Antigen-binding molecule containing two interconnected antigen-binding domains

Abstract

The present invention provides an antigen-binding molecule having the activity to control the interaction between two or more antigen molecules, or a method for producing or using such an antigen-binding molecule. [Solution] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domain includes a hinge region, the first and second antigen-binding domains contain an antibody fragment that binds to a specific antigen, the first and second antigen-binding domains are linked to each other via two or more bonds where amino acid residues at specific positions are linked, at least one of which is a covalent bond, at least one of the amino acid residues that serve as the starting point for the binding between the antigen-binding domains is located within the CH1 region and / or CL region of the antibody fragment, and the antigen-binding molecule exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule, and has one fewer bond between the two antigen-binding domains.

Description

[Technical Field]

[0001] This disclosure relates to an antigen-binding molecule comprising a first and 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. Furthermore, this disclosure relates to a method for increasing the resistance of the antigen-binding molecule to protease cleavage. [Background technology]

[0002] Antibodies are proteins that bind specifically to antigens with high affinity. A wide variety of molecules, from small molecules to proteins, are known to act as antigens. Since the development of monoclonal antibody production techniques, antibody modification techniques have advanced, making it easier to obtain antibodies that recognize specific molecules. Furthermore, antibody modification techniques have expanded beyond simply modifying the protein itself to include fields aiming to add new functions through conjugation with small molecules. For example, cysteine-modified antibodies, which contain free cysteine ​​amino acids in their heavy or light chains, are used in medical applications as antibody-drug conjugates (ADCs) (Patent Document 1). On the other hand, antibody modification technology has not only contributed to the development of antibody engineering as a tool for the detection, analysis, and purification of proteins, but has also contributed to the development of protein engineering in general, such as improving the function of proteins other than antibodies, with the antibody molecule itself serving as a model protein.

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

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

[0005] However, natural IgG antibodies may not fully exhibit the expected effects. Therefore, second-generation antibody drugs have been developed by artificially enhancing or adding, or weakening or deleting, the function of natural IgG antibodies, thereby enhancing or adding, weakening or deleting, the function of which is tailored to the intended use of the antibody. Examples of second-generation antibody drugs include antibodies with enhanced or deleting effector function (Non-Patent Literature 3), antibodies that bind to antigens in a pH-dependent manner (Non-Patent Literature 4), and antibodies that bind to two or more antigens with a single molecule (antibodies that bind to two antigens are generally called "bispecific antibodies") (Non-Patent Literature 5).

[0006] Bispecific antibodies are expected to become more effective pharmaceuticals. For example, antibodies with enhanced antitumor activity have been developed by cross-linking cytotoxic T cells and cancer cells, using one antigen as a protein expressed on the cell membrane of T cells and the other antigen as a cancer antigen (Non-Patent Documents 7, 8, and 2). Examples of bispecific antibodies that have been reported include molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules with antigen-binding sites 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 with the loop region of the CH3 region used as a new antigen-binding site (Fcab) (Non-Patent Document 9), and molecules with Fab-Fab sequences in series (Non-Patent Document 10).

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

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

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

Patent document 6

Non-licensed literature

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

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0011] Attempts to enhance or weaken the agonist or effector functions of antibody drugs, as described above, are still in their early stages of development, and further efforts are expected. This invention was made in view of these circumstances, and aims to provide a novel antigen-binding molecule having the activity to control the interaction between two or more antigen molecules, or a method for producing or using such an antigen-binding molecule. This invention is useful for the screening and development of antibody drugs, and is also considered applicable to various other areas of protein engineering. [Means for solving the problem]

[0012] In a non-limiting embodiment, the inventors introduced amino acid mutations into the antigen-binding domains of an antigen-binding molecule (e.g., an antibody) containing two antigen-binding domains (e.g., Fab moieties) that have agonist activity, and created a molecule in which the antigen-binding domains are linked together, finding that the agonist activity was greatly improved. Also in a non-limiting embodiment, the inventors found an antigen-binding molecule that acquired resistance to protease digestion by linking the antigen-binding domains.

[0013] This disclosure is based on such findings and specifically includes the embodiments described below as illustrative examples. [1] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other via one or more binding sites. [2] The antigen-binding molecule according to [1], wherein at least one of the bonds linking the two antigen-binding domains is a covalent bond. [3] The antigen-binding molecule described in [2], wherein a covalent bond is formed by direct crosslinking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. [4] The antigen-binding molecule described in [3], wherein the type of amino acid residue to be crosslinked is cysteine. [5] The antigen-binding molecule described in [4], wherein the covalent bond formed is a disulfide bond. [6] The antigen-binding molecule described in [2], wherein a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain via a crosslinking agent. [7] The antigen-binding molecule described in [6], wherein the crosslinking agent is an amine-reactive crosslinking agent. [8] The antigen-binding molecule described in [7], wherein the type of amino acid residue crosslinked is lysine. [9] The antigen-binding molecule according to [1], wherein at least one of the bonds linking the two antigen-binding domains is a non-covalent bond.

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

[11] The antigen-binding molecule described in

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

[12] The antigen-binding molecule described in

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

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

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

[14] The antigen-binding molecule described in

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

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

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

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

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

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

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

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

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

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

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

[20] The antigen-binding molecule described in

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

[21] An antigen-binding molecule according to

[19] or

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

[22] The antigen-binding molecule described in

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

[23] The antigen-binding molecule described in

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

[24] The antigen-binding molecule described in

[22] or

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

[25] The antigen-binding molecule described in

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

[26] The antigen-binding molecule described in

[24] or

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

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

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

[28] The antigen-binding molecule described in

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

[29] The antigen-binding molecule described in

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

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

[24] to

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

[31] The antigen-binding molecule according to

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

[32] The antigen-binding molecule according to

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

[33] The antigen-binding molecule according to

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

[34] The antigen-binding molecule according to

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

[35] The antigen-binding molecule according to

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

[36] The antigen-binding molecule according to

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

[37] The antigen-binding molecule according to

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

[38] The antigen-binding molecule according to

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

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

[30] to

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

[40] The antigen-binding molecule according to

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

[41] The antigen-binding molecule according to

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

[42] The antigen-binding molecule according to

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

[43] The antigen-binding molecule according to

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

[44] The antigen-binding molecule according to

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

[45] The antigen-binding molecule according to

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

[46] The antigen-binding molecule according to

[22] or

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

[47] The antigen-binding molecule described in

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

[48] ​​The antigen-binding molecule described in

[46] or

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

[49] The antigen-binding molecule described in

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

[50] The antigen-binding molecule described in

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

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

[46] to

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

[52] The antigen-binding molecule according to

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

[53] The antigen-binding molecule according to

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

[54] The antigen-binding molecule according to

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

[55] The antigen-binding molecule according to

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

[56] The antigen-binding molecule according to

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

[57] The antigen-binding molecule according to

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

[58] The antigen-binding molecule according to

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

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

[51] to

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

[60] The antigen-binding molecule according to

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

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

[24] to

[29] or

[46] to

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

[62] The antigen-binding molecule according to

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

[63] The antigen-binding molecule according to

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

[64] The antigen-binding molecule described in

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

[65] The antigen-binding molecule described in

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

[66] The antigen-binding molecule described in

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

[67] The antigen-binding molecule described in

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

[68] The antigen-binding molecule described in

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

[69] The antigen-binding molecule described in

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

[70] The antigen-binding molecule described in

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

[71] The antigen-binding molecule described in

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

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

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

[73] The antigen-binding molecule described in

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

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

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

[75] The antigen-binding molecule described in

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

[76] The antigen-binding molecule described in

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

[77] An antigen-binding molecule as described in

[74] or

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

[78] The antigen-binding molecule described in

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

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

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

[80] The antigen-binding molecule described in

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

[81] The antigen-binding molecule described in

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

[82] The antigen-binding molecule described in

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

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

[80] to

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

[84] The antigen-binding molecule described in

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

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

[79] to

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

[86] The antigen-binding molecule according to

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

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

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

[88] An antigen-binding molecule as described in

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

[89] The antigen-binding molecule according to

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

[90] The antigen-binding molecule described in

[88] or

[89] , wherein the polymerization is hexamerization.

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

[87] to

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

[0014]

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

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

[93] The antigen-binding molecule according to

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

[94] The antigen-binding molecule according to

[92] , wherein the first and second antigen-binding domains bind to different epitopes on the antigen.

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

[91] , wherein the first and second antigen-binding domains bind to antigens of different types from each other.

[96] The antigen-binding molecule described in

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

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

[93] to

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

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

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

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

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

[0015]

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

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

[101] An antigen-binding molecule according to

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

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

[102] The antigen-binding molecule according to

[100] or

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

[103] The antigen-binding molecule according to

[100] or

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

[104] An antigen-binding molecule according to

[100] or

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

[105] The antigen-binding molecule according to

[104] , wherein the cell expressing the first antigen is a cell having cytotoxic activity, and the cell expressing the second antigen is a target cell, and the damage to the target cell by the cell having cytotoxic activity is promoted.

[106] The antigen-binding molecule described in

[105] , wherein the cell having cytotoxic activity is one of T cells, NK cells, monocytes, or macrophages.

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

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

[108] An antigen-binding molecule as described in

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

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

[109] An antigen-binding molecule as described in

[107] or

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

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

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

[111] An antigen-binding molecule according to

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

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

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

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

[113] An antigen-binding molecule as described in

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

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

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

[99] that is resistant to protease cleavage.

[115] An antigen-binding molecule as described in

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

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

[116] The antigen-binding molecule according to

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

[117] The antigen-binding molecule according to

[115] or

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

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

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

[119] An antigen-binding molecule as described in

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

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

[120] An antigen-binding molecule as described in

[118] or

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

[121] An antigen-binding molecule described in any of

[101] to

[106] ,

[108] to

[109] ,

[111] ,

[113] ,

[115] to

[117] , or

[119] to

[120] , wherein the bond with one less location is formed starting from a mutated amino acid residue.

[122] The antigen-binding molecule described in

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

[0016]

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

[122] and a pharmaceutically acceptable carrier.

[0017]

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

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

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

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

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

[0018]

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

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

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

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

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

[0019]

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

[129] to

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

[135] The method according to

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

[0020] [Figure 1] Figure 1 shows an example of a modified antibody in which Fabs are crosslinked, as described in Example 1. Here, the structural differences between the wild-type antibody (WT), the modified antibody in which the CH1 regions of the antibody H chain are crosslinked (HH type), the modified antibody in which the CL regions of the antibody L chain are crosslinked (LL type), and the modified antibodies in which the CH1 region of the antibody H chain and the CL region of the antibody L chain are crosslinked (HL type, LH type) are schematically shown. [Figure 2] Figure 2 shows the results of measuring the CD3-mediated agonist activity of a native anti-CD3ε antibody molecule (CD3-G4s) and modified antibody molecules (CD3-G4sLL, CD3-G4sHH) in which the Fab-Fab bonds of the native anti-CD3ε antibody molecule (CD3-G4s) were linked by additional disulfide bonds, as described in Example 4-3. [Figure 3] Figure 3 shows the results of measuring the CD3-mediated agonist activity of a native anti-CD3ε antibody molecule (OKT3-G1s) and modified antibody molecules (OKT3-G1sLL, OKT3-G1sHH) in which the Fab-Fab bonds of the native anti-CD3ε antibody molecule (OKT3-G1s) were linked by additional disulfide bonds, as described in Example 4-3. [Figure 4]Figure 4 shows the results of measuring the agonist activity via CD3 and / or CD28 for the following: a native anti-CD3ε antibody molecule (CD3-G1s), an anti-CD28 antibody molecule (CD28-G1s), an anti-CD3ε × anti-CD28 bispecific antibody (CD3 / / CD28-G1s), and modified antibody molecules (CD3 / / CD28-G1sLL, CD3 / / CD28-G1sHH, CD3 / / CD28-G1sLH, CD3 / / CD28-G1sHL) in which the Fab-Fab bonds of the native anti-CD3ε antibody molecule (CD3-G1s), an anti-CD28 antibody molecule (CD28-G1s), as described in Example 4-3. [Figure 5] Figure 5 shows the results of measuring the CD3 and / or CD28-mediated agonist activity for the following: a native anti-CD3ε antibody molecule (OKT3-G1s), an anti-CD28 antibody molecule (CD28-G1s), an anti-CD3ε × anti-CD28 bispecific antibody (OKT3 / / CD28-G1s), and modified antibody molecules (OKT3 / / CD28-G1sHH, OKT3 / / CD28-G1sHL) in which the Fab-Fab bonds of the native anti-CD3ε antibody molecule (OKT3-G1s), as described in Example 4-3. [Figure 6] Figure 6 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (1 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 7] Figure 7 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (2 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 8]Figure 8 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (3 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 9] Figure 9 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (4 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 10] Figure 10 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (5 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 11] Figure 11 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (6 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 12]Figure 12 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (7 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 13] Figure 13 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAH.xxx-G1T4) with cysteine ​​substitution in the heavy chain variable region, and modified antibody (MRAH-G1T4.xxx) with cysteine ​​substitution in the heavy chain constant region, as described in Example 5-2 (8 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 14] Figure 14 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) with cysteine ​​substitution in the light chain variable region, and modified antibody (MRAL-k0.xxx) with cysteine ​​substitution 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 band detection was performed using an anti-kappa chain antibody. [Figure 15] Figure 15 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (2 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 16]Figure 16 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) with cysteine ​​substitution in the light chain variable region, and modified antibody (MRAL-k0.xxx) with cysteine ​​substitution in the light chain constant region, as described in Example 6-2 (3 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 17] Figure 17 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (4 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 18] Figure 18 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (5 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 19] Figure 19 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (6 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 20]Figure 20 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (7 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 21] Figure 21 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (8 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 22] Figure 22 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody (MRAL.xxx-k0) with cysteine ​​substitution in the light chain variable region, and modified antibody (MRAL-k0.xxx) with cysteine ​​substitution 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 band detection was performed using an anti-kappa chain antibody. [Figure 23] Figure 23 shows the results of protease treatment of anti-IL6R antibody (MRA), modified antibody with cysteine ​​substitution in the light chain variable region (MRAL.xxx-k0), and modified antibody with cysteine ​​substitution in the light chain constant region (MRAL-k0.xxx), as described in Example 6-2 (10 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody. [Figure 24]Figure 24 shows the results of protease treatment of the anti-IL6R antibody (MRA) and its modified antibody (MRAL-k0.K126C), which had cysteine ​​substitution in its light chain constant region, as described in Example 7-2. Each protease-treated antibody was applied to non-reducing capillary electrophoresis, and band detection was performed using an anti-kappa chain antibody or an anti-human Fc antibody. [Figure 25] Figure 25 shows the correspondence between the molecular weight of each band obtained as a result of protease treatment of the antibody sample, as described in Example 7-2, and its assumed structure. Below the structure of each molecule, it is also indicated whether the molecule can react with anti-kappa chain antibody or anti-Fc antibody (whether the band is detected in the electrophoresis in Figure 24). [Figure 26] Figure 26 shows the results of measuring the CD3-mediated agonist activity of the anti-CD3 antibody molecule (OKT3), modified antibody molecules (H_T135C, H_S136C, H_S191C, L_K126C) in which the Fab-Fab bonds of the anti-CD3 antibody molecule (OKT3) were linked by additional disulfide bonds, and the anti-KLH antibody molecule (IC17) (negative control), as described in Example 13-4. [Figure 27] Figure 27 shows the results of measuring CD3-mediated agonist activity for the following: an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (OKT3_KiH) in which a Knobs-into-Holes (KiH) modification promoting heterodimerization is introduced into the heavy chain constant region of OKT3, modified antibody molecules (H_S191C_KiH, H_S191C / V188C_KiH, H_S191C / P189C_KiH, H_S191C / S190C_KiH, H_S191C / S192C_KiH, H_S191C / L193C_KiH, H_S191C / G194C_KiH) in which the Fab-Fab segments are linked by additional disulfide bonds, as described in Example 14-4, and an anti-KLH antibody molecule (IC17) (negative control). [Figure 28]Figure 28 shows the following modified antibody molecules as described in Example 15-4: an anti-CD3 antibody molecule (OKT3), a modified antibody molecule (H_S191C) in which the Fab-Fab of OKT3 is linked by an additional disulfide bond, a modified antibody molecule (OKT3_KiH) in which a Knobs-into-Holes (KiH) modification promoting heterodimerization is introduced into the heavy chain constant region of OKT3, a modified antibody molecule (H_S191C_KiH) in which the Fab-Fab of OKT3_KiH is linked by an additional disulfide bond, and one of the heavy chain constant regions of OKT3_KiH This figure shows the results of measuring CD3-mediated agonist activity for modified antibody molecules (0004 / / 0004, 0004 / / 0006) in which a positively charged amino acid substitution was introduced in one heavy chain constant region and a negatively charged amino acid substitution in the other heavy chain constant region; modified antibody molecules (0004 / / OKT3, OKT3 / / 0004, OKT3 / / 0006) in which a positive or negatively charged amino acid substitution was introduced in one heavy chain constant region of OKT3_KiH; and the anti-KLH antibody molecule (IC17) (negative control). [Figure 29] Figure 29 shows the results of measuring the CD3-mediated agonist activity of the anti-CD3 antibody molecule (OKT3), modified antibody molecules (dh1, dh2, dh3) in which the disulfide bond in the hinge region of the anti-CD3 antibody molecule (OKT3), modified antibody molecules (H_S191C_dh1, H_S191C_dh2, H_S191C_dh3) in which the Fab-Fab of these molecules is linked by additional disulfide bonds, and the anti-KLH antibody molecule (IC17) (negative control), as described in Example 16-4. [Figure 30]Figure 30 shows the results of measuring CD3-mediated agonist activity for the following: anti-CD3 monospecific antibody molecule (OKT3-G1s), modified antibody molecule (OKT3-G1sHH) in which the Fab-Fab of the anti-CD3 monospecific antibody (CD3-G1s) is linked by an additional disulfide bond, modified antibody molecule (CD3-G1sLL) in which the Fab-Fab of the anti-CD3 monospecific antibody (CD3-G1s) is linked by an additional disulfide bond, anti-CD3 biparatopic antibody molecule (CD3 / / OKT3-G1s), modified antibody molecules (CD3 / / OKT3-G1sHH, CD3 / / OKT3-G1sLH) in which the Fab-Fab of the anti-CD3 monospecific antibody (CD3 / / OKT3-G1s) is 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-D show the results of measuring the agonist activity via CD3 and / or PD1 for anti-CD3 × anti-PD1 bispecific antibody and modified antibody molecules in which the Fab-Fab bonds of the anti-CD3 × anti-PD1 bispecific antibody and the Fab-Fab bonds of the anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 117-G1silent) composed of anti-CD3 antibody (OKT3) and anti-PD1 antibody (117), and modified antibody molecules (OKT3 / / 117-G1silentHH, OKT3 / / 117-G1silentHL, OKT3 / / 117-G1silentLL) in which the Fab-Fab bonds of the anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 117-G1silent) are linked by additional disulfide bonds. [Figure 31B] (B) The agonist activity of the anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 10-G1silent), which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (10), and the modified antibody molecules (OKT3 / / 10-G1silentHH, OKT3 / / 10-G1silentHL) in which the Fab-Fab bonds are linked by additional disulfide bonds is shown. [Figure 31C](C) This shows the agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (CD3 / / 949-G1silent) composed of an anti-CD3 antibody (CD3) and an anti-PD1 antibody (949), as well as modified antibody molecules (CD3 / / 949-G1silentLH, CD3 / / 949-G1silentHH, CD3 / / 949-G1silentLL, CD3 / / 949-G1silentHL) in which the Fab-Fab bonds are linked by additional disulfide bonds. [Figure 31D] (D) The agonist activity of the anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent), which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), and the modified antibody molecules (OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentLL) in which the Fab-Fab bonds are linked by additional disulfide bonds is shown. [Figure 32] Figure 32 shows the results of measuring the CD3 and / or PD1-mediated agonist activity of an anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent) composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), as described in Example 22-2, as well as modified antibody molecules (OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentLH, OKT3 / / 949-G1silentLL) in which the Fab-Fab bonds of the anti-CD3 × anti-PD1 bispecific antibody molecule (OKT3 / / 949-G1silent), and modified antibody molecules (OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentLH, OKT3 / / 949-G1silentLL) were linked by additional disulfide bonds. [Figure 33A]Figures 33A and 33B show the results of evaluating the T cell-dependent inhibitory effect on cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Example 23-1. When the above-mentioned CD28 / CD3 clamping bispecific antibody and 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 proximity, while the CD28 / CD3 clamping bispecific antibody activates the effector cells. (A) This shows the inhibitory effect on cancer cell proliferation when using the GPC3 / binding attenuation CD3 bispecific antibody molecule (GPC3 / attCE115) as an antibody to target T cells to cancer cells, and the GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), 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 bonds of these molecules are linked by additional disulfide bonds, respectively, as antibodies to activate T cells. [Figure 33B] (B) The inhibitory effects on cancer cell proliferation are shown when using 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, as an antibody for targeting T cells to cancer cells; and when using a GPC3 / CD3 clamping bispecific antibody molecule (GPC3 / clamp CD3), a KLH / CD3 clamping bispecific antibody molecule (KLH / clamp CD3), a CD28 / CD3 clamping bispecific antibody molecule (CD28 / clamp CD3), and a modified antibody molecule (CD28 / clamp CD3_HH) in which the Fab-Fab of the same molecule is linked by an additional disulfide bond, as antibodies for activating T cells. [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 / attenuated CD3 bispecific antibody were used in combination, as described in Example 23-2. When the above-mentioned CD28 / CD3 clamping bispecific antibody and GPC3 / attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells), the GPC3 / attenuated CD3 bispecific antibody brings the target cells and effector cells into close proximity, while the CD28 / CD3 clamping bispecific antibody activates the effector cells. (A) This shows the amount of IL-6 produced when using a GPC3 / binding attenuation 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 by an additional disulfide bond, either alone or in combination, in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 34B] (B) The amount of IL-6 produced when using the GPC3 / 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, either alone or in combination, in the presence of effector cells (T cells) alone. [Figure 34C] (C) This shows the inhibitory effect on cancer cell proliferation when using a GPC3 / binding attenuation 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 by an additional disulfide bond, either individually 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 illustrating the mechanism of action of T cell-dependent inhibition of cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Example 23-1 (ε in the figure represents CD3ε). (A) This shows the mechanism of action of inhibition of cancer cell proliferation when a CD28 / CD3 clamping bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 35B] (B) The mechanism of action of cancer cell proliferation inhibition when a modified antibody molecule, which has been 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 attenuation CD3 bispecific antibody in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 36A] Figures 36A and 36B are schematic diagrams illustrating 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) This shows the mechanism of cytokine production when a modified antibody molecule, which has been modified to introduce an additional disulfide bond between the Fab-Fab of the CD28 / CD3 clamping bispecific antibody, is used in combination with a GPC3 / binding attenuated CD3 bispecific antibody in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells). [Figure 36B] (B) This shows the mechanism of cytokine production when a modified antibody molecule, which has been modified to introduce an additional disulfide bond between the Fab-Fab of a CD28 / CD3 clamping bispecific antibody, is used in combination with a GPC3 / binding attenuation CD3 bispecific antibody in the presence of effector cells (T cells) alone. [Figure 37A]Figures 37A and 37B show the results of measuring the agonist activity of the CD8 / CD28 bispecific antibody molecule (CD8 / CD28-P587) and modified antibody molecules (CD8 / CD28-P587(HH), CD8 / CD28-P587(LL), CD8 / CD28-P587(HL), CD8 / CD28-P587(LH)) in which the Fab-Fab bonds were linked by additional disulfide bonds, as described in Example 24. The anti-KLH antibody molecule (KLH-P587) was used as a negative control. Results using peripheral blood mononuclear cells (PBMCs) from two different donors are shown (top: Donor A, bottom: Donor B). (A) Shows the percentage of divided regulatory T (Treg) cells contained in the PBMCs. [Figure 37B] (B) Shows the percentage of divided CD8α-positive T cells contained in PBMCs. [Modes for carrying out the invention]

[0021] I. Definition In this specification, the term "antigen-binding molecule" refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In another embodiment, the antigen-binding molecule is a non-antibody protein, a fragment thereof, or a derivative thereof.

[0022] In this specification, “antigen-binding domain” means a region that specifically binds to and is complementary to a part or all of an antigen. In this specification, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of the antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. This specific portion is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain may be provided from the variable domains of one or more antibodies. In one 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 particular embodiment, the antigen-binding domain comprises a hinge region.

[0023] In this specification, "specifically binds" means that one molecule of a specifically binding molecule binds without showing any significant binding to any molecules other than the one or more molecules it binds to. It is also used when the antigen-binding domain is specific to a particular epitope among several epitopes contained in a given antigen. Furthermore, if the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing that epitope.

[0024] In this disclosure, "binding to the same epitope" means that the epitopes to which the two antigen-binding domains bind overlap to at least a portion. The degree of overlap is not limited to, but is at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, particularly preferably 90%, and most preferably 100%.

[0025] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0026] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0027] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0028] The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

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

[0030] In one embodiment of the present invention, the constant region is preferably an antibody constant region, more preferably an antibody constant region of type IgG1, IgG2, IgG3, or IgG4, and even more preferably a human antibody constant region of type IgG1, IgG2, IgG3, or IgG4. 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, human IgG2, human IgG3, and human IgG4 constant regions are known. As for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, but any of them may be used in the present invention. The constant region of the present invention with modified amino acids may include other amino acid mutations or modifications, as long as it includes the amino acid mutations of the present invention.

[0031] The term "hinge region" refers to the antibody heavy chain polypeptide portion that links the CH1 and CH2 domains in the wild-type antibody heavy chain, for example, from around position 216 to 230 in the EU numbering system, or from around position 226 to 243 in the Kabat numbering system. In natural-type IgG antibodies, it is known that the cysteine ​​residue at EU numbering position 220 in the hinge region forms a disulfide bond with the cysteine ​​residue at position 214 in the antibody light chain. Furthermore, it is known that disulfide bonds are formed between the cysteine ​​residues at EU numbering positions 226 and 229 in the hinge region of two antibody heavy chains. In this specification, the hinge region includes not only the wild type but also modified versions in which amino acid residues have been substituted, added, or deleted from the wild type.

[0032] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0033] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0034] An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0035] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0036] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0037] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0038] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0039] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0040] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0041] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0042] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0043] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0044] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-chain Fab (scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0045] Fv (variable fragment) In this specification, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that Fv could be prepared from the periplasmic fraction of E. coli in a homogeneous and active state by inserting the antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction had VH and VL associated in a manner that allowed for binding to the antigen.

[0046] scFv, single-chain antibody, or sc(Fv)2 In this specification, the terms “scFv,” “monochain antibody,” or “sc(Fv)2” refer to an antibody fragment that contains variable regions derived from both the heavy and light chains within a single polypeptide chain, but lacks a constant region. Generally, monochain antibodies further include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is expected to allow antigen binding. Monochain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg, and Moore (ed.), Springer-Verlag, New York, 269–315 (1994). See also International Patent Application Publication WO1988 / 001649 and U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monochain antibodies may also be bispecific and / or humanized.

[0047] scFv is an antigen-binding domain in which the VH and VL components of Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). This peptide linker allows VH and VL to be kept in close proximity.

[0048] sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and VLs may originate from different monoclonal antibodies. For example, bispecific sc(Fv)2, which recognizes two different epitopes present in the same antigen, is also preferred, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.

[0049] In this specification, the antigen-binding domain configuration of sc(Fv)2 is characterized by two VHs and two VLs arranged in the order VH, VL, VH, VL ([VH]linker[VL]linker[VH]linker[VL]) starting from the N-terminus of the single-chain polypeptide. 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 configuration can also be given. [VL] Linker [VH] Linker [VH] Linker [VL] [VH] Linker [VL] Linker [VL] Linker [VH] [VH] Linker [VH] Linker [VL] Linker [VL] [VL] Linker [VL] Linker [VH] Linker [VH] [VL] Linker [VH] Linker [VL] Linker [VH]

[0050] Fab, F(ab')2, or Fab' "Fab" consists of a light chain and a heavy chain comprising a CH1 region and a variable region. The heavy chain of the wild-type Fab molecule cannot form disulfide bonds with other heavy-chain molecules. This specification includes not only wild-type Fab but also modified Fab molecules in which amino acid residues have been substituted, added, or deleted from the wild-type. In certain embodiments, mutant amino acid residues in a modified Fab (e.g., substituted, added, or inserted cysteine ​​or lysine residues) can form disulfide bonds with other heavy-chain molecules or parts thereof (e.g., Fab molecules).

[0051] scFab is an antigen-binding domain in which the CH1 region and variable region of one light chain and one heavy chain constituting the Fab are linked by a peptide linker. This peptide linker allows the CH1 region and variable region of the light chain and heavy chain to be kept in close proximity.

[0052] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin or papain, and digesting them before and after the disulfide bond between the two H chains in the hinge region. For example, by treating IgG with papain, the disulfide bond between the two H chains in the hinge region is cleaved upstream, producing two homologous antibody fragments: an L chain consisting of VL (variable L chain region) and CL (constant L chain region), and an H chain fragment consisting of VH (variable H chain region) and CHγ1 (γ1 region in the constant H chain region), which are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab'.

[0053] "F(ab')2" comprises two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain 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 having a desired antigen-binding domain with a protease such as pepsin, and then removing the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited as long as it can digest a full-length antibody to produce F(ab')2 restrictively by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be cited.

[0054] Single-domain antibody (also called monodomain antibody) In this specification, the term "single-domain antibody" is used without regard to its structure, as long as the domain alone can exhibit antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL domains, single-domain antibodies are known to exhibit antigen-binding activity solely through their own domain structure, without pairing with other domains. Single-domain antibodies typically have a relatively low molecular weight and exist in monomeric form. Examples of single-domain antibodies, though not limited to them, include, for example, VHH antibodies for camelids and V antibodies for sharks. NAR Examples include antigen-binding molecules that congenitally lack a light chain, or antibody fragments containing all or part of the VH domain or all or part of the VL domain of an antibody. Examples of single-domain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, single-domain antibodies artificially produced starting from human antibody VH or human antibody VL, as described in, for example, U.S. Patent No. 6,248,516B1. In some embodiments of the present invention, one single-domain antibody has three CDRs (CDR1, CDR2, and CDR3). Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single-domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single-domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized single-domain antibodies can also be obtained by using a human germline sequence or a similar sequence as the framework sequence of a single-domain antibody obtained from an animal. Humanized single-domain antibodies (e.g., humanized VHH) are also one embodiment of the single-domain antibody of the present invention. Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), 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) is one example.

[0055] "Binding activity" refers to the sum of non-covalent interactions between one or more binding sites on a molecule (e.g., an antibody) and its 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, if the members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). If the members of a binding pair are capable of both monovalent and polyvalent binding, binding activity is the sum of these binding forces. The binding activity of molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or "analyte binding 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 "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, “blocking” antigen-binding molecules or “blocking” antibodies, or “antagonist” antigen-binding molecules or “antagonist” antibodies, are antigen-binding molecules or antibodies that significantly inhibit (either partially or completely) the biological activity of the antigen to which they bind.

[0058] As used herein, the expressions “substantially reduced” or “substantially different” mean that the difference between two numbers (usually between one relating to a molecule and one relating to a reference / comparative molecule) is sufficiently large that a person skilled in the art would consider the difference between the two numbers to be statistically significant in terms of the biological characteristic measured by the number (e.g., the KD value).

[0059] As used herein, the terms “substantially similar” or “substantially identical” mean that the similarity between two numerical values ​​(e.g., between the antibody of the present invention and the reference / comparative antibody) is sufficiently high that a person skilled in the art would consider the difference between the two numerical values ​​to be little or no biological and / or statistically significant in terms of the biological characteristics measured by the numerical value (e.g., the KD value).

[0060] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations in which the biological activity of the active ingredients contained herein can exert its effect, and which do not contain additional elements that are toxic to an extent unacceptable to the subject to whom the preparation is administered.

[0061] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0062] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0063] II. Antigen-binding molecules In one aspect, this disclosure is based in part on the finding that antigen-binding molecules comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via one or more bindings, exhibit enhanced or reduced activity compared to control antigen-binding molecules comprising antigen-binding domains linked via no or fewer bindings. In a particular embodiment, an antigen-binding molecule is provided having the activity of holding two or more antigen molecules in spatially close proximity. The antigen-binding molecule of this disclosure is useful, for example, in that it can control the activation of two antigen molecules that are activated by association with each other. In another particular embodiment, an antigen-binding molecule is provided that has acquired resistance to protease digestion by linkage between antigen-binding domains.

[0064] A. Exemplary antigen-binding molecules <Structure of antigen-binding molecule> In one aspect, the present disclosure provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via one or more bonds.

[0065] In one aspect of the above, at least one of the one or more bonds linking the two antigen-binding domains is a covalent bond. In a particular aspect, a covalent bond is formed by direct cross-linking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. The type of amino acid residue to be cross-linked 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 crosslinking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the type of amino acid residue to be crosslinked is, for example, lysine.

[0066] In one aspect of the above, at least one of the one or more bonds linking the antigen-binding domain is a non-covalent bond. In a particular aspect, the non-covalent bond is one of an ionic bond, a hydrogen bond, or a hydrophobic bond. An ionic bond is formed, for example, between an acidic amino acid and a basic amino acid. 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 binding between antigen-binding domains (the binding that links two antigen-binding domains) is formed by linking amino acid residues that serve as the starting point for binding in the first and second antigen-binding domains, respectively. In one embodiment of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is an artificially introduced mutant amino acid residue, for example, an artificially introduced cysteine ​​residue. Such mutant amino acid residues can be introduced into wild-type antigen-binding domains by methods such as amino acid substitution. When the antigen-binding domain includes, for example, an antibody fragment, the sites of amino acid residues that can serve as the starting point for binding between antigen-binding domains are disclosed herein in the constant regions (CH1 region, CL region, and hinge region) and the variable regions (VH region, VL region, and VHH region), and for example, cysteine ​​residues can be introduced into these sites.

[0068] In one aspect of the above, at least one of the first and second antigen-binding domains has the activity to bind to an antigen on its own (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular aspect, both the first and second antigen-binding domains have the activity to bind to an antigen on their own.

[0069] In one aspect of the above, the first and second antigen-binding domains are both antigen-binding domains of the same type. As will be described later, examples of proteins constituting antigen-binding domains include polypeptides derived from antibodies or non-antibody proteins and their fragments (e.g., Fab, Fab', scFab, Fv, scFv, single-domain antibodies, etc.). From the perspective of such molecular shapes, if the structures of the proteins constituting the first and second antigen-binding domains are identical, they are judged to be of the same type.

[0070] In one embodiment of the above aspect, the at least one link between the first antigen-binding domain and the second antigen-binding domain may be formed by linking amino acid residues located at the same position on the first antigen-binding domain and the second antigen-binding domain, respectively, or by linking amino acid residues located at different positions on the first antigen-binding domain and the second antigen-binding domain.

[0071] The positions of amino acid residues on antigen-binding domains can be indicated according to the Kabat numbering or EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. For example, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at the same corresponding position on each antigen-binding domain, the positions of those amino acid residues can be indicated by the same number according to the Kabat numbering or EU numbering system. Conversely, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at different, non-corresponding positions on 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 includes an antibody fragment that binds to a specific antigen. In a particular embodiment, the antibody fragment is one of Fab, Fab', scFab, Fv, scFv, or a single-domain antibody. In a particular embodiment, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is present within the antibody fragment.

[0073] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the constant region. In a particular aspect, the amino acid residue is located within the CH1 region, for example, at EU numbering positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, or 218 to 219 of the CH1 region. According to certain sources, the amino acid residues in question are located at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, and 165 in the CH1 region. The amino acid residue is located in one of the groups selected from positions 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218, and 219. In a particular embodiment, the amino acid residue is located at EU numbering positions 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 in the CH1 region. In a particular embodiment, the amino acid residue is located at EU numbering positions 135, 136, or 191 in the CH1 region. In one embodiment of the above, the steady-state region is of human origin. In a particular embodiment, the subclass of the heavy chain steady-state region is one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a particular embodiment, the subclass of the CH1 region is one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[0074] In one aspect of the above, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CH1 region of the second antigen-binding domain. In a particular aspect, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123. In a particular aspect, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140. In a particular embodiment, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 148, 149, and 150, respectively. In a particular embodiment, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167, respectively. In a particular embodiment, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178, respectively. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 218 and 219.

[0075] In one embodiment of the above aspect, the difference in the positions of the amino acid residues that serve as the starting point for binding in the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. This means that when comparing the position of the amino acid residue that serves as the starting point for binding in the CH1 region of the first antigen-binding domain and the position of the amino acid residue that serves as the starting point for binding in the CH1 region of the second antigen-binding domain using EU numbering, the difference is within 3 amino acids. In a particular embodiment, at least one binding linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 135 in the CH1 region of the first antigen-binding domain with any amino acid residue at EU numbering positions 132 to 138 in the CH1 region of the second antigen-binding domain. In a particular embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain with any amino acid residue from EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain. In a particular embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with any amino acid residue from EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at EU numbering position 135 in the CH1 regions of the two antigen-binding domains. In one exemplary embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at EU numbering position 136 in the CH1 region of the two antigen-binding domains.In one exemplary embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at EU numbering position 191 in the CH1 region of the two antigen-binding domains.

[0076] In one aspect of the aforementioned situation, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the CL region, for example, at one of the following Kabat numbering positions within the CL region: 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, or 208 to 213. In a particular embodiment, the amino acid residue is located at any of the group selected from positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213 of the Kabat numbering in the CL region. In a particular embodiment, the amino acid residue is located at position 126 of the Kabat numbering in the CL region. In one embodiment of the above-mentioned aspect, the steady-state region is of human origin. In a particular embodiment, the subclass of the CL region is κ or λ.

[0077] In one embodiment of the above aspect, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CL region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 108, 109, 110, 111, and 112. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 151, 152, 153, 154, 155, and 156. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 184, 185, 186, 187, 188, 189, and 190. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 195 and 196. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 200, 201, 202, and 203. In a particular embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of Kabat numbering positions 208, 209, 210, 211, 212, and 213.

[0078] In one embodiment of the above aspect, the difference in the positions of the amino acid residues that serve as the starting point for binding in the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. This means that when comparing the positions of the amino acid residues that serve as the starting point for binding in the CL region of the first antigen-binding domain and the positions of the amino acid residues that serve as the starting point for binding in the CL region of the second antigen-binding domain using EU numbering, the difference is within 3 amino acids. In one exemplary embodiment, at least one binding linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at Kabat numbering position 126 in the CL regions of the two antigen-binding domains.

[0079] In one embodiment of the above aspect, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a particular embodiment, the amino acid residue in the CH1 region of the first antigen-binding domain is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region of the second antigen-binding domain is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. In one exemplary embodiment, at least one linkage between the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain with the amino acid residue at Kabat numbering position 126 in the CL region of the second antigen-binding domain.

[0080] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within a variable region. In a particular aspect, the amino acid residue is located within the VH region, for example, in any of the group selected from the Kabat numbering positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b of the VH region. In a particular aspect, the amino acid residue is located within the VL region, for example, in any of the group selected from the Kabat numbering positions 21, 27, 58, 77, 100, 105, and 107 of the VL region (subclass κ), and Kabat numbering positions 6, 19, 33, and 34 of the VL region (subclass λ). In a particular embodiment, the amino acid residue is located within a VHH region, for example, at any of the group selected from positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 of the VHH region's Kabat numbering.

[0081] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the non-antibody protein is either a pair of ligands and / or receptors that bind specifically to each other. Examples of receptors here include receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, C. difficile antigens, costimulatory molecules, and cell adhesion molecules.

[0082] In one embodiment of the above aspect, the first and / or second antigen-binding domains include a hinge region. In a particular embodiment, at least one of the cysteine ​​residues present in the wild-type hinge region is substituted with another amino acid residue. Such cysteine ​​residues are located, for example, at EU numbering positions 226 and / or 229 of the wild-type hinge region. In a particular embodiment, at least one of the amino acid residues that initiate binding between the antigen-binding domains is located within the hinge region and is located, for example, at any of the group selected from EU numbering positions 216, 218, and 219 of the hinge region.

[0083] In one aspect of the above-mentioned configuration, the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more binding sites.

[0084] In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is an amino acid residue present in the wild-type sequence, for example, a cysteine ​​residue in the wild-type hinge region. In certain embodiments, at least one binding site linking the first antigen-binding domain and the second antigen-binding domain is a disulfide bond formed by cross-linking of cysteine ​​residues present in the wild-type hinge region. Such cysteine ​​residues are located, for example, at EU numbering positions 226 and / or 229 in the wild-type hinge region.

[0085] In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the antibody fragment, and at least one is located within the hinge region. In one exemplary embodiment, the antigen-binding molecule of the present disclosure is F(ab')2, in which both the first and second antigen-binding domains include a Fab and a hinge region.

[0086] In one aspect of the foregoing, the antigen-binding molecule of the Disclosure further comprises an Fc region and is, for example, a full-length antibody. In certain aspects, the Fc region of the antigen-binding molecule of the Disclosure is introduced with one or more amino acid mutations that promote multimerization of the Fc region. Such amino acid mutations include, for example, amino acid mutations at at least one site selected from the group consisting of EU numberings 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 aspects, the multimerization is hexamerization.

[0087] <Types of antigens to which antigen-binding molecules bind> In one aspect of the above, the first and second antigen-binding domains both bind to the same type of antigen. In a particular aspect, the first and second antigen-binding domains bind to the same epitope on the same type of antigen. In another particular aspect, the first and second antigen-binding domains bind to different epitopes on the same type of antigen. In a particular aspect, the antigen-binding molecule of the present disclosure is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets one specific type of antigen. In another embodiment of the aforementioned aspect, the first and second antigen-binding domains bind to antigens of different types from each other. In another aspect of the foregoing, the antigen-binding molecule of the Disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). In this Specification, a clamping antigen-binding molecule means an antigen-binding molecule that specifically binds to an antigen-antigen complex formed from an antigen A and an antigen-binding molecule that binds to that antigen A, thereby increasing the binding activity of the antigen-binding molecule to that antigen A (or stabilizing the antigen-antigen complex formed from the antigen A and the antigen-binding molecule that binds to that antigen A). For example, a CD3 clamping antibody can specifically bind to an antigen-antibody complex formed from CD3 and an antibody with reduced binding ability to CD3 (a CD3-attenuated antibody), thereby increasing the binding activity of the CD3-attenuated antibody to CD3 (or stabilizing the antigen-antibody complex formed from CD3 and the CD3-attenuated antibody). In certain aspects, the first and / or second antigen-binding domains in the antigen-binding molecule of the Disclosure may be antigen-binding domains derived from a clamping antigen-binding molecule (clamping antigen-binding domains). In one embodiment of the above aspect, the first and second antigen-binding domains both have the same amino acid sequence. In another embodiment, 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 to which the first and second antigen-binding domains bind is a soluble protein or a membrane protein.

[0089] <Function of antigen-binding molecules> In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one less bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). In another aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has activity to control the interaction between two antigen molecules. While not bound by any particular theory, this interaction-controlling activity is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In certain aspects, the antigen-binding molecule of the present disclosure can enhance or reduce the interaction between two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are a ligand and its receptor, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the activation of the receptor by the ligand. In another particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are an enzyme and its substrate, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the catalytic reaction of the enzyme with respect to the substrate. In another specific embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the Disclosure has the activity to facilitate interaction between a cell expressing the first antigen and a cell expressing the second antigen. The cell expressing the first antigen and the cell expressing the second antigen are, for example, a cytotoxic cell and a target cell, respectively, and the antigen-binding molecule of the Disclosure facilitates damage to the target cell by the cytotoxic cell. The cytotoxic cell is, for example, a T cell, NK cell, monocyte, or macrophage.

[0090] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has an activity that controls the activation of two antigen molecules that are activated by association with each other. Although not bound by any particular theory, this activity that controls the activation is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can enhance or reduce the activation of two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The aforementioned antigen molecule is selected from the group consisting of, for example, receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0091] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has two antigen-binding domains located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced. In a particular aspect, the antigen-binding molecule of the present disclosure has two antigen-binding domains located in closer proximity and / or the mobility of the two antigen-binding domains is further reduced compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0092] In one aspect of the aforementioned aspect, the antigen-binding molecule of the Disclosure is resistant to protease cleavage. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the Disclosure only in that it has one fewer bond between two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular aspect, the antigen-binding molecule of the Disclosure exhibits an increased proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment compared to a control antigen-binding molecule. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits a decreased proportion of specific fragments (e.g., Fab monomers) generated after protease treatment compared to a control antigen-binding molecule.

[0093] In one embodiment of the aforementioned aspect, when the antigen-binding molecule of the Disclosure is treated with a protease, a dimer of the antigen-binding domain or a fragment thereof (e.g., a crosslinked Fab dimer) is cleaved. In a particular embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the Disclosure in that it has one fewer bond between two antigen-binding domains is treated with the protease, a monomer of the antigen-binding domain or a fragment thereof is cleaved. In a further embodiment, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue 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 this disclosure only in that it has one fewer bond between two antigen-binding domains, and this one less bond is formed starting from a mutant amino acid residue. The mutant amino acid residue is, for example, an artificially introduced cysteine ​​residue.

[0095] <Pharmaceutical composition> In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule 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 spatially close 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 produced in (b) with the two antigen molecules. In a particular embodiment, the antigen-binding molecule in (a) may have two antigen-binding domains linked together via one or more bonds, in which case some or all of the one or more bonds are bonds in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) is a bond in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The Disclosure also provides a method for holding two antigen molecules in spatial proximity, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides the antigen-binding molecule or pharmaceutical composition of the Disclosure for holding two antigen molecules in spatial proximity.

[0097] In another aspect, the present disclosure provides a method for controlling the 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. In a particular embodiment, the antigen-binding molecule in (a) above may have the two antigen-binding domains linked together via one or more bonds, in which case some or all of the one or more bonds are bonds in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one bond in (b) above is a bond in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The 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 Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for controlling the interaction between two antigen molecules.

[0098] In another aspect, the Disclosure provides a method for controlling the activity of two antigen molecules that are activated by association, 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 made in (b) with the two antigen molecules. In a particular embodiment, the antigen-binding molecule in (a) above may have the two antigen-binding domains linked together via one or more bonds, in which case some or all of the one or more bonds are bonds in which the amino acid residues that initiate the binding between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). The Disclosure also provides a method for controlling the activity of two antigen molecules that are activated by association, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for controlling the activity of two antigen molecules that are activated by association.

[0099] In another aspect, the disclosure provides a method for positioning two antigen-binding domains in 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 linkage to the antigen-binding molecule that links the two antigen-binding domains together. In a particular embodiment, the antigen-binding molecule in (a) above may have the two antigen-binding domains linked together via one or more linkages, in which case some or all of the one or more linkages are linkages in which the amino acid residue that initiates the linkage between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that initiates the linkage between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0100] In another aspect, the Disclosure provides a method for increasing the resistance of an antigen-binding molecule to protease cleavage, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains; and (b) adding at least one linkage to the antigen-binding molecule that links the two antigen-binding domains together. In a particular embodiment, the antigen-binding molecule in (a) above may have the two antigen-binding domains linked together via one or more linkages, in which case some or all of the one or more linkages are linkages in which the amino acid residue that initiates the linkage between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that initiates the linkage between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

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

[0102] <Method for producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having the activity of holding two antigen molecules in spatially close proximity, 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 such that at least one linkage 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 to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more linkages. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0103] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule having activity to control 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 such that at least one linkage 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 to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more linkages. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0104] In another aspect, the Disclosure provides a method for producing an antigen-binding molecule having activity to control the activation of two antigen molecules activated by association with each other, comprising: (a) providing a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one linkage 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 to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more linkages. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0105] In another aspect, the Disclosure provides a method for producing an antigen-binding molecule in which two antigen-binding domains are located in spatially close proximity and / or the mobility of the two antigen-binding domains is reduced, comprising: (a) 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 linkage connecting the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more linkages. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0106] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) providing a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains such that at least one linkage between the two antigen-binding domains is added; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via one or more linkages. In a particular embodiment, each of the two antigen-binding domains in (a) above may contain one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains, in which case some or all of the one or more amino acid residues that serve as the starting point for linking between the antigen-binding domains are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the at least one linkage in (b) above is a linkage in which the amino acid residue that serves as the starting point for linking between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

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

[0108] <Screening method for antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel pair of protein molecules that are activated by association with each other, comprising: (a) providing any two protein molecules; (b) producing an antigen-binding molecule comprising two antigen-binding domains that bind to each of the two protein molecules by a manufacturing method of the present disclosure; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) measuring whether the two protein molecules are activated. In a particular embodiment, at least one of the two protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0109] A. Exemplary antigen-binding molecules <Structure of antigen-binding molecule> In one aspect, the present disclosure provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via two or more bonds. In one embodiment, at least one of the first and second antigen-binding domains has antigen-binding activity on its own (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular embodiment, both the first and second antigen-binding domains have antigen-binding activity on their own.

[0110] In one aspect of the above, at least one of the first and second antigen-binding domains includes an antibody fragment that binds to a specific antigen. In a particular aspect, the first and / or second antigen-binding domains include a hinge region. The binding between the antigen-binding domains is formed by linking amino acid residues that serve as the starting point for binding in the first and second antigen-binding domains, respectively. In a particular aspect, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is located within the antibody fragment. In a particular aspect, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is located within the hinge region. In a particular aspect, at least one of the amino acid residues that serve as the starting point for binding between the antigen-binding domains is located within the antibody fragment, and at least one is located within the hinge region.

[0111] In one aspect of the above, in at least one of the first and second antigen-binding domains, there are multiple amino acid residues that serve as the starting point for binding between the antigen-binding domains, located at positions 7 amino acids or more apart from each other on the primary structure. This means that there are 6 or more amino acid residues other than those amino acid residues between any two of the above multiple amino acid residues. In a particular aspect, the combination of multiple amino acid residues that serve as the starting point for binding between the antigen-binding domains may include pairs of amino acid residues located at positions less than 7 amino acids apart on the primary structure. In a particular aspect, if the first and second antigen-binding domains are linked to each other via three or more bindings, three or more amino acid residues, including pairs of amino acid residues located at positions 7 amino acids or more apart from each other on the primary structure, can serve as the starting point for binding between the antigen-binding domains. In certain embodiments, amino acid residues located at the same positions on the first antigen-binding domain and the second antigen-binding domain link together to form a bond. In certain embodiments, amino acid residues located at different positions on the first antigen-binding domain and the second antigen-binding domain link together to form a bond.

[0112] The positions of amino acid residues on antigen-binding domains can be indicated according to the Kabat numbering or EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. For example, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at the same corresponding position on each antigen-binding domain, the positions of those amino acid residues can be indicated by the same number according to the Kabat numbering or EU numbering system. Conversely, if the amino acid residues that initiate binding between the first and second antigen-binding domains are located at different, non-corresponding positions on 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 aspect of the above, at least one of the two or more bonds linking the antigen-binding domains is a covalent bond. In a particular aspect, a covalent bond is formed by direct cross-linking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. The type of amino acid residue to be cross-linked is, for example, cysteine, and the covalent bond formed is, for example, a disulfide bond. At least one of the cysteine ​​residues to be cross-linked may be located within the hinge region. In another specific embodiment, a covalent bond is formed by crosslinking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the type of amino acid residue to be crosslinked is, for example, lysine.

[0114] In one aspect of the above, at least one of the two or more bonds linking the antigen-binding domain is a non-covalent bond. In a particular aspect, the non-covalent bond is one of an ionic bond, a hydrogen bond, or a hydrophobic bond.

[0115] In one aspect of the aforementioned situation, the antibody fragment is one of Fab, Fab', scFab, Fv, scFv, or a single-domain antibody.

[0116] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the constant region. In a particular aspect, the amino acid residue is located within the CH1 region, for example, at any of the group selected from positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213, and 214 of the EU numbering of the CH1 region. In one exemplary embodiment, the amino acid residue is located at EU numbering position 191 of the CH1 region, and the amino acid residues at EU numbering position 191 in the CH1 regions of the two antigen-binding domains are linked together to form a bond. In a particular embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the hinge region, for example, in one of the group selected from positions 216, 218, and 219 of the EU numbering of the hinge region. In a particular embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is located within the CL region and is selected from the group consisting of EU numbering positions 109, 112, 121, 126, 128, 151, 152, 153, 156, 184, 186, 188, 190, 200, 201, 202, 203, 208, 210, 211, 212, and 213 of the CL region. In an exemplary embodiment, the amino acid residue is located at EU numbering position 126 of the CL region, and the amino acid residues at EU numbering position 126 in the CL regions of the two antigen-binding domains link together to form a binding. In a particular embodiment, an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain are linked to form a bond. In an exemplary embodiment, an amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and an amino acid residue at EU numbering position 126 in the CL region of the second antigen-binding domain are linked to form a bond.

[0117] In one embodiment of the above, the steady-state region is of human origin. In a particular embodiment, the subclass of the heavy-chain steady-state region is one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a particular embodiment, the subclass of the CH1 region is one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε. In a particular embodiment, the subclass of the CL region is κ or λ.

[0118] In one aspect of the above, at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within a variable region. In a particular aspect, the amino acid residue is located within the VH region, for example, in any of the group selected from the Kabat numbering positions 8, 16, 28, 74, and 82b of the VH region. In a particular aspect, the amino acid residue is located within the VL region, for example, in any of the group selected from the Kabat numbering positions 100, 105, and 107 of the VL region.

[0119] In one embodiment of the above aspect, both the first and second antigen-binding domains include a Fab and a hinge region. In certain embodiments, at least one of the amino acid residues that initiate binding between antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region, for example, a cysteine ​​residue in the hinge region. Examples of such cysteine ​​residues include those at EU numbering positions 226 and 229. In another specific embodiment, at least one of the amino acid residues that initiate binding between antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region, for example, a cysteine ​​residue not present in the wild-type Fab or hinge region. Such mutant amino acid residues can be introduced into the wild-type Fab or hinge region by means of, for example, amino acid substitution. In each of the CH1 region, hinge region, CL region, VH region, and VL region, sites of amino acid residues that can initiate binding between antigen-binding domains are disclosed herein, and for example, cysteine ​​residues can be introduced into these sites. Alternatively, in another embodiment, amino acid residues present in the wild-type Fab or hinge region that may be involved in the binding between antigen-binding domains (e.g., cysteine ​​residues) may be substituted or deleted with other amino acid residues. Examples of such cysteine ​​residues include the cysteine ​​residues at EU numbering positions 220, 226, and 229 in the hinge region, and the cysteine ​​residue at position 214 in the CL region. In certain embodiments, the antigen-binding molecule of the present disclosure is F(ab')2, wherein both the first and second antigen-binding domains include a Fab and a hinge region.

[0120] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the non-antibody protein is either a pair of ligands and / or receptors that bind specifically to each other. Examples of receptors here include receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, C. difficile antigens, costimulatory molecules, and cell adhesion molecules.

[0121] In one aspect of the foregoing, the antigen-binding molecule of the Disclosure further comprises an Fc region and is, for example, a full-length antibody. In certain aspects, the Fc region of the antigen-binding molecule of the Disclosure is introduced with one or more amino acid mutations that promote multimerization of the Fc region. Such amino acid mutations include, for example, amino acid mutations at at least one site selected from the group consisting of EU numberings 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 aspects, the multimerization is hexamerization.

[0122] <Types of antigens to which antigen-binding molecules bind> In one aspect of the above, the first and second antigen-binding domains both bind to the same type of antigen. In a particular aspect, the first and second antigen-binding domains bind to the same epitope on the same type of antigen. In another particular aspect, the first and second antigen-binding domains bind to different epitopes on the same type of antigen. In a particular aspect, the antigen-binding molecule of the present disclosure is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets one specific type of antigen. In another embodiment of the aforementioned aspect, the first and second antigen-binding domains bind to antigens of different types from each other. In another aspect of the foregoing, the antigen-binding molecule of the Disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). In this Specification, a clamping antigen-binding molecule means an antigen-binding molecule that specifically binds to an antigen-antigen complex formed from an antigen A and an antigen-binding molecule that binds to that antigen A, thereby increasing the binding activity of the antigen-binding molecule to that antigen A (or stabilizing the antigen-antigen complex formed from the antigen A and the antigen-binding molecule that binds to that antigen A). For example, a CD3 clamping antibody can specifically bind to an antigen-antibody complex formed from CD3 and an antibody with reduced binding ability to CD3 (a CD3-attenuated antibody), thereby increasing the binding activity of the CD3-attenuated antibody to CD3 (or stabilizing the antigen-antibody complex formed from CD3 and the CD3-attenuated antibody). In certain aspects, the first and / or second antigen-binding domains in the antigen-binding molecule of the Disclosure may be antigen-binding domains derived from a clamping antigen-binding molecule (clamping antigen-binding domains). In one embodiment of the above aspect, the first and second antigen-binding domains both have the same amino acid sequence. In another embodiment, 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 to which the first and second antigen-binding domains bind is a soluble protein or a membrane protein.

[0124] <Function of antigen-binding molecules> In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has the activity of holding two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can hold two antigen molecules in closer proximity than a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one less bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0125] In another aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has activity to control the interaction between two antigen molecules. While not bound by any particular theory, this interaction-controlling activity is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In certain aspects, the antigen-binding molecule of the present disclosure can enhance or reduce the interaction between two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

[0126] In a particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are a ligand and its receptor, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the activation of the receptor by the ligand. In another particular embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are an enzyme and its substrate, respectively, and the antigen-binding molecule of the Disclosure has the activity to promote the catalytic reaction of the enzyme with respect to the substrate.

[0127] In another specific embodiment, the two antigen molecules to which the antigen-binding molecule of the Disclosure binds are both antigens (e.g., proteins) present on the cell surface, and the antigen-binding molecule of the Disclosure has the activity to facilitate interaction between a cell expressing the first antigen and a cell expressing the second antigen. The cell expressing the first antigen and the cell expressing the second antigen are, for example, a cytotoxic cell and a target cell, respectively, and the antigen-binding molecule of the Disclosure facilitates damage to the target cell by the cytotoxic cell. The cytotoxic cell is, for example, a T cell, NK cell, monocyte, or macrophage.

[0128] In one aspect of the aforementioned aspect, the antigen-binding molecule of the present disclosure has an activity that controls the activation of two antigen molecules that are activated by association with each other. Although not bound by any particular theory, this activity that controls the activation is thought to result from the antigen-binding molecule of the present disclosure holding the two antigen molecules in spatially close proximity. In a particular aspect, the antigen-binding molecule of the present disclosure can enhance or reduce the activation of two antigen molecules compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the present disclosure only in that it has one fewer bond between the two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The aforementioned antigen molecule is selected from the group consisting of, for example, receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0129] In one aspect of the aforementioned aspect, the antigen-binding molecule of the Disclosure is resistant to protease cleavage. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differing from the antigen-binding molecule of the Disclosure only in that it has one fewer bond between two antigen-binding domains. In a further aspect, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). In a particular aspect, the antigen-binding molecule of the Disclosure exhibits an increased proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment compared to a control antigen-binding molecule. In a particular aspect, the antigen-binding molecule of the Disclosure exhibits a decreased proportion of specific fragments (e.g., Fab monomers) generated after protease treatment compared to a control antigen-binding molecule.

[0130] In one embodiment of the aforementioned aspect, when the antigen-binding molecule of the Disclosure is treated with a protease, a dimer of the antigen-binding domain or a fragment thereof (e.g., a crosslinked Fab dimer) is cleaved. In a particular embodiment, when a control antigen-binding molecule that differs from the antigen-binding molecule of the Disclosure in that it has one fewer bond between two antigen-binding domains is treated with the protease, a monomer of the antigen-binding domain or a fragment thereof is cleaved. In a further embodiment, the one fewer bond can be selected from a bond in which the amino acid residue that initiates the bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue 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 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 spatially close proximity, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains wherein the two antigen-binding domains are linked to each other by one or more bindings; (b) adding another binding to the antigen-binding molecule which links the two antigen-binding domains to each other; and (c) contacting the antigen-binding molecule produced in (b) with the two antigen molecules. In a particular embodiment, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The Disclosure also provides a method for holding two antigen molecules in spatial proximity, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides the antigen-binding molecule or pharmaceutical composition of the Disclosure for holding two antigen molecules in spatial proximity.

[0133] In another aspect, the Disclosure provides a method for controlling the interaction between two antigen molecules, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked to each other by one or more bindings; (b) adding another binding to the antigen-binding molecule that links the two antigen-binding domains to each other; and (c) contacting the antigen-binding molecule produced in (b) with the two antigen molecules. In a particular embodiment, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other binding in (b) above is binding in which the amino acid residue that initiates the binding between the antigen-binding domains is derived from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The 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 Disclosure. The Disclosure further provides an antigen-binding molecule or pharmaceutical composition of the Disclosure for controlling the interaction between two antigen molecules.

[0134] In another aspect, the Disclosure provides a method for controlling the activity of two antigen molecules activated by association, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains wherein the two antigen-binding domains are linked to each other by one or more bindings; (b) adding another binding to the antigen-binding molecule which links the two antigen-binding domains to each other; and (c) contacting the antigen-binding molecule made in (b) with the two antigen molecules. In a particular embodiment, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue that is not present in the wild-type Fab or hinge region). The Disclosure also provides a method for controlling the activity of two antigen molecules that are activated by association, comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the Disclosure. The Disclosure further provides the antigen-binding molecule or pharmaceutical composition of the Disclosure for controlling the activity of two antigen molecules that are activated by association.

[0135] In another aspect, the Disclosure provides 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 wherein the two antigen-binding domains are linked to each other by one or more bindings; and (b) adding another binding to the antigen-binding molecule which links the two antigen-binding domains to each other. In a particular embodiment, some or all of the one or more bindings in (a) above are bindings in which the amino acid residue that initiates the binding between the antigen-binding domains is an amino acid residue present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region).

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

[0137] <Method for producing antigen-binding molecules> In one aspect, the present disclosure provides a method for producing an antigen-binding molecule having the activity of holding two antigen molecules in spatially close proximity, 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 starting point for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so that another linkage is added to the two antigen-binding domains; (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 the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via two or more links. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region).

[0138] In another aspect, the Disclosure provides a method for producing an antigen-binding molecule having activity to control 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, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so that another linking bond is added to the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via two or more bonds. In a particular embodiment, some or all of the one or more amino acid residues that serve as the starting point for linking 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 binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (for example, a cysteine ​​residue that is not present in the wild-type Fab or hinge region).

[0139] In another aspect, the present disclosure provides a method for producing an antigen-binding molecule having activity to control the activation of two antigen molecules activated by association, 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 starting point for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so that another linking bond is added to the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via two or more bonds. In a particular embodiment, some or all of the one or more amino acid residues that initiate the binding between antigen-binding domains in (a) above are amino acid residues present in the wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In a further embodiment, the other binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains originates from a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., cysteine ​​residues not present in the wild-type Fab or hinge region).

[0140] In another aspect, the Disclosure provides a method for producing an antigen-binding molecule with increased resistance to protease cleavage, comprising: (a) providing a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains contains one or more amino acid residues that serve as the starting point for linking the two antigen-binding domains; (b) introducing a mutation into the nucleic acid encoding the two antigen-binding domains so that another linking bond is added to the two antigen-binding domains; (c) introducing the nucleic acid produced in (b) into a host cell; (d) culturing the host cell to express the two polypeptides; and (e) obtaining an antigen-binding molecule which is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked to each other via two or more bonds. In a particular embodiment, some or all of the one or more amino acid residues that serve as the starting point for linking 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 binding in (b) above is a binding in which the amino acid residue that initiates the binding between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (for example, a cysteine ​​residue that is not present in the wild-type Fab or hinge region). The antigen-binding molecules produced in these various situations may have the characteristics of antigen-binding molecules described herein.

[0141] <Screening method for antigen-binding molecules> In one aspect, the present disclosure provides a method for identifying a novel pair of protein molecules that are activated by association with each other, comprising: (a) providing any two protein molecules; (b) producing an antigen-binding molecule comprising two antigen-binding domains that bind to each of the two protein molecules by a manufacturing method of the present disclosure, the antigen-binding molecule having the activity to hold the two protein molecules in close proximity; (c) contacting the antigen-binding molecule produced in (b) with the two protein molecules; and (d) measuring whether the two protein molecules are activated. In a particular embodiment, at least one of the two protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-binding receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0142] <Linking of antigen-binding domains> In a non-limiting embodiment, two or more antigen-binding domains contained in the antigen-binding molecule of the Disclosure are linked to each other via one or more bonds. In a preferred embodiment, the antigen-binding domains contained in the antigen-binding molecule of the Disclosure have activity to bind to an antigen on their own. In such embodiment, the antigen-binding molecule of the Disclosure containing two antigen-binding domains can bind to two or more antigen molecules, the antigen-binding molecule of the Disclosure containing three antigen-binding domains can bind to three or more antigen molecules, the antigen-binding molecule of the Disclosure containing four antigen-binding domains can bind to four or more antigen molecules, and the antigen-binding molecule of the Disclosure containing n antigen-binding domains can bind to n or more antigen molecules.

[0143] In certain embodiments, at least one of the bindings between antigen-binding domains contained in the antigen-binding molecule of this disclosure is different from those found in native antibodies (e.g., in the Fab or hinge region of wild-type antibodies). Examples of bindings found between antigen-binding domains in native antibodies (e.g., native IgG antibodies) include disulfide bonds in the hinge region. Bindings between amino acid residues located outside the hinge region may also be bindings between amino acid residues within an antibody fragment (e.g., Fab), and include bindings between heavy chains (HH form), bindings between light chains (LL form), and bindings between heavy chains and light chains (HL or LH form) (see Figure 1). Examples of amino acid residues in the heavy or light chain that serve as the starting point for bindings between antigen-binding domains include amino acid residues at the aforementioned positions within the variable region (VH or VL region) or the constant region (CH1 region, hinge region, or CL region).

[0144] In one non-limiting embodiment, a plurality of amino acid residues located at distant positions in the primary structure of at least one of the two or more antigen-binding domains contained in the antigen-binding molecule of the present disclosure serve as the starting point for binding between the antigen-binding domains. The distance between the plurality of amino acid residues is such that, as a result of linking the antigen-binding domains by binding starting from each amino acid residue, a structure of two or more antigen-binding domains that are sufficiently close together is achieved, and the plurality of amino acid residues may be separated by, for example, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 11 or more amino acids, 12 or more amino acids, 13 or more amino acids, 14 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 30 or more amino acids, 35 or more amino acids, 40 or more amino acids, 45 or more amino acids, 50 or more amino acids, 60 or more amino acids, 70 or more amino acids, 80 or more amino acids, 90 or more amino acids, 100 or more amino acids, 110 or more amino acids, 120 or more amino acids, 130 or more amino acids, 140 or more amino acids, 150 or more amino acids, 160 or more amino acids, 170 or more amino acids, 180 or more amino acids, 190 or more amino acids, 200 or more amino acids, 210 or more amino acids, or 220 or more amino acids. Furthermore, the number of bindings between antigen-binding domains, and the number of amino acid residues that initiate such bindings, are such that, as a result of the linkage between antigen-binding domains by such bindings, structures of two or more sufficiently close antigen-binding domains are achieved. For example, there 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 particular embodiment, insofar as the linkage between antigen-binding domains by three or more bindings originating from three or more amino acid residues on the antigen-binding domain results in the formation of structures of two or more antigen-binding domains that are sufficiently close together, the distance between any two amino acid residues selected from the three or more amino acid residues on the primary structure may be seven amino acids or more for at least one pair of amino acid residues, and less than seven amino acids for the remaining pairs of amino acid residues.

[0145] In relation to the antigen-binding domains contained in the antigen-binding molecules of this disclosure, "sufficiently close" means that two or more antigen-binding domains are close enough to achieve the desired function (activity) of the antigen-binding molecules of this disclosure. Examples of such desired functions (activities) include the activity of holding two antigen molecules in spatially close proximity, the activity of controlling the interaction between two antigen molecules, the activity of promoting the activation of a receptor by a ligand, the activity of promoting the catalytic reaction of an enzyme to a substrate, the activity of promoting the interaction between a cell expressing a first antigen and a cell expressing a second antigen, the activity of promoting the damage of a target cell by a cell with cytotoxic activity (e.g., T cells, NK cells, monocytes, macrophages, etc.), the activity of controlling the activation of two antigen molecules that are activated by association with each other, and the resistance of the antigen-binding molecule to protease cleavage.

[0146] In one non-limiting embodiment, the bond between antigen-binding domains in the antigen-binding molecule of the present disclosure may be a covalent bond or a non-covalent bond. Such a covalent bond may be formed by the direct crosslinking of an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain, for example, a disulfide bond between cysteine ​​residues. The amino acid residues that are directly crosslinked may be located on an antibody fragment such as Fab, or within a hinge region. In another embodiment, a covalent bond is formed when an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain are crosslinked via a crosslinking agent. For example, when crosslinking is performed using an amine-reactive crosslinking agent, crosslinking can be performed via the free amino group of the N-terminal amino acid of the antigen-binding domain or via the primary amine of the side chain of the lysine residue on the antigen-binding domain. Amine-reactive crosslinking agents include functional groups that form chemical bonds with primary amines (e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, fluoroesters, etc.). Typical examples include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl) suberate), DSP (dithiobis(succinimidyl propionate)), DTSSP (3,3'-dithiobis (sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), BSOCOES (bis(2-(succinimidooxycarbonyloxy) ethyl)sulfone), and EGS (ethylene glycol bis(succinimidyl) Examples of crosslinking agents include succinate, sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), and DDFNB (1,5-difluoro-2,4-dinitrobenzene). Other examples of crosslinking agents include carboxylamine-reactive, sulfhydryl-reactive, aldehyde-reactive, and photoreactive crosslinking agents. The non-covalent bond connecting the antigen-binding domain may be an ionic bond, a hydrogen bond, or a hydrophobic bond.

[0147] Whether the binding between antigen-binding domains is more than that in a control antigen-binding molecule (for example, an antigen-binding molecule having a structure substantially similar to a native antibody structure) can be evaluated, for example, by the following method. First, an antigen-binding molecule of interest and a control antigen-binding molecule are treated with a protease that excises the antigen-binding domain (for example, a protease that cleaves the N-terminal side from the site where hinge regions such as papain and Lys-C are crosslinked), and then subjected to non-reducing electrophoresis. Next, an antibody that recognizes a part of the antigen-binding domain (for example, an anti-kappa chain HRP-labeled antibody) is used to detect the fragments present after protease treatment. When only monomers of the antigen-binding domain (for example, Fab monomers) of the control antigen-binding molecule are detected, while multimers of the antigen-binding domain (for example, Fab dimers) of the antigen-binding molecule of interest are detected, it can be evaluated that the antigen-binding molecule of interest has more binding between antigen-binding domains than the control antigen-binding molecule. The formation of disulfide bonds between cysteines in a modified antigen-binding molecule in which cysteine is introduced into a control antigen-binding molecule can be evaluated, for example, by the following method. First, an antigen-binding molecule of interest is incubated with chymotrypsin in 20 mM phosphate buffer (pH 7.0), and the mass of peptides that are assumed to be generated from the amino acid sequences of each antibody is detected by LC / MS. When a component corresponding to the theoretical mass of the peptide generated when newly introduced cysteines form disulfide bonds is detected, it can be evaluated that the introduced cysteines have formed disulfide bonds. Furthermore, when the above-mentioned component is not detected when a drug having an effect of reducing disulfide bonds (for example, tris(2-carboxyethyl)phosphine) is added to and analyzed a sample containing the above-mentioned antigen-binding molecule, it is more strongly confirmed that the above evaluation was correct.

[0148] <Resistance to protease cleavage> In one non-limiting embodiment, the antigen-binding molecule of the Disclosure is resistant to protease cleavage. In a particular embodiment, the antigen-binding molecule of the Disclosure exhibits increased resistance to protease cleavage compared to a control antigen-binding molecule (e.g., an antigen-binding molecule having a structure substantially similar to that of a native antibody) having one or more fewer bonds between antigen-binding domains. In a further embodiment, the one fewer bond may be selected from bonds in which the amino acid residue that initiates the bond between antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The antigen-binding molecule can be evaluated as having increased resistance to protease cleavage (improved protease resistance) if, 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 the full-length molecules remaining after protease treatment 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 when viewed in terms of the whole antigen-binding molecule. In another specific embodiment, the proportion of monomers of the antigen-binding domain (e.g., Fab) 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 when viewed in terms of the whole antigen-binding molecule. In another specific embodiment, the proportion of dimers of the antigen-binding domain (e.g., Fab) generated after protease treatment 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 when viewed in terms of the whole antigen-binding molecule. Examples of the protease 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 molecules according to any of the above-described embodiments may incorporate, alone or in combination, any of the features described in the following items 1 to 7.

[0150] 1. Affinity of the antigen-binding molecule 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 to 10 -13M, for example 10 -9 M~10 -13 It has a dissociation constant (KD) of 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. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0152] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 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 molecules provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0154] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually contains one or more variable domains, in which the HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

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

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

[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). A multispecific antigen-binding molecule is a monoclonal antigen-binding molecule having binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is to a particular antigen (e.g., CD3) and the other is to any other antigen (e.g., CD28 or cancer antigens). In certain embodiments, a bispecific antigen-binding molecule may bind to two different epitopes on a single antigen. A bispecific antigen-binding molecule may be prepared as a full-length antibody or as an antibody fragment.

[0158] Methods for producing multispecific antigen-binding molecules are not limited to these, but include, but are: recombinant co-expression of two immunoglobulin heavy-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, for example, U.S. Patent No. 5,731,168). Multispecific antigen-binding molecules can be created by manipulating electrostatic steering effects to produce Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); creating bispecific antibodies using a leucine zipper (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). (See 1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0159] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0160] 7. Antigen-binding molecule variants In certain embodiments, amino acid sequence variants of antigen-binding molecules provided herein are also considered. 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 antigen-binding molecules may be prepared by introducing appropriate modifications to 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 may be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen-binding ability).

[0161] a) Substitution, insertion, and deletion variants In certain embodiments, antigen-binding molecule variants having one or more amino acid substitutions are provided. The target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in the table below under the heading "Preferred Substitutions." More substantial modifications are provided in the same table under the heading "Exemplary Substitutions" and are described in detail below, with reference to the classes of amino acid side chains. Amino acid substitutions may be introduced into the antigen-binding molecule of interest, and the product may be screened for desired activity, such as retained / improved antigen-binding, reduced immunogenicity, or improved ADCC or CDC.

[0162] TIFF2026063391000002.tif160170

[0163] Amino acids can be grouped according to their common side-chain characteristics: (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 affecting chain orientation: Glycine (Gly), Proline (Pro); (6) Aromaticity: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe). Non-conservative substitution refers to the replacement of one member of these classes with one from another class.

[0164] One type of substitution variant comprises substitution of one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study has a modification (e.g., improvement) (e.g., increased affinity, decreased immunogenicity) in certain biological properties compared to the parent antigen-binding molecule, and / or will substantially retain certain biological properties of the parent antigen-binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be suitably made using, for example, phage display-based affinity maturation techniques (such as those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibody is displayed on a phage and screened for a particular biological activity (e.g., binding affinity).

[0165] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve the affinity of antigen-binding molecules. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries 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 variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is constructed. This library is then screened to identify any antigen-binding molecule variants with the desired affinity. Another method for introducing diversity involves an HVR-oriented approach that randomizes several HVR residues (e.g., 4-6 residues at a time). 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 may be made within one or more HVRs, provided that such modifications do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified or contains only one, two, or three amino acid substitutions.

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

[0168] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues within a sequence, but also the fusion of polypeptides ranging in length from one to over 100 residues at the amino-terminus and / or carboxyl-terminus. Examples of terminal insertions include antigen-binding molecules with a methionyl residue at the N-terminus. Other insertion variants of antigen-binding molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antigen-binding molecule is fused to the N- or C-terminus of the antigen-binding molecule.

[0169] b) Glycosylated mutants In certain embodiments, the antigen-binding molecules provided herein are modified to increase or decrease the degree to which the antigen-binding molecule is glycosylated. Adding or removing glycosylation sites to an antigen-binding molecule can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

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

[0171] In one embodiment, antigen-binding molecule variants are provided having carbohydrate structures lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antigen-binding molecule may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence diversity 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 fucosylated mutants may possess improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "defucosylated" or "fucose-deficient" 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 lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0172] Further antigen-binding molecule variants are provided that have a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antigen-binding molecule is bifid 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 U.S.2005 / 0123546 (Umana et al.). Antigen-binding molecule variants having at least one galactose residue in the oligosaccharide attached 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 variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antigen-binding molecule provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0174] In certain embodiments, antigen-binding molecule variants possessing some, but not all, effector functions are also within consideration of the present invention, where such effector functions make the antigen-binding molecule a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antigen-binding molecule lacks FcγR binding ability (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 for evaluating the ADCC activity of the target molecule are described in U.S. Patent 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); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, ACT1® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those 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 cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (see, for example, 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)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, 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 the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581), and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0176] Certain antigen-binding molecule variants with increased or decreased binding affinity to FcRs have been described. (See U.S. Patent 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 includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in 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 cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

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

[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 WO94 / 29351.

[0181] d) Cysteine-modified antigen-binding molecule variants In certain embodiments, it would be desirable to produce cysteine-modified 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 residues to be substituted occur at accessible sites of the antigen-binding molecule. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site of the antigen-binding molecule, and this reactive thiol group may be used to conjugate the antigen-binding molecule to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antigen-binding molecules may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0182] e) Antigen-binding molecule derivative In certain embodiments, the antigen-binding molecules provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the 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 in derivatization are not limited to these, but can be determined based on considerations such as the specific properties or functions of the antigen-binding molecule to be improved, and whether the antigen-binding molecule derivative will be used in therapy under specified conditions.

[0183] In relation to the antigen-binding molecules of this disclosure, the desired properties (activities) are not particularly limited, but examples include binding activity, neutralizing activity, cytotoxic activity, agonist activity, antagonist activity, and enzymatic activity. Agonist activity is the activity by which an antibody binds to an antigen such as a receptor, thereby inducing some kind of physiological activity change, such as the transmission of a signal within the cell. Examples of physiological activity 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, transfer activity, nucleolytic activity, dehydration activity, cell death induction activity, and apoptosis induction activity.

[0184] In another embodiment, a conjugate is provided comprising an antigen-binding molecule and a non-protein portion that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that do not harm normal cells but heat the non-protein portion to a temperature that kills cells adjacent to the antigen-binding molecule-non-protein portion.

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

[0186] For the recombinant production of the antigen-binding molecules of this 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 would be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the 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, especially when glycosylation and Fc effector function are not required. For bacterial expression of antibody fragments and polypeptides, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for a description of antibody fragment expression in Escherichia coli.) After expression, antigen-binding molecules may be isolated from bacterial cell paste into a soluble fraction and further purified.

[0188] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts in which the glycosylation pathway has been "humanized" to produce antigen-binding molecules with a partial or complete human glycosylation pattern, 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] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of 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 for the transformation of Spodoptera frugiperda cells.

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

[0191] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for 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. Measurement Method (Assay) The antigen-binding molecules provided herein may be identified, screened, or have their physical / chemical properties and / or biological activity elucidated by various assay methods known in the art.

[0193] 1. Combined measurement methods and other measurement methods In one aspect, the antigen-binding molecules of this disclosure are tested for their antigen-binding activity by known methods such as ELISA and Western blotting.

[0194] 2.Activity measurement method In one aspect, a method for identifying biologically active antigen-binding molecules is provided. Biological activity may include, for example, the activity of holding two antigen molecules in spatially close proximity, the activity of controlling the interaction between two antigen molecules, the activity of promoting ligand-mediated receptor activation, the activity of promoting the catalytic reaction of an enzyme to its substrate, the activity of promoting the interaction between a cell expressing a first antigen and a cell expressing a second antigen, the activity of promoting damage to a target cell by a cytotoxic cell (e.g., T cells, NK cells, monocytes, macrophages, etc.), the activity of controlling the activation of two antigen molecules activated by association with each other, and resistance to protease cleavage. Antigen-binding molecules having such biological activity in vivo and / or in vitro are also provided.

[0195] Furthermore, the antigen-binding molecules of this disclosure can exert 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 T cell receptor (TCR) complexes (e.g., CD3) and have activity that induces T cell activation (agonist 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 activity that promotes such activation (agonist activity); and so on. In certain embodiments, the biological activity exerted through binding to such antigen molecules is enhanced or diminished by the linkage of two or more antigen-binding domains contained in the antigen-binding molecules of this disclosure. In certain embodiments, such enhancement or diminishing may be achieved by the control of interactions between two or more antigen molecules (e.g., the association of two or more antigen molecules is promoted) by binding to the antigen-binding molecules of this disclosure, although this is not limited to theory.

[0196] In certain embodiments, the antigen-binding molecules of this disclosure are tested for such biological activity. Whether two antigen molecules are spatially close can be evaluated using methods such as crystal structure analysis, electron microscopy, or electron tomography of the complex consisting of the antigen and the antigen-binding molecule. Whether two antigen-binding domains are spatially close, or whether the mobility of the two antigen-binding domains is reduced, can also be evaluated using the same methods as described above. In particular, for methods of analyzing the three-dimensional structure of IgG molecules using electron tomography, please refer to Zhang et al., Sci. Rep. 5:9803 (2015), etc. Electron tomography makes it possible to represent the frequency of occurrence of possible structures of the molecule being measured in a histogram, so structural changes such as a decrease in domain mobility can be evaluated in terms of distribution. For example, if 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 indicates a decrease in the mobility of the two domains. The activity exerted by the interaction of two antigen molecules can be evaluated by selecting an appropriate known activity measurement system depending on the type of antigen molecule. The effect on protease cleavage can be evaluated using methods known to those skilled in the art, or by methods described in the examples below.

[0197] D. Pharmaceutical preparations (pharmaceutical compositions) The pharmaceutical formulations of antigen-binding molecules described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing antigen-binding molecules of 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 the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and Examples of pharmaceutically acceptable carriers herein include: m-cresol, etc.; low molecular weight (less than approximately 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 nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP) (e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Specific exemplary sHASEGPs and their uses (including rHuPH20) are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0198] An exemplary lyophilized antigen-binding molecule formulation is described in U.S. Patent No. 6,267,958. Aqueous aqueous antigen-binding molecule formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0199] The formulations described herein may contain one or more active ingredients if necessary for the specific indication being treated. Preferably, these active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

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

[0201] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing antigen-binding molecules, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0202] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane. [Examples]

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

[0204] [Example 1] Concept of Fab-crosslinked antibody Agonist antibodies possess superior characteristics compared to natural ligands and their fusion proteins in terms of stability, pharmacokinetics, and manufacturing methods, and are being developed as pharmaceuticals. However, obtaining agonist antibodies with potent activity compared to simple conjugating or neutralizing antibodies is generally difficult, and methods to overcome this challenge are needed. The properties required for agonist antibodies are thought to depend on the type of ligand. For agonist antibodies against the TNF receptor superfamily, such as Death receptor (DR), OX40, 4-1BB, and CD40, it has been reported that the activation of agonist antibodies is enhanced by the multimerization of both the antibody and the ligand. Methods to enhance this effect include the use of natural ligands, cross-linking with anti-Fc antibodies, cross-linking via FcγRs, multimerization of the antibody-binding domain, and multimerization via antibody Fc, all of which have been reported to enhance agonist activity. Furthermore, it is known that adjusting the distance of the antigen-binding site using the antibody's Fab structure or scFv, without relying on multimerization, can also lead to enhanced agonist activity depending on the type of antigen. As another approach, examples of bispecific antibodies capable of binding to different epitopes within the same antigen have been reported as agonist antibodies against cytokine receptors. Furthermore, methods have been reported to similarly crosslink two types of Fab 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 to achieve this has been reported. Therefore, the inventors developed a method to crosslink Fabs with minimal mutation introduction, demonstrated that this actually enhances agonist activity, and completed this as an invention. An example of this embodiment is shown in Figure 1.

[0205] [Example 2] Preparation of an expression vector for the modified antibody and expression and purification of the modified antibody The antibody gene inserted into the animal cell expression vector was subjected to amino acid residue sequence substitution using PCR or the Infusion Advantage PCR cloning kit (TAKARA) or other methods known to those skilled in the art, thereby constructing a modified antibody expression vector. The nucleotide sequence of the obtained 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 obtained 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 using the extinction coefficient obtained from the obtained value by the PACE method (Protein Science 1995; 4: 2411-2423). The amount of the modified antibody aggregate was measured using HPLC with Agilent 1260 Infinity® (Agilent Technologies) and a G3000SW gel filtration chromatography column. XL The analysis was performed using (TOSOH) by methods known to those skilled in the art. 10 μL of purified antibody at a concentration of 0.1 mg / mL was injected. The antibodies prepared using this method (anti-CD3ε antibody, anti-CD28 antibody, and anti-CD3ε × anti-CD28 bispecific antibody) are shown in Table 1.

[0206] [Table 1] HH: The EU numbering at position 191 in the two constant regions of the H chain has been changed to Cys. LL: The EU numbering at position 126 in the two L chain constant regions has been changed to Cys. HL, LH: The EU numbering at position 191 in one H chain constant region is changed to Cys, and The EU numbering at position 126 in one L chain constant region was changed to Cys.

[0207] [Example 3] Preparation of bispecific antibodies The purified antibody was dialyzed to TBS (WAKO) buffer to adjust the concentration to 1 mg / mL. Additionally, 250 mM 2-MEA (SIGMA) was prepared as a 10× reaction buffer. Equal volumes of the two homodimeric antibodies prepared in Example 2 were mixed, and 1 / 10 of the volume of the 10× reaction buffer was added and mixed. The mixture was then allowed to stand at 37°C for 90 minutes. After the reaction, the solution was dialyzed to TBS to obtain a bispecific antibody solution in which the two antibodies had heterodimerized. The antibody concentration was measured using the method described above and used for 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 the flask and washed with Assay Buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% MEM Non Essential Amino Acids (Invitrogen), 1 mM Sodium Pyrubate (Invitrogen)). After washing, the cells were placed in Assay Buffer in a 3 × 10⁶ solution. 6 The cells were suspended to a concentration of cells / mL. This cell suspension was used as the Jurkat cells solution for subsequent experiments.

[0209] Example 4-2 Preparation of luminescent 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. This luminescent reagent solution was then 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 above-mentioned Jurkat cells were transformed with a luciferase reporter gene having an NFAT response element, and when stimulated with an anti-TCR / CD3 antibody, activation of the NFAT pathway via an intracellular signal occurred, and expression of luciferase was induced. 10 μL each (3 x 10 4 cells / well) of the Jurkat cells solution prepared above was added to each well of a 384 well Flat bottom white plate. Next, 20 μL each of the antibody solutions prepared at each concentration (150, 15, 1.5, 0.15, 0.015, 0.0015, 0.00015, 0.000015 nM) was added to each well. The plate was allowed to stand in a 37°C, 5% carbon dioxide incubator for 24 hours. Thereafter, the luminescence reagent solution was thawed and 30 μL each was added to each well, and the plate was allowed to stand at room temperature for 10 minutes. The luminescence of luciferase in each well of the plate was measured with a luminometer. As a result, as shown in FIGS. 2 and 3, the modified molecule in which the Fab-Fab regions of the anti-CD3ε antibody were linked by an additional disulfide bond showed a variation in signal transduction via CD3 as compared with the native molecule (the molecule before modification). Further, as shown in FIGS. 4 and 5, even in the modified molecule in which the Fab-Fab regions of the bispecific antibody consisting of an anti-CD3ε antibody and an anti-CD28 antibody were linked by an additional disulfide bond, the signal transduction via CD3 and / or CD28 varied greatly as compared with the native molecule. From the above results, it was considered that by introducing the modification in the present invention, it is possible to enhance or attenuate the agonist activity of an antigen-binding molecule such as an antibody.

[0211] 〔Example 5〕Evaluation of antibodies with cysteine substituted at various positions in the heavy chain Example 5-1 Evaluation of antibodies in which cysteine ​​is substituted at various positions in the heavy chain. Among the variable region and the constant region 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, any amino acid residue structurally exposed on the surface was considered to be substituted with cysteine. The amino acid residues within the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) were replaced with cysteine ​​to create modified MRA heavy chain variable regions as shown in Table 2. These modified MRA heavy chain variable regions were each linked to the MRA heavy chain constant region (G1T4, SEQ ID NO: 18) to create MRA heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, amino acid residues within the MRA heavy chain constant region (G1T4, SEQ ID NO: 18) were substituted with cysteine ​​to create modified MRA heavy chain constant region variants shown in Table 3. These modified MRA heavy chain constant region variants were linked to the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) to create MRA heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to the art. The MRA heavy chain variants and MRA light chains prepared as described above were combined to produce the MRA variants shown in Table 4. These variants were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0212] [Table 2] TIFF2026063391000005.tif146170

[0213] [Table 3] TIFF2026063391000007.tif234170TIFF2026063391000008.tif98170

[0214] [Table 4] TIFF2026063391000010.tif234170TIFF2026063391000011.tif234170TIFF2026063391000012.tif214170

[0215] Example 5-2 Evaluation of Fabogenesis by protease of antibodies in which cysteine ​​is substituted at various positions in the heavy chain. We investigated whether the MRA variant prepared in Example 5-1 acquired protease resistance and thus inhibited fragmentation by using a protease that cleaves the heavy chain hinge region of the antibody and performs Fab fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease, and the mixture was reacted with 2 ng / μL of protease, 100 μg / mL of antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS at 35°C for 2 hours, or with 2 ng / μL of protease, 20 μg / mL of antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS at 35°C for 1 hour, followed by 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). The results are shown in Figures 6 to 13. In the MRA after Lys-C treatment, the IgG band around 150 kDa disappeared due to cleavage of the heavy chain hinge region, and a Fab band appeared around 50 kDa. On the other hand, in the modified MRA prepared in Example 5-1, some modified versions showed a Fab dimer band around 96 kDa after protease treatment, and others 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 percentage of the band area of ​​undigested IgG and generated Fab dimers was calculated. The calculated percentages of each band are shown in Table 5.

[0216] [Table 5] TIFF2026063391000014.tif234170TIFF2026063391000015.tif234170TIFF2026063391000016.tif195170

[0217] These results confirm that in the MRA variants shown in Table 6, the substitution of cysteine ​​in the heavy chain variable or heavy chain constant region improved the protease resistance of the heavy chain hinge region. Alternatively, it was suggested that a Fab dimer was formed by covalent bonding between Fab molecules.

[0218] [Table 6] TIFF2026063391000018.tif92170

[0219] [Example 6] Evaluation of antibodies in which cysteine ​​is substituted at various positions in the light chain. Example 6-1 Evaluation of antibodies in which cysteine ​​was substituted at various positions in the light chain. We investigated the substitution of any structurally surface-exposed amino acid residue with cysteine ​​in 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. The amino acid residues within the MRA light chain variable region (MRAL, SEQ ID NO: 19) were replaced with cysteine ​​to create modified MRA light chain variable regions as shown in Table 7. These modified MRA light chain variable regions were each linked to the MRA light chain constant region (k0, SEQ ID NO: 20) to create MRA light chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, amino acid residues within the MRA light chain constant region (k0, SEQ ID NO: 20) were substituted with cysteine ​​to create modified MRA light chain constant region variants shown in Table 8. These modified MRA light chain constant region variants were then linked to the MRA light chain variable region (MRAL, SEQ ID NO: 19) to create 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 and MRA heavy chains prepared as described above were combined to produce the MRA variants shown in Table 9. These variants were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0220] Table 7 TIFF2026063391000020.tif190170

[0221] Table 8 TIFF2026063391000022.tif234170TIFF2026063391000023.tif176170

[0222] Table 9 TIFF2026063391000025.tif234170TIFF2026063391000026.tif234170TIFF2026063391000027.tif234170TIFF2026063391000028.tif104170

[0223] Example 6-2 Evaluation of Fabogenesis by protease of antibodies in which cysteine ​​is substituted at various positions in the light chain. We investigated whether the MRA variant prepared in Example 6-1 acquired protease resistance and thus inhibited fragmentation by using a protease that cleaves the heavy chain hinge region of the antibody and performs Fab fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease, and the mixture was reacted with 2 ng / μL of protease, 100 μg / mL of antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS at 35°C for 2 hours, or with 2 ng / μL of protease, 20 μg / mL of antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS at 35°C for 1 hour, followed by 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). The results are shown in Figures 14 to 23. In the MRA after Lys-C treatment, the IgG band around 150 kDa disappeared due to cleavage of the heavy chain hinge region, and a Fab band appeared around 50 kDa. On the other hand, in the modified MRA prepared in Example 6-1, some modified versions showed a Fab dimer band around 96 kDa after protease treatment, and others 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 percentage of the band area of ​​undigested IgG and generated Fab dimers was calculated. The calculated percentages of each band are shown in Table 10.

[0224] [Table 10] TIFF2026063391000030.tif234170TIFF2026063391000031.tif234170TIFF2026063391000032.tif234170TIFF2026063391000033.tif91170

[0225] These results confirm that in the MRA variants shown in Table 11, the substitution of cysteine ​​in the light chain variable region or the light chain constant region improved the protease resistance of the heavy chain hinge region. Alternatively, it was suggested that a Fab dimer was formed by covalent bonding between Fab molecules.

[0226] [Table 11]

[0227] [Example 7] Verification of evaluation method for cysteine-substituted antibodies Example 7-1 Preparation of antibodies in which cysteine ​​is substituted into the light chain. A modified version of the MRA light chain constant region, k0.K126C (sequence number 231), was created by substituting cysteine ​​at the 126th amino acid residue in the constant region (k0, sequence number 20) of the light chain (heavy chain: MRAH-G1T4 (sequence number 15), light chain: MRAL-k0 (sequence number 16)) with the Kabat numbering. This modified MRA light chain constant region was ligated with the variable region (MRAL, sequence number 19) to create 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 and MRA heavy chain prepared above were combined to produce the MRA variant MRAL-k0.K126C (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain variable region: MRAL (SEQ ID NO: 19), light chain constant region: k0.K126C (SEQ ID NO: 231)). This variant was expressed transiently using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and then purified using a method known to those skilled in the art with protein A.

[0228] Example 7-2 Evaluation of capillary electrophoresis of antibodies with cysteine ​​substituted in the light chain using protease We investigated whether the MRA light chain variant prepared in Example 7-1 acquired protease resistance and thus inhibited fragmentation by using a protease that cleaves the heavy chain hinge region of the antibody and performs Fab fragmentation. Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001) was used as the protease, and the mixture was reacted for 2 hours under the conditions of 0.1, 0.4, 1.6, and 6.4 ng / μL of protease, 100 μg / mL of antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS, at 35°C, followed by non-reducing capillary electrophoresis. Capillary electrophoresis was performed using Wes (Protein Simple), and detection was performed using either 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. In MRA after Lys-C treatment, when detecting anti-kappa chain antibodies, the band around 150 kDa disappeared and a new band appeared around 50 kDa, and a small band also appeared at 113 kDa at low Lys-C concentrations. Similarly, when detecting anti-human Fc antibodies, the band around 150 kDa disappeared and a new band appeared around 61 kDa, and a small band also appeared at 113 kDa at low Lys-C concentrations. On the other hand, in the modified MRA prepared in Example 7-1, the band around 150 kDa hardly disappeared, and a new band appeared around 96 kDa. In the detection of anti-human Fc antibodies, the band around 150 kDa hardly disappeared, a new band appeared around 61 kDa, and a small band also appeared at 113 kDa at low Lys-C concentrations. Based on these results, as shown in Figure 25, the band around 150 kDa was thought to be IgG, the band around 113 kDa was thought to be the one-arm form with one cleavage of the heavy chain hinge, the band around 96 kDa was thought to be the Fab dimer, the band around 61 kDa was thought to be Fc, and the band around 50 kDa was thought to be Fab.

[0229] [Example 8] Evaluation of antibodies in which cysteine ​​substitutions have been introduced at various positions in IgG1. Example 8-1 Production of antibodies in which cysteine ​​substitutions are introduced at various positions in IgG1 We investigated the substitution of any structurally surface-exposed amino acid residues in 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. Modified versions of the MRA-IgG1 heavy chain variable region (MRAH, SEQ ID NO: 17) were created by substituting cysteine ​​into the amino acid residues within the MRA-IgG1 heavy chain variable region (MRAH, SEQ ID NO: 17), as shown in Table 12. These modified versions of the MRA-IgG1 heavy chain variable region were each linked to the MRA-IgG1 heavy chain constant region (G1T4, SEQ ID NO: 18) to create MRA-IgG1 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, modified versions of the MRA-IgG1 heavy chain constant region (G1T4, SEQ ID NO: 18) were created by substituting cysteine ​​into the amino acid residues within the MRA-IgG1 heavy chain constant region (G1T4, SEQ ID NO: 18), as shown in Table 13. These modified versions of the MRA-IgG1 heavy chain constant region were each linked to the MRA-IgG1 heavy chain variable region (MRAH, SEQ ID NO: 17), as created MRA-IgG1 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art.

[0230] [Table 12] TIFF2026063391000036.tif72170

[0231] [Table 13] TIFF2026063391000038.tif229170

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

[0233] [Table 14] TIFF2026063391000040.tif110170

[0234] [Table 15] TIFF2026063391000042.tif229170TIFF2026063391000043.tif66170

[0235] The MRA-IgG1 heavy chain variants and MRA-IgG1 light chain variants prepared as described above, or the MRA-IgG1 heavy chain variants and MRA-IgG1 light chain variants shown in Tables 16 and 17, were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0236] [Table 16] TIFF2026063391000045.tif229170TIFF2026063391000046.tif229170TIFF2026063391000047.tif66170

[0237] [Table 17] TIFF2026063391000049.tif229170TIFF2026063391000050.tif229170TIFF2026063391000051.tif169170

[0238] Example 8-2 Evaluation of electrophoretic mobility in polyacrylamide gel of antibodies in which cysteine ​​substitutions have been introduced at various positions of IgG1. Non-reducing SDS-PAGE was used to verify whether the MRA-IgG1 variant prepared in Example 8-1 exhibited different electrophoretic mobility from MRA-IgG1. Sample Buffer Solution (2ME-) (x4) (Wako; 198-13282) was used to prepare the electrophoretic samples. The samples were treated at a concentration of 50 μg / mL and 70°C for 10 minutes, and then subjected to non-reducing SDS-PAGE. For non-reducing SDS-PAGE, electrophoresis was performed at 125 V for 90 minutes using a 4% SDS-PAGE mini 15well 1.0 mm 15well (TEFCO; Cat#01-052-6). The gel was then stained with CBB stain, and gel images were acquired using 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 (bands in a lower molecular weight range than MRA-IgG1), HMW (bands in a higher molecular weight range than MRA-IgG1), and Faint (bands were blurry and indistinguishable). For the MRA-IgG1 variants classified as Double, one of the two bands showed the same electrophoretic mobility as MRA-IgG1, while the other band showed slightly faster or slightly slower electrophoretic mobility. Therefore, for the MRA-IgG1 variants classified as Double, the percentage of bands showing different electrophoretic mobility from MRA-IgG1 (percentage of new bands (%)) was also calculated. Tables 18 and 19 show the grouping of band patterns and the calculation of band percentages for MRA-IgG1 heavy chain variants and MRA-IgG1 light chain variants, respectively. Table 20 shows the variants classified into the Double and Triple groups from Tables 18 and 19. In these variants, structural changes such as cross-linking between Fabs occur due to cysteine ​​substitution, and it is highly likely that this has resulted in changes in electrophoretic mobility. Although MRAL.K107C-IgG1 is listed as "no data" in Table 19, the cysteine ​​substitution site in this variant, Kabat numbering position 107, is a hinge region where a residue is structurally exposed to the surface. Therefore, it is highly likely that structural changes such as cross-linking between Fabs occur in this variant due to cysteine ​​substitution, resulting in changes in electrophoretic mobility.

[0239] [Table 18] TIFF2026063391000053.tif229170TIFF2026063391000054.tif229170TIFF2026063391000055.tif66170

[0240] [Table 19] TIFF2026063391000057.tif229170TIFF2026063391000058.tif229170TIFF2026063391000059.tif164170

[0241] [Table 20] TIFF2026063391000061.tif55170

[0242] [Example 9] Evaluation of antibodies in which cysteine ​​substitutions have been introduced at various positions in IgG4. Example 9-1 Production of antibodies in which cysteine ​​substitutions are introduced at various positions of IgG4 We investigated the substitution of any structurally surface-exposed amino acid residues with cysteine ​​in the heavy chain and light chain 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. Modified versions of the MRA-IgG4 heavy chain variable region (MRAH, SEQ ID NO: 17) were created by substituting cysteine ​​into the MRA-IgG4 heavy chain variable region (MRAH, SEQ ID NO: 17) as shown in Table 21. These modified versions of the MRA-IgG4 heavy chain variable region were each linked to the MRA-IgG4 heavy chain constant region (G4T1, SEQ ID NO: 311) to create MRA-IgG4 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, modified versions of the MRA-IgG4 heavy chain constant region (G4T1, SEQ ID NO: 311) were created by substituting cysteine ​​into the MRA-IgG4 heavy chain constant region (G4T1, SEQ ID NO: 311) as shown in Table 22. These modified versions of the MRA-IgG4 heavy chain constant region were each linked to the MRA-IgG4 heavy chain variable region (MRAH, SEQ ID NO: 17) to create MRA-IgG4 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art.

[0243] [Table 21] TIFF2026063391000063.tif77170

[0244] [Table 22] TIFF2026063391000065.tif224170

[0245] The MRA-IgG4 heavy chain variants prepared above and the MRA-IgG4 light chain, or the MRA-IgG4 heavy chain and the MRA-IgG4 light chain variant prepared in Example 8-1, were combined and expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0246] [Table 23] TIFF2026063391000067.tif229170TIFF2026063391000068.tif229170TIFF2026063391000069.tif55170

[0247] [Table 24] TIFF2026063391000071.tif229170TIFF2026063391000072.tif229170TIFF2026063391000073.tif169170

[0248] Example 9-2 Evaluation of electrophoretic mobility in polyacrylamide gel of antibodies in which cysteine ​​substitutions have been introduced at various positions of IgG4. Similar to Example 8-2, non-reducing SDS-PAGE was performed using the MRA-IgG4 modified compound prepared in Example 9-1 to acquire gel images and quantify the bands. 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 (bands in a lower molecular weight range than MRA-IgG4), HMW (bands in a higher molecular weight range than MRA-IgG4), and Faint (bands were blurry and indistinguishable). For the MRA-IgG4 variants classified as Double, one of the two bands showed the same electrophoretic mobility as MRA-IgG4, while the other band showed slightly faster or slightly slower electrophoretic mobility. Therefore, for the MRA-IgG4 variants classified as Double, the percentage of bands showing different electrophoretic mobility from MRA-IgG4 (novel band percentage (%)) was also calculated. Tables 25 and 26 show the grouping of band patterns and the calculation of band percentages for MRA-IgG4 heavy chain variants and MRA-IgG4 light chain variants, respectively. Table 27 shows the variants classified into the Double and Triple groups from Tables 25 and 26. In these variants, structural changes such as cross-linking between Fabs occur due to cysteine ​​substitution, and it is highly likely that this has resulted in changes in electrophoretic mobility. Although MRAL.K107C-IgG4 is listed as "no data" in Table 26, the cysteine ​​substitution site in this variant, Kabat numbering position 107, is a hinge region where a residue is structurally exposed to the surface. Therefore, it is highly likely that structural changes such as cross-linking between Fabs occur in this variant due to cysteine ​​substitution, resulting in changes in electrophoretic mobility.

[0249] [Table 25] TIFF2026063391000075.tif229170TIFF2026063391000076.tif229170TIFF2026063391000077.tif55170

[0250] [Table 26] TIFF2026063391000079.tif229170TIFF2026063391000080.tif229170TIFF2026063391000081.tif164170

[0251] [Table 27] TIFF2026063391000083.tif153170

[0252] [Example 10] Evaluation of antibodies in which cysteine ​​substitutions have been introduced at various positions in IgG2. Example 10-1 Production of antibodies in which cysteine ​​substitutions are introduced at various positions in IgG2 We investigated the substitution of any structurally exposed amino acid residues on the surface of the heavy chain and light chain 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. Modified versions of the MRA-IgG2 heavy chain variable region (MRAH, SEQ ID NO: 17) were created by substituting cysteine ​​into the amino acid residues within the MRA-IgG2 heavy chain variable region (MRAH, SEQ ID NO: 17), as shown in Table 28. These modified versions of the MRA-IgG2 heavy chain variable region were each linked to the MRA-IgG2 heavy chain constant region (G2d, SEQ ID NO: 313) to create MRA-IgG2 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, modified versions of the MRA-IgG2 heavy chain constant region (G2d, SEQ ID NO: 313) were created by substituting cysteine ​​into the amino acid residues within the MRA-IgG2 heavy chain constant region (G2d, SEQ ID NO: 313), as shown in Table 29. These modified versions of the MRA-IgG2 heavy chain constant region were each linked to the MRA-IgG2 heavy chain variable region (MRAH, SEQ ID NO: 17), as created MRA-IgG2 heavy chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to the art.

[0253] [Table 28] TIFF2026063391000085.tif77170

[0254] [Table 29] TIFF2026063391000087.tif224170

[0255] The MRA-IgG2 heavy chain variants prepared as described above, along with the MRA-IgG2 light chain, or the MRA-IgG2 heavy chain and the MRA-IgG2 light chain variant prepared in Example 8-1, were combined and expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0256] [Table 30] TIFF2026063391000089.tif229170TIFF2026063391000090.tif229170TIFF2026063391000091.tif55170

[0257] [Table 31] TIFF2026063391000093.tif229170TIFF2026063391000094.tif229170TIFF2026063391000095.tif169170

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

[0259] [Table 32] TIFF2026063391000097.tif229170TIFF2026063391000098.tif229170TIFF2026063391000099.tif50170

[0260] [Table 33] TIFF2026063391000101.tif229170TIFF2026063391000102.tif229170TIFF2026063391000103.tif164170

[0261] [Table 34] TIFF2026063391000105.tif229170TIFF2026063391000106.tif229170TIFF2026063391000107.tif229170TIFF2026063391000108.tif224170

[0262] [Example 11] Evaluation of antibodies in which cysteine ​​substitutions have been introduced at various positions in the Lambda chain. Example 11-1 Production of antibodies in which cysteine ​​substitutions are introduced at various positions in the Lambda chain. We investigated the substitution of any structurally exposed amino acid residues 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 within the G7-IgG1 light chain variable region (G7L, SEQ ID NO: 317) were replaced with cysteine ​​to create modified G7-IgG1 light chains shown in Table 35. These modified G7-IgG1 light chain variable regions were each ligated to the G7-IgG1 light chain constant region (LT0, SEQ ID NO: 318) to create modified G7-IgG1 light chains, and expression vectors encoding the corresponding genes were prepared by methods known to the art. Furthermore, the amino acid residues within the G7-IgG1 light chain constant region (LT0, SEQ ID NO: 318) were replaced with cysteine ​​to create modified G7-IgG1 light chains shown in Table 36. These modified G7-IgG1 heavy chain constant regions were each ligated to the G7-IgG1 light chain variable region (G7L, SEQ ID NO: 317) to create modified G7-IgG1 light chains, and expression vectors encoding the corresponding genes were prepared by methods known to the art.

[0263] [Table 35] TIFF2026063391000110.tif115170

[0264] [Table 36] TIFF2026063391000112.tif229170TIFF2026063391000113.tif55170

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

[0266] [Table 37] TIFF2026063391000115.tif229170TIFF2026063391000116.tif229170TIFF2026063391000117.tif153170

[0267] Example 11-2 Evaluation of electrophoretic mobility in polyacrylamide gel of antibodies with cysteine ​​substitutions introduced at various positions in the Lambda chain. Similar to Example 8-2, non-reducing SDS-PAGE was performed using the G7-IgG1 modified compound prepared in Example 11-1 to acquire gel images and quantify the bands. Based on the acquired gel images, each G7-IgG1 variant was classified into seven groups according to its band pattern: 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 (bands in a lower molecular weight range than G7-IgG1), HMW (bands in a higher molecular weight range than G7-IgG1), and Faint (bands are blurry and indistinguishable). For G7-IgG1 variants classified as Double, one of the two bands showed the same electrophoretic mobility as G7-IgG1, while the other band showed slightly faster or slightly slower electrophoretic mobility. Therefore, for G7-IgG1 variants classified as Double, the percentage of bands showing different electrophoretic mobility from G7-IgG1 (percentage of new bands (%)) was also calculated. Table 38 shows the grouping of band patterns and the calculation of band percentages for G7-IgG1 light chain variants. Table 39 shows the variants classified into the Double and Triple groups from Table 38. In these variants, structural changes such as crosslinking between Fabs occur due to cysteine ​​substitution, and it is highly likely that this resulted in a change in electrophoretic mobility. In this example, the variant in which the amino acid residue at position 107a (Kabat numbering) is substituted with cysteine ​​has not been evaluated. However, position 107a (Kabat numbering) is a hinge region where a residue is structurally exposed on the surface. Therefore, it is highly likely that structural changes such as crosslinking between Fabs occur in this variant as well, resulting in a change in electrophoretic mobility.

[0268] [Table 38] TIFF2026063391000119.tif229170TIFF2026063391000120.tif229170TIFF2026063391000121.tif148170

[0269] [Table 39]

[0270] [Example 12] Evaluation of antibodies in which cysteine ​​substitutions have been introduced at various positions in VHH Example 12-1 Preparation of antibodies with cysteine ​​substitutions introduced at various positions in VHH We created IL6R90-Fc (IL6R90-G1T3dCH1dC, SEQ ID NO: 321) by fusing IL6R90 (SEQ ID NO: 319), a neutralizing VHH for human IL6R, to the Fc region (G1T3dCH1dC, SEQ ID NO: 320) of human IgG1, and investigated the possibility of substituting any structurally exposed amino acid residue in the IL6R90 region with cysteine. The amino acid residues within the IL6R90 region were replaced with cysteine, and expression vectors encoding modified IL6R90-Fc VHH region genes, as shown in Table 40, were prepared by methods known to those skilled in the art. These modified IL6R90-Fc VHH region genes were each ligated to the Fc region of human IgG1 (G1T3dCH1dC, SEQ ID NO: 320) to create modified IL6R90-Fc genes, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art.

[0271] [Table 40] The IL6R90-Fc variants shown in Table 41, prepared as described above, were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and purified using methods known to those skilled in the art with Protein A.

[0272] [Table 41] TIFF2026063391000126.tif251170

[0273] Example 12-2 Evaluation of electrophoretic mobility in polyacrylamide gel of antibodies with cysteine ​​substitutions introduced at various positions in VHH Non-reducing SDS-PAGE was used to verify whether the IL6R90-Fc modified molecule prepared in Example 12-1 exhibited different electrophoretic mobility from IL6R90-Fc. Sample preparation involved using Sample Buffer Solution (2ME-) (x4) (Wako; 198-13282), which was treated at a sample concentration of 50 μg / mL and 70°C for 10 minutes before being subjected to non-reducing SDS-PAGE. For non-reducing SDS-PAGE, electrophoresis was performed at 200 V for 2.5 hours using Mini-PROTEAN TGX Precast gel 4-20% 15well (BIORAD; 456-1096). Subsequently, the gel was stained with CBB stain, and gel images were acquired using ChemiDocTouchMP (BIORAD), while band quantification was performed using Image Lab (BIORAD). Based on the acquired gel images, each IL6R90-Fc variant was classified into seven groups according to its band pattern: 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 (bands in a lower molecular weight range than IL6R90-Fc), HMW (bands in a higher molecular weight range than IL6R90-Fc), and Faint (bands were blurry and indistinguishable). For the IL6R90-Fc variants classified as Double, one of the two bands showed the same electrophoretic mobility as IL6R90-Fc, while the other band showed slightly faster or slightly slower electrophoretic mobility. Therefore, for the IL6R90-Fc variants classified as Double, the percentage of bands showing different electrophoretic mobility from IL6R90-Fc (percentage of new bands (%)) was also calculated. The grouping of band patterns and the calculation results of band percentages for the IL6R90-Fc variants are shown in Table 42. Table 43 shows the variants classified into Double and Triple groups from Table 42. In these variants, it is highly likely that structural changes such as crosslinking between VHH molecules occurred due to cysteine ​​substitution, resulting in changes in electrophoretic mobility.

[0274] [Table 42] TIFF2026063391000128.tif251170

[0275] [Table 43]

[0276] [Example 13] Evaluation of CD3 agonist activity of an antibody with cysteine ​​substitution introduced into the Fab. Example 13-1 Preparation of antibodies with cysteine ​​substitution introduced into the constant region We investigated the substitution of any structurally exposed amino acid residue with cysteine ​​in OKT3 (heavy chain: OKT3VH0000-G1T4 (SEQ ID NO: 1007), light chain: OKT3VL0000-KT0 (SEQ ID NO: 1008)), an agonist antibody against human CD3. The amino acid residues within the constant region of the OKT3 heavy chain (G1T4, SEQ ID NO: 1009) were replaced with cysteine ​​to create modified OKT3 heavy chain regions as shown in Table 44. These modified OKT3 heavy chain regions were each linked to the variable region of the OKT3 heavy chain (OKT3VH0000, SEQ ID NO: 1010) to create 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 within the constant region of the OKT3 light chain (KT0, SEQ ID NO: 1011) were replaced with cysteine ​​to create modified OKT3 light chain regions as shown in Table 45. These modified OKT3 light chain regions were each linked to the variable region of the OKT3 light chain (OKT3VL0000, SEQ ID NO: 1012) to create 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 and light chain variants prepared above were combined with the OKT3 light chain and OKT3 heavy chain, respectively. The OKT3 variants shown in Table 46 were expressed transiently 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. In addition, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was prepared in the same manner as a negative control.

[0281] [Table 46]

[0282] Example 13-2 Preparation of Jurkat cell solution Jurkat cells (TCR / CD3 Effector Cells (NFAT), Promega) were collected from the flask and washed with Assay Buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% MEM Non Essential Amino Acids (Invitrogen), 1 mM Sodium Pyrubate (Invitrogen)). After washing, the cells were placed in Assay Buffer in a 3 × 10⁶ solution. 6 The cells were suspended to a concentration of cells / mL. This cell suspension was used as the Jurkat cell solution for subsequent experiments.

[0283] Example 13-3 Preparation of luminescent 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 luminescence reagent solution was then used in subsequent experiments.

[0284] Example 13-4 Evaluation of T cell activation of antibodies with cysteine ​​substitution introduced into the constant region T cell activation induced by agonist signaling was evaluated by the fold change of luciferase luminescence. The Jurkat cells described above were transformed with a luciferase reporter gene containing an NFAT response element. Upon stimulation with an anti-TCR / CD3 antibody, the NFAT pathway was activated via intracellular signaling, 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 were added to each well. 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 to stand in a 37°C, 5% carbon dioxide incubator for 24 hours. After this, the luminescence reagent solution was melted and 30 μL was added to each well, and the plate was left to stand at room temperature for 10 minutes. The luminescence of luciferase in each well of the plate was measured using a luminometer. The amount of luminescence (fold) was calculated by dividing the amount of luminescence in the antibody-added wells by the amount of luminescence in the antibody-free wells. As a result, as shown in Figure 26, among the OKT3 variants in which cysteine ​​substitution was introduced into the constant region, several variants significantly increased the T cell activation state compared to OKT3. This indicates that there are multiple cysteine ​​modifications that can crosslink Fab cells and enhance CD3 agonist activity.

[0285] [Example 14] Evaluation of CD3 agonist activity of antibodies with different cysteine ​​substitutions introduced from two Fabs. Example 14-1 Preparation of antibodies with heterogeneous cysteine ​​substitutions introduced into the constant region We investigated the substitution of any structurally exposed amino acid residue with cysteine ​​in OKT3 (heavy chain: OKT3VH0000-G1T4 (SEQ ID NO: 1007), light chain: OKT3VL0000-KT0 (SEQ ID NO: 1008)), an agonist antibody against human CD3. Modified OKT3 heavy chain constant region 1 (G1T4k, SEQ ID NO: 1015) was prepared by substituting an amino acid residue with cysteine, as shown in Table 47. These modified OKT3 heavy chain constant region 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 the art. Similarly, modified OKT3 heavy chain constant region 2 (G1T4h, SEQ ID NO: 1016) was prepared by substituting an amino acid residue with cysteine, as shown in Table 48. These modified OKT3 heavy chain constant region 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 the art. Furthermore, in the heavy chain steady regions 1 and 2 here, a Knobs-into-Holes (KiH) modification has been introduced to the CH3 region to promote heterodimerization.

[0286] [Table 47]

[0287] [Table 48]

[0288] The OKT3 heavy chain variants 1 and 2 prepared above were combined with the OKT3 light chain to produce the OKT3 variants shown in Table 49. These were expressed transiently 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. In addition, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was prepared in the same manner as a negative control.

[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 luminescent reagent solution A luminescent 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 heterocysteine ​​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 OKT3 variant in which different cysteine ​​substitutions were introduced between two constant regions within the antibody significantly increased T cell activation compared to OKT3. This indicates that even with different cysteine ​​substitutions between Fabs, cross-linking between Fabs can be achieved, enhancing CD3 agonist activity.

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

[0294] [Table 50]

[0295] [Table 51]

[0296] The OKT3 heavy chain variants 1 and 2 prepared above were combined with the OKT3 light chain to produce the OKT3 variants shown in Table 52. These were expressed transiently 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. In addition, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was prepared in the same manner as a negative control.

[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 luminescent reagent solution A luminescent 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, the OKT3 variant in which a positively charged amino acid substitution was introduced in one constant region and a negatively charged amino acid substitution in the other constant region significantly increased the T cell activation state compared to OKT3. On the other hand, the OKT3 variant in which a positive or negatively charged amino acid substitution was introduced in one constant region and no modification was introduced in the other constant region hardly changed the T cell activation state compared to OKT3. This indicates that not only cysteine ​​substitutions but also charged amino acid substitutions can crosslink Fab cells via non-covalent bonds, thereby enhancing CD3 agonist activity.

[0301] [Example 16] Evaluation of CD3 agonist activity of an antibody in which the disulfide bond in the hinge region was removed and a cysteine ​​substitution was introduced within the Fab. Example 16-1 Preparation of an antibody by removing the disulfide bond in the hinge region and introducing a cysteine ​​substitution within the Fab. We investigated the removal of the disulfide bond in the hinge region of the heavy chain of the human CD3 agonist antibody OKT3 (heavy chain: OKT3VH0000-G1T4 (SEQ ID NO: 1007), light chain: OKT3VL0000-KT0 (SEQ ID NO: 1008)) and the substitution of structurally exposed amino acid residues with cysteine. Modified OKT3 heavy chain constant region (G1T4, SEQ ID NO: 1009) was prepared by substituting cysteine ​​with serine in the hinge region to create the OKT3 heavy chain constant region shown in Table 53. For these modified OKT3 heavy chain constant region, the amino acid residue at position 191 (EU numbering) was substituted with cysteine ​​to create the OKT3 heavy chain constant region shown in Table 54. These modified OKT3 heavy chain constant region were then linked to the OKT3 heavy chain variable region (OKT3VH0000, SEQ ID NO: 1010) to create OKT3 heavy chain modifiers, 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 heavy chain variants prepared above were combined with the OKT3 light chain to produce the OKT3 variants shown in Table 55. These were expressed transiently 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. In addition, the anti-KLH antibody IC17 (heavy chain: IC17HdK-G1T4 (SEQ ID NO: 1013), light chain: IC17L-k0 (SEQ ID NO: 1014)) was prepared in the same manner as a negative control.

[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 luminescent reagent solution A luminescent reagent solution was prepared in the same manner as in Example 13-3.

[0308] Example 16-4 Evaluation of T cell activation of an antibody in which the disulfide bond in the hinge region was removed and a cysteine ​​substitution was introduced within 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 OKT3 variant in which only the disulfide bond in the hinge region was removed showed a decrease in T cell activation status compared to OKT3, or hardly any change. On the other hand, the OKT3 variant in which the disulfide bond in the hinge region was removed and a cysteine ​​substitution was introduced in the constant region showed a significant increase in T cell...

Claims

1. An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, The antigen-binding domain includes a hinge region. The first and second antigen-binding domains contain antibody fragments that bind to a specific antigen. The first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more binding sites, and at least one of these binding sites is a covalent bond. At least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the hinge region. At least one of the bindings linking the two antigen-binding domains is, It 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. The amino acid residue in the CH1 region is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. Antigen-binding molecules.

2. The antigen-binding molecule according to claim 1, wherein the antibody fragment is one of Fab, Fab', or scFab.

3. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding domain includes an Fc region.

4. An antigen-binding molecule according to any one of claims 1 to 3, having the activity to control the interaction between two antigen molecules.

5. An antigen-binding molecule according to any one of claims 1 to 4, wherein resistance to protease cleavage is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule only in that the number of bonds between the two antigen-binding domains is one less.

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

7. A method for controlling the interaction between two antigen molecules, comprising the following: (a) To provide an antigen-binding molecule comprising two antigen-binding domains, wherein the antigen-binding domains comprise a hinge region, and the first and second antigen-binding domains comprise an antibody fragment that binds to a specific antigen. (b) Adding at least one bond to the antigen-binding molecule that links two antigen-binding domains together, 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 starting point for binding between antigen-binding domains is located within the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the hinge region. At least one of the bindings linking the two antigen-binding domains is, It 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. The amino acid residue in the CH1 region is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. and (c) The antigen-binding molecule prepared in (b) is brought into contact with the two antigen molecules.

8. A method for producing an antigen-binding molecule having activity to control the interaction between two antigen molecules, comprising the following: (a) To provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein the antigen-binding domain comprises a hinge region, and the first and second antigen-binding domains comprise an antibody fragment that binds to a specific antigen. (b) Introducing mutations into the nucleic acids encoding the two antigen-binding domains such that at least one bond is added linking the two antigen-binding domains, so that the first antigen-binding domain and the second antigen-binding domain are linked to each other by 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 starting point for binding between antigen-binding domains is located within the CH1 region and / or CL region of the antibody fragment, and at least one of the amino acid residues that serve as the starting point for binding between antigen-binding domains is located within the hinge region. At least one of the bindings linking the two antigen-binding domains is, It 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. The amino acid residue in the CH1 region is selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, and the amino acid residue in the CL region is selected from the group consisting of Kabat numbering positions 121, 122, 123, 124, 125, 126, 127, and 128. (c) Introducing the nucleic acids prepared in (b) into host cells, (d) Culturing host cells so that the two polypeptides are expressed, (e) To obtain an antigen-binding molecule which is a polypeptide comprising a first and a second antigen-binding domain, wherein the two antigen-binding domains are linked to each other by two or more binding sites.

Images