] [Example 2] Construction of expression vector for modified antibodies and expression and purification of modified antibodies The antibody gene inserted into an animal cell expression vector was subjected to amino acid residue sequence substitution using PCR or an Infusion Advantage PCR cloning kit (TAKARA) or other methods known to those skilled in the art to construct a modified antibody expression vector. The nucleotide sequence of the resulting expression vector was determined using methods known to those skilled in the art. The constructed expression vector was transiently introduced into FreeStyle293® or Expi293® cells (Invitrogen) to express the modified antibody in the culture supernatant. The modified antibody was purified from the resulting culture supernatant using rProtein A Sepharose® Fast Flow (GE Healthcare) using methods known to those skilled in the art. The absorbance at 280 nm was measured using a spectrophotometer, and the antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423). The amount of aggregates of the modified antibodies was measured using an HPLC system, Agilent 1260 Infinity (registered trademark) (Agilent Technologies), and a gel filtration chromatography column, G3000SW. XL (TOSOH) was used for analysis by a method known to those skilled in the art. The purified antibody concentration was 0.1 mg / mL, and 10 μL was injected. The antibodies prepared by this method (anti-CD3ε antibody, anti-CD28 antibody, and anti-CD3ε x anti-CD28 bispecific antibody) are shown in Table 1.

[0206] [Table 1] HH: Position 191 (EU numbering) in both H chain constant regions was changed to Cys LL: EU numbering position 126 in both L chain constant regions is changed to Cys HL, LH: Position 191 (EU numbering) in one H chain constant region is changed to Cys, and One L chain constant region had position 126 (EU numbering) changed to Cys

[0207] [Example 3] Preparation of bispecific antibodies The purified antibody was dialyzed against TBS (WAKO) buffer to a concentration of 1 mg / mL. 250 mM 2-MEA (SIGMA) was also prepared as a 10x reaction buffer. Equal amounts of the two homodimeric antibodies prepared in Example 2 were mixed, and 1 / 10 the amount of 10x reaction buffer was added and mixed, followed by incubation at 37°C for 90 minutes. After the reaction, the mixture was dialyzed against TBS to obtain a solution of a bispecific antibody in which the two antibodies were heterodimerized. The antibody concentration was measured as described above and used in subsequent experiments.

[0208] [Example 4] Evaluation of agonist activity Example 4-1 Preparation of Jurkat cell solution Jurkat cells (TCR / CD3 Effector Cells (NFAT), Promega) were collected from flasks and washed with Assay Buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% MEM Non-Essential Amino Acids (Invitrogen), 1 mM Sodium Pyrubate (Invitrogen)). The cells were then diluted to 3 × 10 cells in Assay Buffer. 6 The cells were suspended at 1000 cells / mL. This cell suspension was used as a Jurkat cell solution in subsequent experiments.

[0209] Example 4-2 Preparation of luminescence reagent solution 100 mL of Bio-Glo Luciferase Assay Buffer (Promega) was added to a bottle of Bio-Glo Luciferase Assay Substrate (Promega) and mixed by inversion. The bottle was protected from light and frozen at -20°C. The resulting luminescent reagent solution was used in subsequent experiments.

[0210] Example 4-3 T cell activation assay T cell activation by agonist signals was evaluated by the fold change in luciferase luminescence. The Jurkat cells were transfected with a luciferase reporter gene containing an NFAT response element, and upon stimulation with anti-TCR / CD3 antibodies, the NFAT pathway was activated via intracellular signals, inducing luciferase expression. 10 μL of the Jurkat cell solution prepared above was placed in each well of a 384-well flat-bottom white plate (3 x 10 4 Cells / well) were added. Next, 20 μL of antibody solution prepared at various concentrations (150, 15, 1.5, 0.15, 0.015, 0.0015, 0.00015, 0.000015 nM) was added to each well. The plate was left to stand in a 37°C, 5% carbon dioxide incubator for 24 hours. After this, 30 μL of the luminescence reagent solution was thawed and added to each well, and the plate was left to stand at room temperature for 10 minutes. Luciferase luminescence in each well of the plate was measured using a luminometer. As a result, as shown in Figures 2 and 3, modified molecules in which the Fab-Fab pairs of anti-CD3ε antibodies were linked by an additional disulfide bond exhibited altered CD3-mediated signaling compared to the native molecule (molecule before modification). Furthermore, as shown in Figures 4 and 5, modified molecules in which the Fab-Fab pairs of bispecific antibodies consisting of anti-CD3ε antibodies and anti-CD28 antibodies were linked by an additional disulfide bond also exhibited greater alterations in CD3- and / or CD28-mediated signaling compared to the native molecule. From the above results, it was considered that the agonist activity of antigen-binding molecules such as antibodies can be enhanced or attenuated by introducing the modifications of the present invention.

[0211] [Example 5] Evaluation of antibodies with cysteine ​​substitutions at various positions in the heavy chain Example 5-1 Evaluation of antibodies with cysteine ​​substitutions at various positions in the heavy chain We investigated the substitution of any amino acid residue structurally exposed on the surface of the variable and constant regions of the heavy chain of MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, with cysteine. The amino acid residues in the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) were substituted with cysteine ​​to prepare the variants of the MRA heavy chain variable region shown in Table 2. These variants of the MRA heavy chain variable region were each linked to the MRA heavy chain constant region (G1T4, SEQ ID NO: 18) to prepare the MRA heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Additionally, amino acid residues in the MRA heavy chain constant region (G1T4, SEQ ID NO: 18) were substituted with cysteine ​​to prepare the variants of the MRA heavy chain constant region shown in Table 3. These variants of the MRA heavy chain constant region were each linked to the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) to prepare the MRA heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. The MRA heavy chain variants and MRA light chains prepared above were combined to produce the MRA variants shown in Table 4. These variants were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using Protein A by methods known to those skilled in the art.

[0212] [Table 2] TIFF0007811967000004.tif146170

[0213] [Table 3] TIFF0007811967000006.tif234170TIFF0007811967000007.tif98170

[0214] [Table 4] TIFF0007811967000009.tif234170TIFF0007811967000010.tif234170TIFF0007811967000011.tif214170

[0215] Example 5-2 Evaluation of protease-mediated Fab formation of antibodies with cysteine ​​substitutions at various positions in the heavy chain We used a protease that cleaves the hinge region of the antibody heavy chain to fragment Fab fragments to determine whether the modified MRA prepared in Example 5-1 acquires protease resistance and inhibits fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease. The MRA samples were incubated at 35°C for 2 hours with 2 ng / μL of protease and 100 μg / mL of antibody in 80% 25 mM Tris-HCl pH 8.0, 20% PBS, or at 35°C for 1 hour with 2 ng / μL of protease and 20 μg / mL of antibody in 80% 25 mM Tris-HCl pH 8.0, 20% PBS, and then subjected to non-reducing capillary electrophoresis. Capillary electrophoresis was performed using Wes (Protein Simple) and an anti-kappa chain HRP-labeled antibody (abcam; ab46527) was used for detection. The results are shown in Figures 6 to 13. After Lys-C treatment, the heavy chain hinge region of the MRA was cleaved, resulting in the disappearance of the IgG band around 150 kDa and the appearance of a Fab band around 50 kDa. On the other hand, among the MRA variants prepared in Example 5-1, some variants showed a Fab dimer band around 96 kDa after protease treatment, and some variants showed an undigested IgG band around 150 kDa. The area of ​​each band obtained after protease treatment was output using Wes-specific software (Compass for SW; Protein Simple), and the band area ratios of undigested IgG, Fab dimers, etc. were calculated. The calculated band ratios are shown in Table 5.

[0216] [Table 5] TIFF0007811967000013.tif234170TIFF0007811967000014.tif234170TIFF0007811967000015.tif195170

[0217] These results confirmed that the protease resistance of the heavy chain hinge region of the MRA variants shown in Table 6 was improved by the cysteine ​​substitution in the heavy chain variable region or heavy chain constant region. Alternatively, they suggested that Fab dimers were formed by covalent bonds between Fabs.

[0218] [Table 6] TIFF0007811967000017.tif92170

[0219] [Example 6] Evaluation of antibodies with cysteine ​​substitutions at various positions in the light chain Example 6-1 Evaluation of antibodies with cysteine ​​substitutions at various positions in the light chain We investigated the substitution of any amino acid residue structurally exposed on the surface of the variable and constant regions of the light chain of MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, with cysteine. The amino acid residues in the MRA light chain variable region (MRAL, SEQ ID NO: 19) were substituted with cysteine ​​to prepare the MRA light chain variable region variants shown in Table 7. These MRA light chain variable region variants were each linked to the MRA light chain constant region (k0, SEQ ID NO: 20) to prepare MRA light chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Additionally, amino acid residues in the MRA light chain constant region (k0, SEQ ID NO: 20) were substituted with cysteine ​​to prepare the variants of the MRA light chain constant region shown in Table 8. These variants of the MRA light chain constant region were each linked to the MRA light chain variable region (MRAL, SEQ ID NO: 19) to prepare the MRA light chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. The MRA light chain variants prepared above were combined with the MRA heavy chain to produce the MRA variants shown in Table 9. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using Protein A by methods known to those skilled in the art.

[0220] Table 7 TIFF0007811967000019.tif190170

[0221] Table 8 TIFF0007811967000021.tif234170TIFF0007811967000022.tif176170

[0222] Table 9 TIFF0007811967000024.tif234170TIFF0007811967000025.tif234170TIFF0007811967000026.tif234170TIFF0007811967000027.tif104170

[0223] Example 6-2 Evaluation of protease-mediated Fab formation of antibodies with cysteine ​​substitutions at various positions in the light chain We used a protease that cleaves the heavy chain hinge region of an antibody to fragment Fab fragments to determine whether the modified MRA prepared in Example 6-1 acquires protease resistance and inhibits fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease. The MRA samples were incubated at 35°C for 2 hours with 2 ng / μL of protease and 100 μg / mL of antibody in 80% 25 mM Tris-HCl pH 8.0, 20% PBS, or at 35°C for 1 hour with 2 ng / μL of protease and 20 μg / mL of antibody in 80% 25 mM Tris-HCl pH 8.0, 20% PBS, and then subjected to non-reducing capillary electrophoresis. Capillary electrophoresis was performed using Wes (Protein Simple) and an anti-kappa chain HRP-labeled antibody (abcam; ab46527) was used for detection. The results are shown in Figures 14 to 23. After Lys-C treatment, the heavy chain hinge region of the MRA was cleaved, resulting in the disappearance of the IgG band around 150 kDa and the appearance of a Fab band around 50 kDa. On the other hand, among the MRA variants prepared in Example 6-1, some variants showed a Fab dimer band around 96 kDa after protease treatment, and some variants showed an undigested IgG band around 150 kDa. The area of ​​each band obtained after protease treatment was output using Wes-specific software (Compass for SW; Protein Simple), and the band area ratios of undigested IgG, Fab dimers, etc. were calculated. The calculated band ratios are shown in Table 10.

[0224] [Table 10] TIFF0007811967000029.tif234170TIFF0007811967000030.tif234170TIFF0007811967000031.tif234170TIFF0007811967000032.tif91170

[0225] These results confirmed that the protease resistance of the heavy chain hinge region was improved by the cysteine ​​substitution in the light chain variable region or light chain constant region in the MRA variants shown in Table 11. Alternatively, they suggested that Fab dimers were formed by covalent bonds between Fabs.

[0226] [Table 11]

[0227] [Example 7] Verification of the evaluation method for cysteine-substituted antibodies Example 7-1: Preparation of an antibody with cysteine ​​substitution in the light chain The 126th amino acid residue (Kabat numbering) in the light chain constant region (k0, SEQ ID NO: 20) of MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, was substituted with cysteine ​​to generate a modified MRA light chain constant region, k0.K126C (SEQ ID NO: 231). This modified MRA light chain constant region was linked to the MRA light chain variable region (MRAL, SEQ ID NO: 19) to generate a modified MRA light chain, and an expression vector encoding the corresponding gene was prepared by a method known to those skilled in the art. The MRA light chain variant prepared above was combined with the MRA heavy chain to produce the MRA variant MRAL-k0.K126C (heavy chain: MRAH-G1T4 (sequence number: 15), light chain variable region: MRAL (sequence number: 19), light chain constant region: k0.K126C (sequence number: 231)). This was transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0228] Example 7-2 Evaluation of capillary electrophoresis using proteases of antibodies with cysteine ​​substitutions in the light chain We used a protease that cleaves the hinge region of antibody heavy chains to fragment Fab fragments to determine whether the modified MRA light chains prepared in Example 7-1 acquire protease resistance and inhibit fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease. The samples were incubated at 35°C for 2 hours in 80% 25 mM Tris-HCl (pH 8.0), 20% PBS, with 0.1, 0.4, 1.6, or 6.4 ng / μL of protease and 100 μg / mL of antibody. The samples were then subjected to non-reducing capillary electrophoresis. Capillary electrophoresis was performed using Wes (Protein Simple), and detection was performed using an anti-kappa chain HRP-labeled antibody (abcam; ab46527) or an anti-human Fc HRP-labeled antibody (Protein Simple; 043-491). The results are shown in Figure 24. For MRA after Lys-C treatment, when detected with the anti-kappa chain antibody, the band around 150 kDa disappeared, and a new band appeared around 50 kDa. At low Lys-C concentrations, a faint band also appeared at 113 kDa. When detected with the anti-human Fc antibody, the band around 150 kDa disappeared, and a new band appeared around 61 kDa. At low Lys-C concentrations, a faint band also appeared at 113 kDa. On the other hand, for the modified MRA prepared in Example 7-1, the band around 150 kDa was barely lost, and a new band appeared around 96 kDa. In the detection of anti-human Fc antibody, the band around 150 kDa hardly disappeared, but a new band around 61 kDa appeared, and a faint band at 113 kDa also appeared at low Lys-C concentrations. From these results, as shown in Figure 25, it was concluded that the band around 150 kDa corresponds to IgG, the band around 113 kDa corresponds to a one-arm form in which the heavy chain hinge is cleaved once, the band around 96 kDa corresponds to Fab dimer, the band around 61 kDa corresponds to Fc, and the band around 50 kDa corresponds to Fab.

[0229] [Example 8] Evaluation of antibodies with cysteine ​​substitutions introduced at various positions in IgG1 Example 8-1: Preparation of antibodies with cysteine ​​substitutions at various positions in IgG1 We investigated the substitution of any amino acid residue structurally exposed on the surface of the heavy and light chains of MRA-IgG1 (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, with cysteine. The amino acid residues in the MRA-IgG1 heavy chain variable region (MRAH, SEQ ID NO: 17) were substituted with cysteine ​​to prepare the modified MRA-IgG1 heavy chain variable region shown in Table 12. These modified MRA-IgG1 heavy chain variable region variants were each linked to the MRA-IgG1 heavy chain constant region (G1T4, SEQ ID NO: 18) to prepare the modified MRA-IgG1 heavy chain, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, the amino acid residues in the MRA-IgG1 heavy chain constant region (G1T4, SEQ ID NO: 18) were substituted with cysteine ​​to prepare the modified MRA-IgG1 heavy chain constant region shown in Table 13. These modified MRA-IgG1 heavy chain constant region variants were each linked to the MRA-IgG1 heavy chain variable region (MRAH, SEQ ID NO: 17) to prepare the modified MRA-IgG1 heavy chain, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0230] [Table 12] TIFF0007811967000035.tif72170

[0231] [Table 13] TIFF0007811967000037.tif229170

[0232] Similarly, amino acid residues in the MRA-IgG1 light chain variable region (MRAL, SEQ ID NO: 19) were substituted with cysteine ​​to prepare the modified MRA-IgG1 light chain variable region shown in Table 14. These modified MRA-IgG1 light chain variable region variants were each linked to the MRA-IgG1 light chain constant region (k0, SEQ ID NO: 20) to prepare MRA-IgG1 light chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, amino acid residues in the MRA-IgG1 light chain constant region (k0, SEQ ID NO: 20) were substituted with cysteine ​​to prepare the modified MRA-IgG1 light chain constant region shown in Table 15. These modified MRA-IgG1 heavy chain constant region variants were each linked to the MRA-IgG1 light chain variable region (MRAL, SEQ ID NO: 19) to prepare the MRA-IgG1 light chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0233] [Table 14] TIFF0007811967000039.tif110170

[0234] [Table 15] TIFF0007811967000041.tif229170TIFF0007811967000042.tif66170

[0235] The modified MRA-IgG1 heavy chain and MRA-IgG1 light chain, or the modified MRA-IgG1 heavy chain and MRA-IgG1 light chain, prepared above were combined to produce the modified MRA-IgG1 heavy chain and MRA-IgG1 light chain shown in Tables 16 and 17. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0236] [Table 16] TIFF0007811967000044.tif229170TIFF0007811967000045.tif229170TIFF0007811967000046.tif66170

[0237] [Table 17] TIFF0007811967000048.tif229170TIFF0007811967000049.tif229170TIFF0007811967000050.tif169170

[0238] Example 8-2 Evaluation of polyacrylamide gel mobility of antibodies with cysteine ​​substitutions introduced at various positions in IgG1 Non-reducing SDS-PAGE was used to verify whether the MRA-IgG1 variants prepared in Example 8-1 exhibited a different electrophoretic mobility from MRA-IgG1. Samples for electrophoresis were prepared using Sample Buffer Solution (2ME-) (x4) (Wako; 198-13282) at a sample concentration of 50 μg / mL at 70°C for 10 minutes, followed by non-reducing SDS-PAGE. Non-reducing SDS-PAGE was performed at 125 V for 90 minutes using a 4% SDS-PAGE mini 15well 1.0mm 15well (TEFCO; Cat#01-052-6). The gel was then stained with CBB staining solution, and gel images were captured using a ChemiDocTouchMP (BIORAD), and bands were quantified using Image Lab (BIORAD). Based on the acquired gel images, each MRA-IgG1 variant was classified into seven groups according to its band pattern: Single (one band in the same molecular weight range as MRA-IgG1), Double (two bands in the same molecular weight range as MRA-IgG1), Triple (three bands in the same molecular weight range as MRA-IgG1), Several (four or more bands in the same molecular weight range as MRA-IgG1), LMW (a band in a lower molecular weight range than MRA-IgG1), HMW (a band in a higher molecular weight range than MRA-IgG1), and Faint (the bands were faint and indistinguishable). Furthermore, for MRA-IgG1 variants classified as Double, one of the two bands exhibited the same electrophoretic mobility as MRA-IgG1, while the other band exhibited a mobility slightly faster or slightly slower than MRA-IgG1. Therefore, the proportion of bands exhibiting a different mobility from MRA-IgG1 (proportion of new bands (%)) was also calculated for MRA-IgG1 variants classified as Double. The grouping of band patterns and the calculated band ratios for the MRA-IgG1 heavy chain variants and MRA-IgG1 light chain variants are shown in Tables 18 and 19, respectively. From Tables 18 and 19, variants classified into the double and triple groups are shown in Table 20. In these variants, the cysteine ​​substitution caused structural changes such as cross-linking between Fabs, which is highly likely to have resulted in changes in electrophoretic mobility. Although there is no data for MRAL.K107C-IgG1 in Table 19, the cysteine ​​substitution position in this variant, position 107 (Kabat numbering), is the position of a residue structurally exposed to the surface in the hinge region. Therefore, it is highly likely that the cysteine ​​substitution also caused structural changes such as cross-linking between Fabs in this variant, which is highly likely to have resulted in changes in electrophoretic mobility.

[0239] [Table 18] TIFF0007811967000052.tif229170TIFF0007811967000053.tif229170TIFF0007811967000054.tif66170

[0240] [Table 19] TIFF0007811967000056.tif229170TIFF0007811967000057.tif229170TIFF0007811967000058.tif164170

[0241] [Table 20] TIFF0007811967000060.tif55170

[0242] [Example 9] Evaluation of antibodies with cysteine ​​substitutions introduced at various positions in IgG4 Example 9-1: Preparation of antibodies with cysteine ​​substitutions introduced at various positions in IgG4 We investigated the substitution of any amino acid residue structurally exposed on the surface of the heavy and light chains of MRA-IgG4 (heavy chain: MRAH-G4T1 (SEQ ID NO: 310), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, with cysteine. The amino acid residues in the MRA-IgG4 heavy chain variable region (MRAH, SEQ ID NO: 17) were substituted with cysteine ​​to prepare the modified MRA-IgG4 heavy chain variable region shown in Table 21. These modified MRA-IgG4 heavy chain variable region variants were each linked to the MRA-IgG4 heavy chain constant region (G4T1, SEQ ID NO: 311) to prepare MRA-IgG4 heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, the amino acid residues in the MRA-IgG4 heavy chain constant region (G4T1, SEQ ID NO: 311) were substituted with cysteine ​​to prepare the modified MRA-IgG4 heavy chain constant region shown in Table 22. These modified MRA-IgG4 heavy chain constant region variants were each linked to the MRA-IgG4 heavy chain variable region (MRAH, SEQ ID NO: 17) to prepare the MRA-IgG4 heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0243] [Table 21] TIFF0007811967000062.tif77170

[0244] [Table 22] TIFF0007811967000064.tif224170

[0245] The modified MRA-IgG4 heavy chain and MRA-IgG4 light chain prepared above, or the MRA-IgG4 heavy chain and the modified MRA-IgG4 light chain prepared in Example 8-1, were combined to produce the modified MRA-IgG4 heavy chain and MRA-IgG4 light chain shown in Table 23 and Table 24, respectively. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0246] [Table 23] TIFF0007811967000066.tif229170TIFF0007811967000067.tif229170TIFF0007811967000068.tif55170

[0247] [Table 24] TIFF0007811967000070.tif229170TIFF0007811967000071.tif229170TIFF0007811967000072.tif169170

[0248] Example 9-2 Evaluation of polyacrylamide gel mobility of antibodies with cysteine ​​substitutions introduced at various positions in IgG4 As in Example 8-2, non-reducing SDS-PAGE was performed using the modified MRA-IgG4 prepared in Example 9-1, and the gel image was captured and the bands were quantified. Based on the acquired gel images, each MRA-IgG4 variant was classified into seven groups according to its band pattern: Single (one band in the same molecular weight range as MRA-IgG4), Double (two bands in the same molecular weight range as MRA-IgG4), Triple (three bands in the same molecular weight range as MRA-IgG4), Several (four or more bands in the same molecular weight range as MRA-IgG4), LMW (a band in a lower molecular weight range than MRA-IgG4), HMW (a band in a higher molecular weight range than MRA-IgG4), and Faint (the bands were vague and indistinguishable). Furthermore, for MRA-IgG4 variants classified as Double, one of the two bands exhibited the same electrophoretic mobility as MRA-IgG4, while the other band exhibited a mobility slightly faster or slightly slower than MRA-IgG4. Therefore, the proportion of bands exhibiting a different mobility from MRA-IgG4 (proportion of new bands (%)) was also calculated for MRA-IgG4 variants classified as Double. The grouping of band patterns and the calculated band ratios for the MRA-IgG4 heavy chain variants and MRA-IgG4 light chain variants are shown in Tables 25 and 26, respectively. From Tables 25 and 26, variants classified into the double and triple groups are shown in Table 27. In these variants, the cysteine ​​substitution causes structural changes such as cross-linking between Fabs, which is highly likely to have resulted in changes in electrophoretic mobility. Although there is no data for MRAL.K107C-IgG4 in Table 26, the cysteine ​​substitution position in this variant, position 107 (Kabat numbering), is the position of a residue structurally exposed to the surface in the hinge region. Therefore, it is highly likely that the cysteine ​​substitution also causes structural changes such as cross-linking between Fabs in this variant, which is highly likely to have resulted in changes in electrophoretic mobility.

[0249] [Table 25] TIFF0007811967000074.tif229170TIFF0007811967000075.tif229170TIFF0007811967000076.tif55170

[0250] [Table 26] TIFF0007811967000078.tif229170TIFF0007811967000079.tif229170TIFF0007811967000080.tif164170

[0251] [Table 27] TIFF0007811967000082.tif153170

[0252] [Example 10] Evaluation of antibodies with cysteine ​​substitutions introduced at various positions in IgG2 Example 10-1: Preparation of antibodies with cysteine ​​substitutions at various positions in IgG2 We investigated the substitution of any amino acid residue structurally exposed on the surface of the heavy and light chains of MRA-IgG2 (heavy chain: MRAH-G2d (SEQ ID NO: 312), light chain: MRAL-k0 (SEQ ID NO: 16)), a neutralizing antibody against human IL6R, with cysteine. The amino acid residues in the MRA-IgG2 heavy chain variable region (MRAH, SEQ ID NO: 17) were substituted with cysteine ​​to prepare the modified MRA-IgG2 heavy chain variable region shown in Table 28. These modified MRA-IgG2 heavy chain variable region variants were each linked to the MRA-IgG2 heavy chain constant region (G2d, SEQ ID NO: 313) to prepare MRA-IgG2 heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, the amino acid residues in the MRA-IgG2 heavy chain constant region (G2d, SEQ ID NO: 313) were substituted with cysteine ​​to prepare the modified MRA-IgG2 heavy chain constant region shown in Table 29. These modified MRA-IgG2 heavy chain constant region variants were each linked to the MRA-IgG2 heavy chain variable region (MRAH, SEQ ID NO: 17) to prepare the MRA-IgG2 heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0253] [Table 28] TIFF0007811967000084.tif77170

[0254] [Table 29] TIFF0007811967000086.tif224170

[0255] The modified MRA-IgG2 heavy chain and MRA-IgG2 light chain prepared above, or the MRA-IgG2 heavy chain and the modified MRA-IgG2 light chain prepared in Example 8-1, were combined to produce the modified MRA-IgG2 heavy chain and MRA-IgG2 light chain shown in Table 30 and Table 31, respectively. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0256] [Table 30] TIFF0007811967000088.tif229170TIFF0007811967000089.tif229170TIFF0007811967000090.tif55170

[0257] [Table 31] TIFF0007811967000092.tif229170TIFF0007811967000093.tif229170TIFF0007811967000094.tif169170

[0258] Example 10-2 Evaluation of polyacrylamide gel mobility of antibodies with cysteine ​​substitutions introduced at various positions in IgG2 As in Example 8-2, non-reducing SDS-PAGE was performed using the modified MRA-IgG2 prepared in Example 10-1, and gel images were captured and bands were analyzed. Based on the acquired gel images, the band patterns of each MRA-IgG2 variant were classified into seven groups: Single (one band in the molecular weight region around 140 kDa), Double (two bands in the molecular weight region around 140 kDa), Triple (three bands in the molecular weight region around 140 kDa), Several (four or more bands in the molecular weight region around 140 kDa), LMW (bands in the molecular weight region lower than around 140 kDa), HMW (bands in the molecular weight region higher than around 140 kDa), and Faint (bands are vague and indistinguishable). The grouping results for the band patterns of the MRA-IgG2 heavy chain variants and MRA-IgG2 light chain variants are shown in Tables 32 and 33, respectively. The variants classified into the Double and Triple groups from Tables 32 and 33 are shown in Table 34. Although there is no data for MRAL.K107C-IgG2 in Table 33, the cysteine ​​substitution position in this variant, 107 (Kabat numbering), is the position of a residue that is structurally exposed to the surface in the hinge region. Therefore, this variant may also be double-mutated.

[0259] [Table 32] TIFF0007811967000096.tif229170TIFF0007811967000097.tif229170TIFF0007811967000098.tif50170

[0260] [Table 33] TIFF0007811967000100.tif229170TIFF0007811967000101.tif229170TIFF0007811967000102.tif164170

[0261] [Table 34] TIFF0007811967000104.tif229170TIFF0007811967000105.tif229170TIFF0007811967000106.tif229170TIFF0007811967000107.tif224170

[0262] [Example 11] Evaluation of antibodies with cysteine ​​substitutions introduced at various positions in the Lambda chain Example 11-1: Preparation of antibodies with cysteine ​​substitutions at various positions in the Lambda chain We investigated the substitution of any amino acid residue structurally exposed on the surface of the light chain (Lambda chain) of G7-IgG1 (heavy chain: G7H-G1T4 (SEQ ID NO: 314), light chain: G7L-LT0 (SEQ ID NO: 316)), a neutralizing antibody against human CXCL10, with cysteine. The amino acid residues in the G7-IgG1 light chain variable region (G7L, SEQ ID NO: 317) were substituted with cysteine ​​to prepare the modified G7-IgG1 light chain variable region shown in Table 35. These modified G7-IgG1 light chain variable region variants were each linked to the G7-IgG1 light chain constant region (LT0, SEQ ID NO: 318) to prepare modified G7-IgG1 light chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, the amino acid residues in the G7-IgG1 light chain constant region (LT0, SEQ ID NO: 318) were substituted with cysteine ​​to prepare the modified G7-IgG1 light chain constant region shown in Table 36. These modified G7-IgG1 heavy chain constant region variants were each linked to the G7-IgG1 light chain variable region (G7L, SEQ ID NO: 317) to prepare modified G7-IgG1 light chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0263] [Table 35] TIFF0007811967000109.tif115170

[0264] [Table 36] TIFF0007811967000111.tif229170TIFF0007811967000112.tif55170

[0265] The G7-IgG1 light chain variants shown in Table 37 were combined with the G7-IgG1 heavy chain prepared above, and were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0266] [Table 37] TIFF0007811967000114.tif229170TIFF0007811967000115.tif229170TIFF0007811967000116.tif153170

[0267] Example 11-2 Evaluation of polyacrylamide gel mobility of antibodies with cysteine ​​substitutions introduced at various positions in the Lambda chain As in Example 8-2, non-reducing SDS-PAGE was performed using the modified G7-IgG1 prepared in Example 11-1, and the gel image was captured and the bands were quantified. Based on the acquired gel images, G7-IgG1 variants were classified into seven groups according to their band patterns: Single (one band in the same molecular weight range as G7-IgG1), Double (two bands in the same molecular weight range as G7-IgG1), Triple (three bands in the same molecular weight range as G7-IgG1), Several (four or more bands in the same molecular weight range as G7-IgG1), LMW (a band in a lower molecular weight range than G7-IgG1), HMW (a band in a higher molecular weight range than G7-IgG1), and Faint (the bands were faint and indistinguishable). Furthermore, for G7-IgG1 variants classified as Double, one of the two bands exhibited the same electrophoretic mobility as G7-IgG1, while the other band exhibited a mobility slightly faster or slightly slower than G7-IgG1. Therefore, the proportion of bands exhibiting a different mobility from G7-IgG1 (proportion of new bands (%)) was also calculated for G7-IgG1 variants classified as Double. Table 38 shows the grouping of band patterns for G7-IgG1 light chain variants and the calculated band ratios. From Table 38, variants classified into the double and triple groups are shown in Table 39. In these variants, the cysteine ​​substitution caused structural changes such as Fab cross-linking, which is likely to have resulted in changes in electrophoretic mobility. In this Example, a variant in which the amino acid residue at position 107a (Kabat numbering) was substituted with cysteine ​​was not evaluated. However, position 107a (Kabat numbering) is the position of a residue structurally exposed to the surface in the hinge region. Therefore, it is likely that the cysteine ​​substitution also caused structural changes such as Fab cross-linking in this variant, which is likely to have resulted in changes in electrophoretic mobility.

[0268] [Table 38] TIFF0007811967000118.tif229170TIFF0007811967000119.tif229170TIFF0007811967000120.tif148170

[0269] [Table 39]

[0270] [Example 12] Evaluation of antibodies with cysteine ​​substitutions introduced at various positions in VHH Example 12-1: Preparation of antibodies with cysteine ​​substitutions introduced at various positions in VHH IL6R90-Fc (IL6R90-G1T3dCH1dC, SEQ ID NO: 321) was prepared by fusing IL6R90 (SEQ ID NO: 319), a neutralizing VHH against human IL6R, to the Fc region of human IgG1 (G1T3dCH1dC, SEQ ID NO: 320). An investigation was then carried out into substituting any amino acid residue structurally exposed on the surface of the IL6R90 region with cysteine. Amino acid residues in the IL6R90 region were substituted with cysteine, and expression vectors encoding the genes of the modified VHH domains of IL6R90-Fc shown in Table 40 were prepared by methods known to those skilled in the art. These modified IL6R90-Fc VHH domains were each linked to the Fc domain of human IgG1 (G1T3dCH1dC, SEQ ID NO: 320) to prepare modified IL6R90-Fc, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0271] [Table 40] TIFF0007811967000123.tif251170 The IL6R90-Fc variants shown in Table 41 prepared above were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art.

[0272] [Table 41] TIFF0007811967000125.tif251170

[0273] Example 12-2 Evaluation of polyacrylamide gel mobility of antibodies with cysteine ​​substitutions introduced at various positions in VHH Non-reducing SDS-PAGE was used to verify whether the IL6R90-Fc variant prepared in Example 12-1 exhibited a different electrophoretic mobility from IL6R90-Fc. Samples were prepared using Sample Buffer Solution (2ME-) (x4) (Wako; 198-13282) at a sample concentration of 50 μg / mL at 70°C for 10 minutes, followed by non-reducing SDS-PAGE. Non-reducing SDS-PAGE was performed at 200 V for 2.5 hours using a Mini-PROTEAN TGX Precast Gel 4-20% 15well (BIORAD; 456-1096). The gel was then stained with CBB staining solution, and gel images were captured using a ChemiDocTouchMP (BIORAD), and bands were quantified using Image Lab (BIORAD). Based on the acquired gel images, IL6R90-Fc variants were classified into seven groups according to their band patterns: Single (one band in the same molecular weight range as IL6R90-Fc), Double (two bands in the same molecular weight range as IL6R90-Fc), Triple (three bands in the same molecular weight range as IL6R90-Fc), Several (four or more bands in the same molecular weight range as IL6R90-Fc), LMW (a band in a lower molecular weight range than IL6R90-Fc), HMW (a band in a higher molecular weight range than IL6R90-Fc), and Faint (the bands were vague and indistinguishable). Furthermore, for IL6R90-Fc variants classified as Double, one of the two bands showed the same electrophoretic mobility as IL6R90-Fc, and the other band showed a slightly faster or slower electrophoretic mobility. Therefore, for IL6R90-Fc variants classified as Double, the proportion of bands that showed electrophoretic mobility different from that of IL6R90-Fc (proportion of new bands (%)) was also calculated. Table 42 shows the grouping of band patterns for IL6R90-Fc variants and the calculated band proportions. From Table 42, variants classified into the Double and Triple groups are shown in Table 43. It is highly likely that these variants have undergone structural changes, such as cross-linking between VHHs, due to cysteine ​​substitution, resulting in changes in electrophoretic mobility.

[0274] [Table 42] TIFF0007811967000127.tif251170

[0275] [Table 43]

[0276] [Example 13] Evaluation of CD3 agonist activity of antibodies with cysteine ​​substitutions introduced into the Fab Example 13-1: Preparation of antibodies with cysteine ​​substitutions in the constant region We investigated the substitution of any amino acid residue structurally exposed on the surface of OKT3 (heavy chain: OKT3VH0000-G1T4 (sequence number: 1007), light chain: OKT3VL0000-KT0 (sequence number: 1008)), an agonistic antibody against human CD3, with cysteine. The amino acid residues in the OKT3 heavy chain constant region (G1T4, SEQ ID NO: 1009) were substituted with cysteine ​​to prepare the modified OKT3 heavy chain constant regions shown in Table 44. These modified OKT3 heavy chain constant regions were each linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to prepare the modified OKT3 heavy chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0277] [Table 44]

[0278] Similarly, amino acid residues in the OKT3 light chain constant region (KT0, SEQ ID NO: 1011) were substituted with cysteine ​​to prepare the modified OKT3 light chain constant regions shown in Table 45. These modified OKT3 light chain constant regions were each linked to the OKT3 light chain variable region (OKT3VL0000, SEQ ID NO: 1012) to prepare the modified OKT3 light chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0279] [Table 45]

[0280] The OKT3 heavy chain variants and OKT3 light chain variants prepared above were combined with the OKT3 light chain and OKT3 heavy chain, respectively, to produce the OKT3 variants shown in Table 46. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art. As a negative control, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was also prepared in the same manner.

[0281] [Table 46]

[0282] Example 13-2 Preparation of Jurkat cell solution Jurkat cells (TCR / CD3 Effector Cells (NFAT), Promega) were collected from flasks and washed with Assay Buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% MEM Non-Essential Amino Acids (Invitrogen), 1 mM Sodium Pyrubate (Invitrogen)). The cells were then diluted to 3 × 10 cells in Assay Buffer. 6 The cells were suspended at a concentration of 1000 cells / mL. This cell suspension was used as a Jurkat cell solution in subsequent experiments.

[0283] Example 13-3 Preparation of luminescence reagent solution 100 mL of Bio-Glo Luciferase Assay Buffer (Promega) was added to a bottle of Bio-Glo Luciferase Assay Substrate (Promega) and mixed by inversion. The bottle was protected from light and frozen at -20°C. The resulting luminescent reagent solution was used in subsequent experiments.

[0284] Example 13-4 Evaluation of T cell activation of antibodies with cysteine ​​substitutions introduced into the constant region T cell activation by agonist signals was evaluated by the fold change in luciferase luminescence. The Jurkat cells were transfected with a luciferase reporter gene containing an NFAT response element, and upon stimulation with anti-TCR / CD3 antibodies, the NFAT pathway was activated via intracellular signals, inducing luciferase expression. 10 μL of the Jurkat cell solution prepared above was placed in each well of a 384-well flat-bottom white plate (3 x 10 4 Cells / well) were added. Next, 20 μL of antibody solution prepared at various concentrations (10,000, 1,000, 100, 10, 1, and 0.1 ng / mL) was added to each well. The plate was left standing in a 37°C, 5% carbon dioxide incubator for 24 hours. After this, 30 μL of the luminescence reagent solution was thawed and added to each well, and the plate was left standing at room temperature for 10 minutes. Luciferase luminescence in each well of the plate was measured using a luminometer. The luminescence intensity (fold) was calculated by dividing the luminescence intensity in the antibody-added wells by the luminescence intensity in the antibody-free wells. As a result, as shown in Figure 26, among the OKT3 variants in which cysteine ​​substitutions were introduced into the constant region, several variants significantly increased the T cell activation state compared to OKT3, indicating the existence of several cysteine ​​modifications that can cross-link Fabs and enhance CD3 agonist activity.

[0285] [Example 14] Evaluation of CD3 agonist activity of antibodies with different cysteine ​​substitutions in two Fabs Example 14-1: Preparation of antibodies with heterologous cysteine ​​substitutions in the constant region We investigated the substitution of any amino acid residue structurally exposed on the surface of OKT3 (heavy chain: OKT3VH0000-G1T4 (sequence number: 1007), light chain: OKT3VL0000-KT0 (sequence number: 1008)), an agonistic antibody against human CD3, with cysteine. The amino acid residues in OKT3 heavy chain constant region 1 (G1T4k, SEQ ID NO: 1015) were substituted with cysteine ​​to prepare the OKT3 heavy chain constant region variants shown in Table 47. These OKT3 heavy chain constant region variants were each linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to prepare OKT3 heavy chain variant 1, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Similarly, the amino acid residues in OKT3 heavy chain constant region 2 (G1T4h, SEQ ID NO: 1016) were substituted with cysteine ​​to prepare the OKT3 heavy chain constant region variants shown in Table 48. These OKT3 heavy chain constant region variants were each linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to prepare OKT3 heavy chain variant 2, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. In addition, heavy chain constant regions 1 and 2 here have Knobs-into-Holes (KiH) modifications introduced into the CH3 region to promote heterodimerization.

[0286] [Table 47]

[0287] [Table 48]

[0288] The OKT3 variants shown in Table 49 were combined with the OKT3 heavy chain variants 1 and 2 prepared above and the OKT3 light chain, and were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art. As a negative control, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was also prepared in the same manner.

[0289] [Table 49]

[0290] Example 14-2 Preparation of Jurkat cell solution A Jurkat cell solution was prepared in the same manner as in Example 13-2.

[0291] Example 14-3 Preparation of luminescence reagent solution A luminescence reagent solution was prepared in the same manner as in Example 13-3.

[0292] Example 14-4 Evaluation of T cell activation of antibodies with heterologous cysteine ​​substitutions introduced into the constant region T cell activation was evaluated in the same manner as in Example 13-4. As a result, as shown in Figure 27, the modified OKT3 in which different cysteine ​​substitutions were introduced between the two constant regions of the antibody significantly increased the T cell activation state compared to OKT3. This indicates that even when the Fabs have different cysteine ​​substitutions, they can cross-link the Fabs and enhance CD3 agonist activity.

[0293] [Example 15] Evaluation of CD3 agonist activity of antibodies with charge modifications introduced into the Fab Example 15-1: Preparation of antibodies with charged amino acid substitutions in the constant region We investigated the substitution of any amino acid residues structurally exposed on the surface of the heavy chain of OKT3 (heavy chain: OKT3VH0000-G1T4 (sequence number: 1007), light chain: OKT3VL0000-KT0 (sequence number: 1008)), an agonistic antibody against human CD3, with charged amino acids. The amino acid residues in OKT3 heavy chain constant region 1 (G1T4k, SEQ ID NO: 1015) were substituted with arginine (R) or lysine (K) to prepare the OKT3 heavy chain constant region variants shown in Table 50. These OKT3 heavy chain constant region variants were each linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to prepare OKT3 heavy chain variant 1, and expression vectors encoding the corresponding genes were prepared using methods known to those skilled in the art. Similarly, the amino acid residues in OKT3 heavy chain constant region 2 (G1T4h, SEQ ID NO: 1016) were substituted with aspartic acid (D) or glutamic acid (E) to prepare the OKT3 heavy chain constant region variants shown in Table 51. These OKT3 heavy chain constant region variants were each linked to an OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to produce OKT3 heavy chain variant 2, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Note that Knobs-into-Holes (KiH) modifications were introduced into the CH3 regions of heavy chain constant regions 1 and 2 to promote heterodimerization.

[0294] [Table 50]

[0295] [Table 51]

[0296] The OKT3 variants shown in Table 52 were combined with the OKT3 heavy chain variants 1 and 2 prepared above and the OKT3 light chain, and were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art. As a negative control, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was also prepared in the same manner.

[0297] [Table 52]

[0298] Example 15-2 Preparation of Jurkat cell solution A Jurkat cell solution was prepared in the same manner as in Example 13-2.

[0299] Example 15-3 Preparation of luminescence reagent solution A luminescence reagent solution was prepared in the same manner as in Example 13-3.

[0300] Example 15-4 Evaluation of T cell activation of antibodies with amino acid substitutions other than cysteine ​​introduced into the constant region T cell activation was evaluated in the same manner as in Example 13-4. As a result, as shown in Figure 28, modified OKT3s in which positively charged amino acid substitutions were introduced into one constant region and negatively charged amino acid substitutions into the other constant region significantly increased the T cell activation state compared to OKT3. On the other hand, modified OKT3s in which positively or negatively charged amino acid substitutions were introduced into one constant region and no modifications were introduced into the other constant region showed almost no change in the T cell activation state compared to OKT3. This indicates that not only cysteine ​​substitutions but also charged amino acid substitutions can cross-link Fabs via non-covalent bonds and enhance CD3 agonist activity.

[0301] [Example 16] Evaluation of the CD3 agonist activity of antibodies in which disulfide bonds in the hinge region have been removed and cysteine ​​substitutions have been introduced into the Fab Example 16-1: Preparation of an antibody in which disulfide bonds in the hinge region have been removed and cysteine ​​substitutions have been introduced into the Fab We investigated the removal of disulfide bonds in the hinge region of the heavy chain of OKT3 (heavy chain: OKT3VH0000-G1T4 (sequence number: 1007), light chain: OKT3VL0000-KT0 (sequence number: 1008)), an agonistic antibody against human CD3, and the substitution of amino acid residues structurally exposed to the surface with cysteines. The cysteine ​​in the hinge region of the OKT3 heavy chain constant region (G1T4, SEQ ID NO: 1009) was substituted with serine to prepare the OKT3 heavy chain constant region variants shown in Table 53. The amino acid residue at position 191 (EU numbering) of these OKT3 heavy chain constant region variants was substituted with cysteine ​​to prepare the OKT3 heavy chain constant region variants shown in Table 54. Each of these OKT3 heavy chain constant region variants was linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to prepare OKT3 heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0302] [Table 53]

[0303] [Table 54]

[0304] The OKT3 variants shown in Table 55 were combined with the OKT3 heavy chain variants prepared above and transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art. As a negative control, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was also prepared in the same manner.

[0305] [Table 55]

[0306] Example 16-2 Preparation of Jurkat cell solution A Jurkat cell solution was prepared in the same manner as in Example 13-2.

[0307] Example 16-3 Preparation of luminescence reagent solution A luminescence reagent solution was prepared in the same manner as in Example 13-3.

[0308] Example 16-4 Evaluation of T cell activation of antibodies in which disulfide bonds in the hinge region have been removed and cysteine ​​substitutions have been introduced into the Fab T cell activation was evaluated in the same manner as in Example 13-4. As a result, as shown in Figure 29, the modified OKT3 in which the disulfide bond in the hinge region was simply removed reduced or barely changed the T cell activation state compared to OKT3. On the other hand, the modified OKT3 in which the disulfide bond in the hinge region was removed and cysteine ​​substitutions were introduced into the constant region significantly increased the T cell activation state compared to OKT3. This indicates that even in the absence of disulfide bonds in the hinge region, cysteine ​​substitutions within Fab can cross-link Fabs and enhance CD3 agonist activity.

[0309] [Example 17] Construction of expression vector for modified antibodies and expression and purification of modified antibodies The antibody gene inserted into an animal cell expression vector was subjected to amino acid residue sequence substitution using PCR or an Infusion Advantage PCR cloning kit (TAKARA) or similar methods known to those skilled in the art to construct a modified antibody expression vector. The nucleotide sequence of the resulting expression vector was determined using methods known to those skilled in the art. The constructed expression vector was transiently introduced into FreeStyle293® or Expi293® cells (Invitrogen) to express the modified antibody in the culture supernatant. The modified antibody was purified from the resulting culture supernatant using Protein A or similar methods known to those skilled in the art. The absorbance at 280 nm was measured using a spectrophotometer, and the antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

[0310] [Example 18] Preparation of bispecific antibodies The purified antibody was dialyzed against TBS or PBS buffer to a concentration of 1 mg / mL. 250 mM 2-MEA (SIGMA) was also prepared as a 10x reaction buffer. Equal amounts of the two homodimeric antibodies prepared in Example 17 were mixed, and 1 / 10 the amount of 10x reaction buffer was added and mixed, followed by incubation at 37°C for 90 minutes. After the reaction, the mixture was dialyzed against TBS or PBS to obtain a solution of a bispecific antibody in which the two antibodies were heterodimerized. The antibody concentration was measured as described above and used in subsequent experiments.

[0311] [Example 19] Evaluation of agonist activity Example 19-1 Preparation of Jurkat cell solution Jurkat cells (TCR / CD3 Effector Cells (NFAT), Promega) were collected from flasks and washed with Assay Buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% MEM Non-Essential Amino Acids (Invitrogen), 1 mM Sodium Pyrubate (Invitrogen)). The cells were then diluted to 3 × 10 cells in Assay Buffer. 6 The cells were suspended at 1000 cells / mL. This cell suspension was used as a Jurkat cell solution in subsequent experiments.

[0312] Example 19-2 Preparation of luminescence reagent solution 100 mL of Bio-Glo Luciferase Assay Buffer (Promega) was added to a bottle of Bio-Glo Luciferase Assay Substrate (Promega) and mixed by inversion. The bottle was protected from light and frozen at -20°C. The resulting luminescent reagent solution was used in subsequent experiments.

[0313] Example 19-3 T cell activation assay T cell activation by agonist signals was evaluated by the fold change in luciferase luminescence. The Jurkat cells were transfected with a luciferase reporter gene containing an NFAT response element, and upon stimulation with anti-TCR / CD3 antibodies, the NFAT pathway was activated via intracellular signals, inducing luciferase expression. 10 μL of the Jurkat c...

Claims

1. An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, the antigen-binding domain comprises a hinge region; the first and second antigen-binding domains comprise antibody fragments that bind to specific antigens; the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more bonds, at least one of which is a covalent bond; at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the hinge region; At least one of the bonds connecting the two antigen-binding domains is formed by linking amino acid residues in the CH1 region of the first antigen-binding domain with amino acid residues in the CL region of the second antigen-binding domain; 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); Antigen-binding molecules.

2. The antigen-binding molecule of claim 1, wherein the antibody fragment is any of Fab, Fab', and scFab.

3. The antigen-binding molecule of claim 1 or 2, wherein the antigen-binding domain comprises an Fc region.

4. The antigen-binding molecule of claim 1 , which has an activity of regulating the interaction between two antigen molecules.

5. An antigen-binding molecule according to any one of claims 1 to 4, 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 only in that the control antigen-binding molecule has one fewer bond between the two antigen-binding domains.

6. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. 1. A method for controlling an interaction between two antigen molecules, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the antigen-binding domain comprises a hinge region, and the first and second antigen-binding domains comprise antibody fragments that bind to a specific antigen; (b) adding at least one bond to the antigen-binding molecule that links the two antigen-binding domains to each other, so that the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more bonds, at least one of which is a covalent bond; at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the hinge region; At least one of the bonds connecting the two antigen-binding domains is formed by linking amino acid residues in the CH1 region of the first antigen-binding domain with amino acid residues in the CL region of the second antigen-binding domain; 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); and (c) contacting the antigen-binding molecule prepared in (b) with the two antigen molecules.

8. A method for producing an antigen-binding molecule having an activity of regulating the interaction between two antigen molecules, 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, wherein the antigen-binding domain comprises a hinge region, and wherein the first and second antigen-binding domains comprise antibody fragments that bind to a specific antigen; (b) introducing mutations into nucleic acids encoding the two antigen-binding domains so as to add at least one bond linking the two antigen-binding domains, thereby allowing the first antigen-binding domain and the second antigen-binding domain to be linked to each other via two or more bonds, at least one of which is a covalent bond; at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the hinge region; At least one of the bonds connecting the two antigen-binding domains is formed by linking amino acid residues in the CH1 region of the first antigen-binding domain with amino acid residues in the CL region of the second antigen-binding domain; 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); (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell so that the two polypeptides are expressed; 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.

Images