Antigen-binding molecule comprising two antigen-binding domains that are linked to each other, pharmaceutical composition, nucleic acid, vector, host cell, and methods for regulating the interaction between two antigen molecules, and for producing an antigen-binding molecule that has regulatory activity on the interaction between two antigen molecules.

Description

Antigen-binding molecule comprising two antigen-binding domains that are linked to each other, pharmaceutical composition, nucleic acid, vector, host cell, and methods for regulating the interaction between two antigen molecules, and for producing an antigen-binding molecule that has regulatory activity on the interaction between two antigen molecules. Separated from BR112021001693-7, filed on August 2, 2019. Technical Field

[001] The present invention relates to antigen-binding molecules containing a first antigen-binding domain and a second antigen-binding domain that are linked together, methods for producing such an antigen-binding molecule, methods for using such an antigen-binding molecule, and pharmaceutical compositions containing such an antigen-binding molecule. The present invention also relates to methods for increasing the resistance of an antigen-binding molecule to cleavage by protease. Antecedent Technique

[002] Antibodies are proteins that bind specifically to an antigen with high affinity. It is known that various molecules ranging from low molecular weight compounds to proteins can be antigens. Since the technique of producing monoclonal antibodies has been developed, antibody modification techniques have advanced, facilitating the production of antibodies that recognize a specific molecule. Now, antibody modification techniques are not only for modifying the proteins themselves, but have also expanded into a field that aims at adding new functions where conjugation with low molecular weight compounds is Petition 870260047205, dated 05 / 18 / 2026, page 10 / 2830 2 / 348 contemplated. For example, cysteine-engineered antibodies, which contain a free cysteine ​​amino acid in the heavy or light chain, are used as antibody-drug conjugates (ADCs) for medical purposes (PTL 1).

[003] Meanwhile, antibody modification techniques have contributed not only to the development of antibody engineering as tools for detecting, analyzing, and purifying proteins, but also to the development of protein engineering in general, such as improving the function of a non-antibody protein using an antibody molecule itself as a model protein.

[004] Antibodies are attracting attention as drugs because they are highly stable in blood plasma and have fewer side effects. Not only do antibodies bind to an antigen and exhibit agonistic or antagonistic effects, but they also induce cytotoxic activity mediated by effector cells (also referred to as effector functions), including ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and CDC (complement-dependent cytotoxicity). Taking advantage of these antibody functions, pharmaceuticals have been developed for cancer, immunological diseases, chronic diseases, infections, etc. (NPL 1).

[005] For example, pharmaceutical products that utilize an agonist antibody against a co-stimulatory molecule that promotes the activation of cytotoxic T cells have been developed as anticancer agents (NPL 2). Recently, it has been discovered that immune checkpoint inhibitor antibodies with antagonistic activity against co-inhibitory molecules are useful as anticancer agents. This discovery led to the launch of a series of antibody drugs that inhibit the interaction of CTLA4 / CD80 or PD-1 / PD-L1: Ipilimu Petition 870260047205, dated 05 / 18 / 2026, page 11 / 2830 3 / 348 mab, Nivolumab, Pembrolizumab and Atezolizumab (NPL 1).

[006] However, these antibodies sometimes do not sufficiently exert the expected effects in their original native IgG form. Therefore, second-generation antibody pharmaceuticals, in which the functions of the native IgG antibody have been artificially increased or added, or decreased or eliminated, depending on the purpose of use, have been developed. Second-generation antibody drugs include, for example, antibodies with increased or eliminated effector functions (NPL 3), antibodies that bind to an antigen in a pH-dependent manner (NPL 4), and antibodies that bind to two or more different antigens per molecule (antibodies that bind to two different antigens are generally referred to as bispecific antibodies (NPL 5)).

[007] Bispecific antibodies are expected to be more effective pharmaceutical products. For example, antibodies with increased antitumor activity that crosslink a cytotoxic T cell with a cancer cell by binding to a protein expressed on the T cell membrane as one antigen and to a cancer antigen as the other antigen have been developed (NPL 7, NPL 8 and PTL 2). Previously reported bispecific antibodies include molecules with two antibody Fab domains, each having a different sequence (common light chain bispecific antibodies and hybrid hybridomas), molecules with an additional antigen-binding site attached to the N- or C-terminus of the antibody (DVD-Ig and scFvIgG), molecules with a Fab domain that binds to two antigens (two-in-one IgG), molecules in which the calibrated loop regions of the CH3 domain have been designed to form novel antigen-binding sites (Fcab) (NPL 9), and molecules with tandem Fab-Fab (NPL 10).

[008] Meanwhile, antibodies with effector functions facilitate Petition 870260047205, dated 05 / 18 / 2026, page 12 / 2830 4 / 348 mind causes side effects, acting even on normal cells that express a target antigen at low levels. Thus, efforts have been made to enable antibody pharmaceuticals to exert their effector functions specifically in the target tissue. Examples reported above are antibodies whose binding activity changes after binding to a cellular metabolite (PTL 3), antibodies that become capable of binding to an antigen after protease cleavage (PTL 4), and a technology that regulates antibody-mediated crosslinking between chimeric antigen receptor T cells and cancer cells by the addition of a compound (ABT737) (NPL 11).

[009] Agonist antibodies can be difficult to obtain depending on the target. In particular, for membrane proteins such as G protein-coupled receptors, many different techniques have been developed (NPL 12). Thus, there is a demand for simple methods to increase the agonistic effect of antibodies on such targets. Known existing methods include, for example, a method for crosslinking an anti-DR4 (Death Receptor 4) or anti-DR5 (Death Receptor 5) antibody (NPL13), a method for multimerizing anti-DR5 (Death Receptor 5) antibody nanobodies (NPL 14), a method for converting an anti-thrombopoietin receptor antibody into a covalent diabody, sc(Fv)2 (NPL 15), a method for altering the IgG subclass of the anti-CD40 antibody (NPL 16), a method for hexamerizing an anti-CD20 antibody (NPL 17), and a method for producing a circular antibody-like molecule (PTL 5).Furthermore, reported methods using bispecific antibodies include, for example, a method using a combination of two appropriate antierythropoietin antibodies against different epitopes as a bispecific antibody (NPL 18), a method using a combination of an antibody for guide functions and an antibody for fun. Petition 870260047205, dated 05 / 18 / 2026, page 13 / 2830 5 / 348 effector actions such as a bispecific antibody (NPL 19), and a method of introducing Cys residues into multiple antibody fragments specific for different epitopes and their conjugation (NPL 20, NPL 21 and PTL 6). List of Citations Patent Literature

[0010]

[0011]

[0012]

[0013]

[0014]

[0015] [PTL 1] WO 2016 / 040856 [PTL 2] WO 2008 / 157379 [PTL 3] WO 2013 / 180200 [PTL 4] WO 2009 / 025846 [PTL 5] WO 2017 / 191101 [PTL 6] WO 2018 / 027204 Non-Patent Literature

[0016]

[0017] 9-19 [NPL 1] Nature Reviews Drug Discovery (2018) 17, 197-223 [NPL 2] Clinical and Experimental Immunology (2009) 157,

[0018] [NPL 3] Current Pharmaceutical Biotechnology (2016) 17, 1298-1314

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028] 1012 [NPL 4] Nature Biotechnology (2010) 28, 1203-1208 [NPL 5] MAbs (2012) 4, 182-197 [NPL 6] Nature Reviews Immunology (2010) 10, 301-316 [NPL 7] Sci Transl Med (2017) 9(410), eaal4291 [NPL 8] Blood (2011) 117(17): 4403-4404 [NPL 9] Protein Eng Des Sel (2010) 23(4), 289-297 [NPL 10] J Immunol (2016) 196(7): 3199-3211 [NPL 11] Nature Chemical Biology (2018) 14, 112-117 [NPL 12] Exp Mol Med (2016) 48(2): e207 [NPL 13] Nature Reviews Drug Discovery (2008) 7, 1001-

[0029] [NPL 14] MAbs (2014) 6(6): 1560-1570 Petição 870260047205, de 18 / 05 / 2026, pág. 14 / 2830 6 / 348

[0030] [NPL 15] Blood (2005) 105(2): 562-566

[0031] [NPL 16] J Biol Chem (2008) 283(23): 16206-16215

[0032] [NPL 17] PLoS Biol (2016) 14(1): e1002344

[0033] [NPL 18] Proc Natl Acad Sci USA (2012) 109(39): 1572815733

[0034] [NPL 19] Scientific Reports (2018) 8, Article number: 766

[0035] [NPL 20] PLoS One (2012) 7(12): e51817

[0036] [NPL 21] Nucleic Acids Res (2010) 38(22): 8188-8195 Summary of the Invention Technical Problem

[0037] The aforementioned efforts to increase or decrease the agonistic action or effector function of antibody pharmaceuticals are still under development, and further efforts are expected. The present invention was developed under these circumstances. An object of the present invention is to provide novel antigen-binding molecules that possess regulatory activity on the interaction between two or more antigen molecules, or methods for producing or utilizing such antigen-binding molecules. The present invention may be useful for the screening and development of antibody pharmaceuticals and may also be applicable to other protein engineering techniques. Solution to the Problem

[0038] In a non-limiting aspect, the present inventors have modified antigen-binding molecules (e.g., antibodies) that possess agonist activity and contain two antigen-binding domains (e.g., Fab parts) by introducing amino acid mutations into the antigen-binding domains, and produced molecules in which the antigen-binding domains were linked together. As a result, the inventors found that the agonist activity of the molecules was greatly improved. In addition, Petition 870260047205, dated 05 / 18 / 2026, p. 15 / 2830 7 / 348 so, in a non-limiting aspect, the present inventors have found antigen-binding molecules that have acquired resistance to protease digestion through linkage between antigen-binding domains.

[0039] The present invention is based on these findings and specifically encompasses the embodiments exemplified below.

[0040] [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 together by one or more bonds.

[0041] [2] The antigen-binding molecule according to [1], in which at least one of the bonds linking the two antigen-binding domains is a covalent bond.

[0042] [3] The antigen-binding molecule according to [2], wherein the 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.

[0043] [4] The antigen-binding molecule according to [3], wherein the cross-linked amino acid residues are cysteine.

[0044] [5] The antigen-binding molecule according to [4], where the covalent bond formed is a disulfide bond.

[0045] [6] The antigen-binding molecule according to [2], wherein the 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 by means of a crosslinking agent.

[0046] [7] The antigen-binding molecule according to [6], wherein the crosslinking agent is an amine-reactive crosslinking agent. Petition 870260047205, dated 05 / 18 / 2026, page 16 / 2830 8 / 348

[0047] [8] The antigen-binding molecule according to [7], wherein the cross-linked amino acid residues are lysine.

[0048] [9] The antigen-binding molecule according to [1], in which at least one of the bonds linking the two antigen-binding domains is a non-covalent bond.

[0049]

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

[0050]

[11] The antigen-binding molecule according to

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

[0051]

[12] The antigen-binding molecule according to

[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).

[0052]

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

[12] , in which at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is an artificially introduced mutated amino acid residue.

[0053]

[14] The antigen-binding molecule according to

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

[0054]

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

[14] , in which at least one of the first and second antigen-binding domains has, individually, antigen-binding activity.

[0055]

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

[15] , wherein the first and second antigen-binding domains are both antigen-binding domains of Petition 870260047205, dated 05 / 18 / 2026, page 17 / 2830 9 / 348 same type.

[0056]

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

[16] , in which at least one of the links connecting the two antigen-binding domains is formed by linking amino acid residues present at the same position in the first antigen-binding domain and in the second antigen-binding domain to each other.

[0057]

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

[16] , in which at least one of the links connecting the two antigen-binding domains is formed by linking amino acid residues present at different positions in the first antigen-binding domain and in the second antigen-binding domain to each other.

[0058]

[19] The 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 particular antigen.

[0059]

[20] The antigen-binding molecule according to

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

[0060]

[21] The antigen-binding molecule according to

[19] or

[20] , in which at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present in the antibody fragment.

[0061]

[22] The antigen-binding molecule according to

[21] , in which the amino acid residue from which the bonds between the antigen-binding domains originate is present within a constant region.

[0062]

[23] The antigen-binding molecule according to

[22] , wherein the constant region is derived from human. Petition 870260047205, dated 05 / 18 / 2026, page 18 / 2830 10 / 348

[0063]

[24] The antigen-binding molecule according to

[22] or

[23] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present within a CH1 region.

[0064]

[25] The antigen-binding molecule according to

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

[0065]

[26] The antigen-binding molecule according to

[24] or

[25] , wherein the amino acid residue from which the links between the antigen-binding domains originate is present in any of the positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214 and 218 to 219, according to EU numbering, in the CH1 region.

[0066]

[27] The antigen-binding molecule according to

[26] , wherein the amino acid residue from which the linkages between the antigen-binding domains originate is present at a position selected from the group consisting of positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218 and 219, according to the EU numbering system, in region CH1.

[0067]

[28] The antigen-binding molecule according to

[27] , wherein the amino acid residue from which the links between the antigen-binding domains originate is present at position 134, 135, 136, 137, 191, 192, 193, 194, 195 or 196, according to EU numbering, in the CH1 region.

[0068]

[29] The antigen-binding molecule according to

[28] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at position 135, 136 or 191, according to EU numbering, in the CH1 region. Petition 870260047205, dated 05 / 18 / 2026, page 19 / 2830 11 / 348

[0069]

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

[24] to

[29] , wherein at least one of the links connecting 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.

[0070]

[31] The antigen-binding molecule according to

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

[0071]

[32] The antigen-binding molecule according to

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

[0072]

[33] The antigen-binding molecule according to

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

[0073]

[34] The antigen-binding molecule according to

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

[0074]

[35] The antigen-binding molecule according to

[30] , Petition 870260047205, dated 05 / 18 / 2026, page 20 / 2830 12 / 348 wherein the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 174, 175, 176, 177, and 178 according to EU numbering.

[0075]

[36] The antigen-binding molecule according to

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

[0076]

[37] The antigen-binding molecule according to

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

[0077]

[38] The antigen-binding molecule according to

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

[0078]

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

[30] to

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

[0079]

[40] The antigen-binding molecule according to

[39] , wherein at least one of the links connecting the two antigen-binding domains is formed by linking an amino acid residue at position 135 according to EU numbering in the re Petition 870260047205, dated 05 / 18 / 2026, page 21 / 2830 13 / 348 CH1 region of the first antigen-binding domain with an amino acid residue at any of the positions 132 to 138 according to EU numbering in the CH1 region of the second antigen-binding domain.

[0080]

[41] The antigen-binding molecule according to

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

[0081]

[42] The antigen-binding molecule according to

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

[0082]

[43] The antigen-binding molecule according to

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

[0083]

[44] The antigen-binding molecule according to

[41] , wherein at least one of the links connecting the two antigen-binding domains is formed by linking amino acid residues at position 136 according to EU numbering in the CH1 region of the two antigen-binding domains to each other. Petition 870260047205, dated 05 / 18 / 2026, page 22 / 2830 14 / 348

[0084]

[45] The antigen-binding molecule according to

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

[0085]

[46] The antigen-binding molecule according to

[22] or

[23] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present within a CL region.

[0086]

[47] The antigen-binding molecule according to

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

[0087]

[48] The antigen-binding molecule according to

[46] or

[47] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present in any of the positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203 and 208 to 213, according to Kabat numbering, in the CL region.

[0088]

[49] The antigen-binding molecule according to

[48] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at a position selected from the group consisting of positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212 and 213 according to Kabat numbering in the CL region.

[0089]

[50] The antigen-binding molecule according to

[49] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at position 126 according to Kabat numbering in the CL region.

[0090]

[51] The antigen-binding molecule according to any of

[46] to

[50] , in which at least one of the bonds that Petition 870260047205, dated 05 / 18 / 2026, page 23 / 2830 The 15 / 348 linkage between 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.

[0091]

[52] The antigen-binding molecule according to

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

[0092]

[53] The antigen-binding molecule according to

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

[0093]

[54] The antigen-binding molecule according to

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

[0094]

[55] The antigen-binding molecule according to

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

[0095]

[56] The antigen-binding molecule according to

[51] , wherein the amino acid residues in the first binding domain to Petition 870260047205, dated 05 / 18 / 2026, page 24 / 2830 16 / 348 antigen and in the second antigen-binding domain are each independently selected from the group consisting of positions 195 and 196 according to Kabat numbering.

[0096]

[57] The antigen-binding molecule according to

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

[0097]

[58] The antigen-binding molecule according to

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

[0098]

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

[51] to

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

[0099]

[60] The antigen-binding molecule according to

[59] , in which at least one of the links connecting the two antigen-binding domains is formed by linking amino acid residues at position 126 according to Kabat numbering in the CL region of the two antigen-binding domains to each other.

[00100]

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

[24] to

[29] and

[46] to

[50] , wherein at least one of the links connecting 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. Petition 870260047205, dated 05 / 18 / 2026, page 25 / 2830 17 / 348

[00101]

[62] The antigen-binding molecule according to

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

[00102]

[63] The antigen-binding molecule according to

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

[00103]

[64] The antigen-binding molecule according to

[21] , in which the amino acid residue from which the bonds between the antigen-binding domains originate is present within a variable region.

[00104]

[65] The antigen-binding molecule according to

[64] , in which the amino acid residue from which the bonds between the antigen-binding domains originate is present within a VH region.

[00105]

[66] The antigen-binding molecule according to

[65] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at a selected position of the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74 and 82b according to Kabat numbering in the VH region.

[00106]

[67] The antigen-binding molecule according to

[64] , in which the amino acid residue from which the bonds between the Petition 870260047205, dated 05 / 18 / 2026, page 26 / 2830 18 / 348 antigen-binding domains originate within a VL region.

[00107]

[68] The antigen-binding molecule according to

[67] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at a selected position of the group consisting of positions 21, 27, 58, 77, 100, 105 and 107 according to Kabat numbering in the VL region (κ subclass).

[00108]

[69] The antigen-binding molecule according to

[67] , wherein the amino acid residue from which the linkages between the antigen-binding domains originate is present in a selected position of the group consisting of positions 6, 19, 33 and 34, according to Kabat numbering in the VL region (λ subclass).

[00109]

[70] The antigen-binding molecule according to

[64] , wherein the amino acid residue from which the linkages between the antigen-binding domains originate is present within a VHH region.

[00110]

[71] The antigen-binding molecule according to

[70] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at a selected position of the group consisting of positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94 and 107 according to Kabat numbering in the VHH region.

[00111]

[72] The 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 that binds to a particular antigen, or a fragment thereof.

[00112]

[73] The antigen-binding molecule according to

[72] , Petition 870260047205, dated 05 / 18 / 2026, page 27 / 2830 19 / 348 where the non-antibody protein is a ligand-receptor pair that specifically binds to each other.

[00113]

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

[73] , wherein the antigen-binding domains comprise a hinge region.

[00114]

[75] The antigen-binding molecule according to

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

[00115]

[76] The antigen-binding molecule according to

[75] , wherein the cysteine ​​residue is present at positions 226 and / or 229, according to EU numbering in the hinge region.

[00116]

[77] The antigen-binding molecule according to

[74] or

[76] , in which at least one of the amino acid residues from which the bonds between the antigen-binding domains originate is present in the hinge region.

[00117]

[78] The antigen-binding molecule according to

[77] , wherein the amino acid residue from which the bonds between the antigen-binding domains originate is present at a position selected from the group consisting of positions 216, 218 and 219 according to EU numbering in the hinge region.

[00118]

[79] The 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 together by means of two or more bonds.

[00119]

[80] The antigen-binding molecule according to

[79] , in which at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is an amino acid residue present in a wild-type sequence.

[00120]

[81] The antigen-binding molecule according to

[80] , Petition 870260047205, dated 05 / 18 / 2026, page 28 / 2830 20 / 348 in which the amino acid residue from which the bonds between the antigen-binding domains originate is present within a hinge region.

[00121]

[82] The antigen-binding molecule according to

[81] , in which the amino acid residue from which the bonds between the antigen-binding domains originate is a cysteine ​​residue in the hinge region.

[00122]

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

[80] to

[82] , wherein at least one of the links connecting the two antigen-binding domains is a disulfide bond formed by cross-linking cysteine ​​residues present in the hinge region with each other.

[00123]

[84] The antigen-binding molecule according to

[83] , wherein cysteine ​​residues are present at positions 226 and / or 229, according to EU numbering in the hinge region.

[00124]

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

[79] to

[84] , in which at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within the antibody fragment, and at least one of the amino acid residues is present in the hinge region.

[00125]

[86] The antigen-binding molecule according to

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

[00126]

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

[86] , wherein the antigen-binding domains comprise an Fc region.

[00127]

[88] The antigen-binding molecule according to

[87] , Petition 870260047205, dated 05 / 18 / 2026, page 29 / 2830 21 / 348 in which one or more amino acid mutations that promote multimerization of the Fc regions are introduced into the Fc region.

[00128]

[89] The antigen-binding molecule according to

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

[00129]

[90] The antigen-binding molecule according to

[88] or

[89] , wherein multimerization is hexamerization.

[00130]

[91] The antigen-binding molecule according to any of

[87] to

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

[00131]

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

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

[00132]

[93] The antigen-binding molecule according to

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

[00133]

[94] The antigen-binding molecule according to

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

[00134]

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

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

[00135]

[96] The antigen-binding molecule according to

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

[00136]

[97] The antigen-binding molecule according to any of

[93] to

[95] , wherein each of the first and second antigen-binding domains has an amino acid sequence Petition 870260047205, dated 05 / 18 / 2026, page 30 / 2830 22 / 348 different.

[00137]

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

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

[00138]

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

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

[00139]

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

[99] , which has regulatory activity of the interaction between two antigen molecules.

[00140]

[101] The antigen-binding molecule according to

[100] , which is capable of increasing or decreasing the interaction between two antigen molecules compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the

[100] antigen-binding molecule only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.

[00141]

[102] The antigen-binding molecule according to

[100] or

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

[00142]

[103] The antigen-binding molecule according to

[100] or

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

[00143]

[104] The antigen-binding molecule according to Petition 870260047205, dated 05 / 18 / 2026, page 31 / 2830 23 / 348

[100] or

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

[00144]

[105] The antigen-binding molecule according to

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

[00145]

[106] The antigen-binding molecule according to

[105] , in which the cell with cytotoxic activity is a T cell, NK cell, monocyte or macrophage.

[00146]

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

[99] , which has regulatory activity of activating two antigen molecules that are activated through association with each other.

[00147]

[108] The antigen-binding molecule according to

[107] , which increases or decreases the activation of two antigen molecules compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule of

[107] only insofar as the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.

[00148]

[109] The antigen-binding molecule according to

[107] or

[108] , wherein antigen molecules are selected from the group consisting of receptors belonging to superfamilies of cytokine receptors, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immunological receptors Petition 870260047205, dated 05 / 18 / 2026, page 32 / 2830 24 / 348 cos of the control point, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[00149]

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

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

[00150]

[111] The antigen-binding molecule according to

[110] , which is able to hold two antigen molecules in closer positions than a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the

[110] antigen-binding molecule only insofar as the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.

[00151]

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

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

[00152]

[113] The antigen-binding molecule according to

[112] , wherein the two antigen-binding domains are in closer positions and / or the two antigen-binding domains have less mobility than a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule of

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

[00153]

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

[99] , which has resistance to protease cleavage.

[00154]

[115] The antigen-binding molecule according to

[114] , which has increased resistance to protease cleavage compared to a control antigen-binding molecule, Petition 870260047205, dated 05 / 18 / 2026, page 33 / 2830 25 / 348 where the control antigen-binding molecule differs from the antigen-binding molecule of

[114] only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.

[00155]

[116] The antigen-binding molecule according to

[115] , in which the proportion of the total length molecule that remains after protease treatment is increased compared to the control antigen-binding molecule.

[00156]

[117] The antigen-binding molecule according to

[115] or

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

[00157]

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

[99] , wherein when the molecule is treated with a protease, a dimer of the antigen-binding domains or fragments thereof is removed.

[00158]

[119] The antigen-binding molecule according to

[118] , wherein when the control antigen-binding molecule is treated with said protease, monomers of the antigen-binding domains or fragments thereof are removed, and wherein the control antigen-binding molecule differs from the antigen-binding molecule of

[118] only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.

[00159]

[120] The antigen-binding molecule according to

[118] or

[119] , in which the protease cleaves the hinge region.

[00160]

[121] The antigen-binding molecule according to any of

[101] to

[106] ,

[108] to

[109] ,

[111] ,

[113] ,

[115] to

[117] and

[119] to

[120] , wherein a minor linkage is a linkage formed that originates from a mutated amino acid residue. Petition 870260047205, dated 05 / 18 / 2026, page 34 / 2830 26 / 348

[00161]

[122] The antigen-binding molecule according to

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

[00162]

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

[122] and a pharmaceutically acceptable carrier.

[00163]

[124] Method for regulating the interaction between two antigen molecules, comprising:

[00164] (a) provide an antigen-binding molecule comprising two antigen-binding domains,

[00165] (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together, and

[00166] (c) bring the antigen-binding molecule produced in (b) into contact with the two antigen molecules.

[00167]

[125] A method for regulating the activity of two antigen molecules that are activated through association with each other, comprising:

[00168] (a) provide an antigen-binding molecule comprising two antigen-binding domains,

[00169] (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together, and

[00170] (c) bring the antigen-binding molecule produced in (b) into contact with the two antigen molecules.

[00171]

[126] Method for maintaining two antigen molecules in spatially close positions, comprising:

[00172] (a) provide an antigen-binding molecule comprising two antigen-binding domains,

[00173] (b) add to the antigen-binding molecule at least Petition 870260047205, dated 05 / 18 / 2026, page 35 / 2830 27 / 348 a link that connects the two antigen-binding domains to each other, and

[00174] (c) bring the antigen-binding molecule produced in (b) into contact with the two antigen molecules.

[00175]

[127] Method for placing two antigen-binding domains in spatially close positions and / or reducing the mobility of the two antigen-binding domains, comprising:

[00176] (a) provide an antigen-binding molecule comprising two antigen-binding domains, and

[00177] (b) add to the antigen-binding molecule at least one link that links the two antigen-binding domains together.

[00178]

[128] Method for increasing the resistance of an antigen-binding molecule to cleavage by protease, comprising:

[00179] (a) provide an antigen-binding molecule comprising two antigen-binding domains, and

[00180] (b) add to the antigen-binding molecule at least one link that links the two antigen-binding domains together.

[00181]

[129] Method for producing an antigen-binding molecule that has regulatory activity on the interaction between two antigen molecules, comprising:

[00182] (a) 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,

[00183] (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,

[00184] (c) introduce the nucleic acids produced in (b) into a host cell, Petition 870260047205, dated 05 / 18 / 2026, page 36 / 2830 28 / 348

[00185] (d) cultivate the host cell in such a way that both polypeptides are expressed, and

[00186] (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.

[00187]

[130] Method for producing an antigen-binding molecule that has activation-regulating activity between two antigen molecules that are activated by association with each other, comprising:

[00188] (a) 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,

[00189] (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,

[00190] (c) introduce the nucleic acids produced in (b) into a host cell,

[00191] (d) cultivate the host cell in such a way that both polypeptides are expressed, and

[00192] (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.

[00193]

[131] Method for producing an antigen-binding molecule that has the activity of holding two antigen molecules in spatially close positions, comprising:

[00194] (a) provide a nucleic acid that codes for a polypeptide Petition 870260047205, dated 05 / 18 / 2026, page 37 / 2830 29 / 348 comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain,

[00195] (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,

[00196] (c) introduce the nucleic acids produced in (b) into a host cell,

[00197] (d) cultivate the host cell in such a way that both polypeptides are expressed, and

[00198] (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.

[00199]

[132] A method for producing an antigen-binding molecule in which two antigen-binding domains are present in spatially close positions and / or the mobility of the two antigen-binding domains is reduced, comprising:

[00200] (a) 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,

[00201] (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,

[00202] (c) introduce the nucleic acids produced in (b) into a host cell,

[00203] (d) cultivate the host cell in such a way that the two po Petition 870260047205, dated 05 / 18 / 2026, page 38 / 2830 30 / 348 lipeptides are expressed, and

[00204] (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.

[00205]

[133] A method for producing an antigen-binding molecule that has increased resistance to protease cleavage, comprising:

[00206] (a) 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,

[00207] (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,

[00208] (c) introduce the nucleic acids produced in (b) into a host cell,

[00209] (d) cultivate the host cell in such a way that both polypeptides are expressed, and

[00210] (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.

[00211]

[134] Method for identifying a new pair of protein molecules that are activated through association with each other, comprising:

[00212] (a) provide two arbitrary protein molecules,

[00213] (b) produce, by the method in accordance with any of

[129] to

[133] , an antigen-binding molecule comprising Petition 870260047205, dated 05 / 18 / 2026, page 39 / 2830 31 / 348 two antigen-binding domains that bind respectively to the two protein molecules,

[00214] (c) bring the antigen-binding molecule produced in (b) into contact with the two protein molecules, and

[00215] (d) evaluate whether the two protein molecules are activated or not.

[00216]

[135] The method of

[134] , in which at least one of the protein molecules is selected from the group consisting of receptors belonging to superfamilies of cytokine receptors, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules and cell adhesion molecules. Brief Description of the Drawings

[00217] Fig. 1 represents examples of modified antibodies in which the Fabs are crosslinked together, as described in Example 1. The figure schematically shows the structural differences between a wild-type antibody (WT) and a modified antibody in which the CH1 regions of the antibody's H chain are crosslinked together (HH type), a modified antibody in which the CL regions of the antibody's L chain are crosslinked together (LL type), and a modified antibody in which the CH1 region of the antibody's H chain is crosslinked with the CL region of the antibody's L chain (HL or LH type).

[00218] Fig. 2 shows the results of the CD3-mediated agonist activity assay of a wild-type anti-CD3ε antibody molecule (CD3-G4s) and modified antibody molecules produced by Fab-Fab linkage of the wild-type molecule via an additional disulfide bond (CD3-G4sLL, CD3G4sHH), as described in Example 4-3.

[00219] Fig. 3 shows the results of the activity test ago Petition 870260047205, dated 05 / 18 / 2026, p. 40 / 2830 32 / 348 CD3-mediated synthesis of a wild-type anti-CD3e antibody molecule (OKT3-G1s) and modified antibody molecules produced by Fab-Fab binding of the wild-type molecule via an additional disulfide bond (OKT3-G1sLL, OKT3G1sHH), as described in Example 4-3.

[00220] Fig. 4 shows the results of the CD3 and / or CD28-mediated agonist activity assay of a wild-type anti-CD3e antibody molecule (CD3-G1s), an anti-CD28 antibody molecule (CD28-G1s), and a bispecific anti-CD3s x anti-CD28 antibody (CD3 / / CD28-G1s) and modified antibody molecules produced by linking the Fab-Fab of the bispecific antibody via an additional disulfide bond (CD3 / / CD28-G1sLL, CD3 / / CD28G1sHH, CD3 / / CD28-G1sLH, CD3 / / CD28-G1sHL), as described in Example 4-3.

[00221] Fig. 5 shows the results of the CD3 and / or CD28-mediated agonist activity assay of a wild-type anti-CD3e antibody molecule (OKT3-G1s), an anti-CD28 antibody molecule (CD28-G1s), and a bispecific anti-CD3e x anti-CD28 antibody (OKT3 / / CD28-G1s) and modified antibody molecules produced by Fab-Fab linkage of the bispecific antibody via an additional disulfide bond (OKT3 / / CD28-G1sHH, OKT3 / / CD28G1sHL), as described in Example 4-3.

[00222] Fig. 6 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the heavy chain of the anti-IL6R antibody (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the heavy chain of the anti-IL6R antibody (MRAH-G1T4.xxx), as described in Example 5-2 (1 / 8). Each protease-treated antibody was applied to non-reactive capillary electrophoresis. Petition 870260047205, dated 05 / 18 / 2026, page 41 / 2830 33 / 348 duct, followed by band detection with an anti-cap chain antibody.

[00223] Fig. 7 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the anti-IL6R antibody heavy chain (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody heavy chain (MRAH-G1T4.xxx), as described in Example 5-2 (2 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00224] Fig. 8 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the heavy chain of the anti-IL6R antibody (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the heavy chain of the anti-IL6R antibody (MRAH-G1T4.xxx), as described in Example 5-2 (3 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00225] Fig. 9 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the heavy chain of the anti-IL6R antibody (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the heavy chain of the anti-IL6R antibody (MRAH-G1T4.xxx), as described in Example 5-2 (4 / 8). Each protease-treated antibody was applied to non-reactive capillary electrophoresis. Petition 870260047205, dated 05 / 18 / 2026, page 42 / 2830 34 / 348 duct, followed by band detection with an anti-cap chain antibody.

[00226] Fig. 10 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the anti-IL6R antibody heavy chain (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody heavy chain (MRAH-G1T4.xxx), as described in Example 5-2 (5 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00227] Fig. 11 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the anti-IL6R antibody heavy chain (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody heavy chain (MRAH-G1T4.xxx), as described in Example 5-2 (6 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00228] Fig. 12 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the heavy chain of the anti-IL6R antibody (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the heavy chain of the anti-IL6R antibody (MRAH-G1T4.xxx), as described in Example 5-2 (7 / 8). Each protease-treated antibody was applied to non-reactive capillary electrophoresis. Petition 870260047205, dated 05 / 18 / 2026, page 43 / 2830 35 / 348 duct, followed by band detection with an anti-cap chain antibody.

[00229] Fig. 13 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the anti-IL6R antibody heavy chain (MRAH.xxx-G1T4), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody heavy chain (MRAH-G1T4.xxx), as described in Example 5-2 (8 / 8). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00230] Fig. 14 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (1 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00231] Fig. 15 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (2 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody. Petition 870260047205, dated 05 / 18 / 2026, page 44 / 2830 36 / 348

[00232] Fig. 16 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (3 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00233] Fig. 17 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (4 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00234] Fig. 18 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (5 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00235] Fig. 19 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of Petition 870260047205, dated 05 / 18 / 2026, page 45 / 2830 37 / 348 anti-IL6R antibody light chain (MRAL.xxx-kO) and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody light chain (MRAL-k0.xxx), as described in Example 6-2 (6 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-capa chain antibody.

[00236] Fig. 20 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (7 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00237] Fig. 21 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (8 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00238] Fig. 22 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable region of the anti-IL6R antibody light chain (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody light chain (MRAL-k0.xxx). Petition 870260047205, dated 05 / 18 / 2026, page 46 / 2830 38 / 348 as described in Example 6-2 (9 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00239] Fig. 23 shows the results of protease treatment of an anti-IL6R antibody (MRA), modified antibodies produced by introducing a cysteine ​​substitution in the variable light chain region of the anti-IL6R antibody (MRAL.xxx-k0), and modified antibodies produced by introducing a cysteine ​​substitution in the constant light chain region of the anti-IL6R antibody (MRAL-k0.xxx), as described in Example 6-2 (10 / 10). Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with an anti-cap chain antibody.

[00240] Fig. 24 shows the results of protease treatment of an anti-IL6R antibody (MRA) and a modified antibody produced by introducing a cysteine ​​substitution in the constant region of the anti-IL6R antibody light chain (MRAL-k0.K126C), as described in Example 7-2. Each protease-treated antibody was applied to non-reducing capillary electrophoresis, followed by band detection with either an anti-capacitance antibody or an anti-human Fc antibody.

[00241] Fig. 25 shows the correspondence between the molecular weight of each band obtained by protease treatment of the antibody sample and its putative structure, as described in Example 7-2. It is also noted below the structure of each molecule whether the molecule can react with an anti-cap chain antibody or an anti-Fc antibody (if a band is detected in the electrophoresis of Fig. 24).

[00242] Fig. 26 shows the results of the CD3-mediated agonist activity assay of an anti-CD3 antibody molecule (OKT3), modified antibody molecules produced by Fab-Fab binding of this antibody molecule via an additional disulfide bond (H_T135C, H_S136C, H_S191C and L_K126C), and Petition 870260047205, dated 05 / 18 / 2026, page 47 / 2830 39 / 348 an anti-KLH antibody molecule (IC17) (negative control), as described in Example 13-4.

[00243] Fig. 27 shows the results of the CD3-mediated agonist activity assay of an anti-CD3 antibody molecule (OKT3), a modified antibody molecule produced by introducing Knobs-into-Holes (KiH) modifications, which facilitate heterodimerization, in the constant region of the OKT3 heavy chain (OKT3_KiH), modified antibody molecules produced by the Fab-Fab linkage of this antibody molecule via an additional disulfide bond (H_S191C_KiH, H_S191C / V188C_KiH, H_S191C / P189C_KiH, H_S191C / S190C_KiH, H_S191C / S192C_KiH, H_S191C / L193C_KiH, H_S191C / G194C_KiH), and an anti-KLH antibody (IC17) (negative control), as described in Example 14-4.

[00244] Fig. 28 shows the results of the CD3-mediated agonist activity assay of an anti-CD3 antibody molecule (OKT3), a modified antibody molecule produced by Fab-Fab linkage of this antibody molecule via an additional disulfide bond (H_S191C), a modified antibody molecule produced by introducing Knobs-into-Holes (KiH) modifications, which facilitate heterodimerization, in the heavy chain constant region of OKT3 (OKT3_KiH), a modified antibody molecule produced by Fab-Fab linkage of this antibody molecule via a disulfide bond (H_S191C_KiH), modified antibody molecules produced by introducing a positively charged amino acid substitution in one of the heavy chain constant regions of OKT3_KiH and introducing a negatively charged amino acid substitution in the other heavy chain constant region (0004 / / 0004, 0004 / / 0006),Modified antibody molecules produced by introducing a positively or negatively charged amino acid substitution into one of the cons regions. Petition 870260047205, dated 05 / 18 / 2026, page 48 / 2830 40 / 348 heavy chain molecules of OKT3_KiH (0004 / / OKT3, OKT3 / / 0004, OKT3 / / 0006) and an anti-KLH antibody molecule (IC17) (negative control), as described in Example 15-4.

[00245] Fig. 29 shows the results of the CD3-mediated agonist activity assay of an anti-CD3 antibody molecule (OKT3), modified antibody molecules produced by removing a disulfide bond in the hinge region of this antibody molecule (dh1, dh2, dh3), modified antibody molecules produced by Fab-Fab binding of these molecules via an additional disulfide bond (H_S191C_dh1, H_S191C_dh2, H_S191C_dh3), and an anti-KLH antibody molecule (IC17) (negative control), as described in Example 16-4.

[00246] Fig. 30 shows the results of the CD3-mediated agonist activity assay of a monospecific anti-CD3 antibody molecule (OKT3-G1s), a modified antibody molecule produced by Fab-Fab binding of this antibody molecule via an additional disulfide bond (OKT3-G1sHH), a modified antibody molecule produced by Fab-Fab binding of a monospecific anti-CD3 antibody (CD3-G1s) via an additional disulfide bond (CD3-G1sLL), a biparatopic anti-CD3 antibody molecule (CD3 / / OKT3-G1s), modified antibody molecules produced by Fab-Fab binding of this antibody molecule via an additional disulfide bond (CD3 / / OKT3G1sHH, CD3 / / OKT3-G1sLH), and a combination of CD3-G1sLL and OKT3-G1s (CD3-G1sLL + OKT3-G1s), as described in Example 20.

[00247] Figs. 31A-D show the results of the assay of CD3 and / or PD1-mediated agonist activity of bispecific anti-CD3 x anti-PD1 antibodies and modified antibody molecules produced by Fab-Fab binding of these antibodies via a linkage Petition 870260047205, dated 05 / 18 / 2026, p. 49 / 2830 41 / 348 additional disulfide bond, as described in Example 22-1. Fig. 31A shows the agonist activity of a bispecific anti-CD3 x anti-PD1 antibody molecule (OKT3 / / 117-G1silent) which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (117), and modified antibody molecules produced by the Fab-Fab linkage of this antibody molecule via an additional disulfide bond (OKT3 / / 117-G1silentHH, OKT3 / / 117-G1silentHL, OKT3 / / 117G1silentLL).

[00248] Fig. 31B shows the agonist activity of a bispecific anti-CD3 x anti-PD1 antibody molecule (OKT3 / / 10-G1silent) which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (10), and modified antibody molecules produced by the Fab-Fab binding of this antibody molecule via an additional disulfide bond (OKT3 / / 10-G1silentHH, OKT3 / / 10-G1silentHL).

[00249] Fig. 31C shows the agonist activity of a bispecific anti-CD3 x anti-PD1 antibody molecule (CD3 / / 949-G1silent) which is composed of an anti-CD3 antibody (CD3) and an anti-PD1 antibody (949), and modified antibody molecules produced by the Fab-Fab binding of this antibody molecule via an additional disulfide bond (CD3 / / 949-G1silentLH, CD3 / / 949-G1silentHH, CD3 / / 949-G1silentLL, CD3 / / 949-G1silentHL).

[00250] Fig. 31D shows the agonist activity of a bispecific anti-CD3 x anti-PD1 antibody molecule (OKT3 / / 949-G1silent) which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), and modified antibody molecules produced by the Fab-Fab binding of this antibody molecule via an additional disulfide bond (OKT3 / / 949-G1silentHL, OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentLL).

[00251] Fig. 32 shows the results of the CD3 and / or PD1-mediated agonist activity assay of a bies antibody molecule. Petition 870260047205, dated 05 / 18 / 2026, p. 50 / 2830 42 / 348 specific anti-CD3 x anti-PD1 (OKT3 / / 949-G1silent) which is composed of an anti-CD3 antibody (OKT3) and an anti-PD1 antibody (949), and modified antibody molecules produced by Fab-Fab linkage of this antibody molecule via an additional disulfide bond (OKT3 / / 949-G1silentHH, OKT3 / / 949-G1silentHL, OKT3 / / 949G1silentLH, OKT3 / / 949-G1silentLL), as described in Example 222.

[00252] Figs. 33A and B show the results of evaluating the T cell-dependent inhibitory effect on cancer cell growth when using a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody in combination, as described in Example 23-1. When the aforementioned CD28 / CD3 binding bispecific antibody and the GPC3 / binding attenuated CD3 bispecific antibody are combined and can act in the presence of target cells (cancer cells expressing GPC3) and effector cells (T cells), the GPC3 / binding attenuated CD3 bispecific antibody brings the target cell and the effector cell closer together, and the CD28 / CD3 binding bispecific antibody activates the effector cell. Fig.Figure 33A shows the inhibitory effect on cancer cell growth when a GPC3 / binding attenuated bispecific CD3 antibody molecule (GPC3 / attCE115) was used as an antibody to target T cells to cancer cells and a GPC3 / CD3 binding bispecific antibody molecule (fixed GPC3 / CD3), a KLH / CD3 binding bispecific antibody molecule (fixed KLH / CD3), a CD28 / CD3 binding bispecific antibody molecule (fixed CD28 / CD3) or a modified antibody molecule produced by Fab-Fab binding of that antibody molecule via an additional disulfide bond (fixed CD28 / CD3_HH) was used as an antibody to activate T cells. Petition 870260047205, dated 05 / 18 / 2026, p. 51 / 2830 43 / 348

[00253] Fig. 33B shows the inhibitory effect on cancer cell growth when a modified antibody molecule produced by Fab-Fab binding of the attenuated CD3 bispecific antibody via an additional disulfide bond (GPC3 / attCE115_LL) was used as an antibody to target T cells to cancer cells and a GPC3 / CD3 bispecific fixation antibody molecule (fixed GPC3 / CD3), a KLH / CD3 bispecific fixation antibody molecule (fixed KLH / CD3), a CD28 / CD3 bispecific fixation antibody molecule (fixed CD28 / CD3) or a modified antibody molecule produced by Fab-Fab binding of that antibody molecule via an additional disulfide bond (fixed CD28 / CD3_HH) was used as an antibody to activate T cells.

[00254] Figs. 34A-C show the results of evaluating cytokine production from T cells when a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody were used in combination as described in Example 23-2. When the aforementioned CD28 / CD3 binding bispecific antibody and the GPC3 / binding attenuated CD3 bispecific antibody are used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells), the GPC3 / binding attenuated CD3 bispecific antibody brings the target cell and the effector cell close together, and the CD28 / CD3 binding bispecific antibody activates the effector cell. Fig.Figure 34A shows the level of IL-6 production when a GPC3 / binding attenuated bispecific CD3 antibody molecule (GPC3 / attCE115) and a modified antibody molecule produced by Fab-Fab binding of the CD28 / CD3 bispecific fixation antibody via an additional disulfide bond (CD28 / CD3_HH fixed) were used individually or in combination. Petition 870260047205, dated 05 / 18 / 2026, page 52 / 2830 44 / 348 action in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells).

[00255] Fig. 34B shows the level of IL-6 production when a GPC3 / binding attenuated bispecific CD3 antibody molecule (GPC3 / attCE115) and a modified antibody molecule produced by Fab-Fab binding of the CD28 / CD3 bispecific fixation antibody via an additional disulfide bond (CD28 / CD3_HH fixed) were used individually or in combination only in the presence of effector cells (T cells).

[00256] Fig. 34C shows the inhibitory effect on cancer cell growth when a GPC3 / binding attenuated bispecific CD3 antibody molecule (GPC3 / attCE115) and a modified antibody molecule produced by Fab-Fab binding of the CD28 / CD3 bispecific fixation antibody via an additional disulfide bond (CD28 / CD3_HH fixed) were used individually or in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells).

[00257] Figs. 35A and B are schematic diagrams showing the mechanism of action of T cell-dependent cancer cell growth inhibition when a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Examples 23-1 (“ε” in the diagrams indicates CD3ε). Fig. 35A shows the mechanism of action of cancer cell growth inhibition when a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination in the presence of target cells (cancer cells expressing GPC3) and effector cells (T cells).

[00258] Fig. 35B shows the mechanism of action of inhibiting the growth of cancer cells when an antibody molecule Petition 870260047205, dated 05 / 18 / 2026, page 53 / 2830 The modified 45 / 348, which has been modified to introduce an additional disulfide linkage in the Fab-Fab of a bispecific CD28 / CD3 binding antibody and a GPC3 / attenuated CD3 bispecific antibody, is used in combination in the presence of target cells (cancer cells expressing GPC3) and effector cells (T cells).

[00259] Figs. 36A and B are schematic diagrams showing the mechanism of action of cytokine production from T cells when a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody are used in combination, as described in Examples 23-2 (ε” in the diagrams indicates CD3ε). Fig. 36A shows the mechanism of action of cytokine production when a modified antibody molecule that has been modified to introduce an additional disulfide bond in the Fab-Fab of a CD28 / CD3 binding bispecific antibody and a GPC3 / binding attenuated CD3 bispecific antibody is used in combination in the presence of target cells (GPC3-expressing cancer cells) and effector cells (T cells).

[00260] Fig. 36B shows the mechanism of action of cytokine production when a modified antibody molecule that has been modified to introduce an additional disulfide bond in the Fab-Fab of a bispecific CD28 / CD3 binding antibody and a GPC3 / binding attenuated CD3 bispecific antibody, is used in combination only in the presence of effector cells (T cells).

[00261] Figs. 37A and B show the assay results of the agonist activity of a bispecific CD8 / CD28 antibody molecule (CD8 / CD28-P587) and modified antibody molecules produced by linking the Fab-Fab of this antibody via an additional disulfide bond (CD8 / CD28-P587(HH), CD8 / CD28-P587(LL), CD8 / CD28-P587(HL), CD8 / CD28-P587(LH)) Petition 870260047205, dated 05 / 18 / 2026, p. 54 / 2830 46 / 348 as described in Example 24. An anti-KLH antibody molecule (KLH-P587) was used as a negative control. The results obtained using peripheral blood mononuclear cells (PBMCs) from two different donors are shown (top panel: donor A, bottom panel: donor B). Fig. 37A shows the proportion of split regulatory T cells (Treg) in PBMCs.

[00262] Fig. 37B shows the proportion of CD8a-positive T cells divided into PBMCs. Description of the Modalities I. Definitions

[00263] Here, the term antigen-binding molecule refers, in its broadest sense, to a molecule that binds specifically to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, antibody fragment, or antibody derivative. In another embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment thereof, or a derivative thereof.

[00264] In this invention, antigen-binding domain refers to a region that specifically binds to and is complementary to all or part of an antigen. Here, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, an antigen-binding domain can only bind to a particular part of the antigen. The particular part is called an epitope. In one embodiment, an antigen-binding domain comprises an antibody fragment that binds to a particular antigen. An antigen-binding domain can be provided from one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domains comprise the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH). Examples of Petition 870260047205, dated 05 / 18 / 2026, page 55 / 2830 47 / 348 Such antigen-binding domains include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In other embodiments, an antigen-binding domain comprises a non-antibody protein that binds to a particular antigen, or a fragment thereof. In a specific embodiment, an antigen-binding domain comprises a hinge region.

[00265] In the present invention, specifically, "binds" means binding in a state where one of the molecules involved in the specific binding does not show any significant binding with molecules other than a single or multiple binding partner molecules. Furthermore, it is also used when an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. When an epitope bound by an antigen-binding domain is contained in several different antigens, the antigen-binding molecules comprising the antigen-binding domain can bind to multiple antigens possessing the epitope.

[00266] In the present invention, recitation binds to the same epitope means that the epitopes to which two antigen-binding domains bind, at least partially, overlap with each other. The degree of overlap is, but is not limited to, at least 10% or more, preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more and 80% or more, particularly preferably 90% or more, and most preferably 100%.

[00267] The term antibody in this invention is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit binding activity. Petition 870260047205, dated 05 / 18 / 2026, p. 56 / 2830 48 / 348 desired antigen response.

[00268] The term monoclonal antibody, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or mutations that arise during the production of a monoclonal antibody preparation, such variants generally being present in smaller quantities. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.Thus, the monoclonal modifier indicates the character of the antibody as being 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, the monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin positions, such methods and other exemplary methods for the production of monoclonal antibodies being described herein.

[00269] Native antibodies refer to naturally occurring immunoglobulin molecules with variable structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-linked. From the N- to the C-terminal, each heavy chain has a variable region (VH), Petition 870260047205, dated 05 / 18 / 2026, page 57 / 2830 49 / 348, also called the variable heavy domain or variable heavy chain domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N- to C-terminal, each light chain has a variable region (VL), also called the variable light domain or variable light chain domain, followed by a constant light domain (CL). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[00270] The term chimeric antibody refers to an antibody in which part of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[00271] The class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different immunoglobulin classes are called alpha, delta, epsilon, gamma, and mu, respectively.

[00272] In one embodiment of the present invention, the constant regions are preferably antibody constant regions, more preferably IgG1, IgG2, IgG3 and IgG4 type antibody constant regions, and even more preferably human IgG1, IgG2, IgG3 and IgG4 type antibody constant regions. Furthermore, in another embodiment of the present invention, the constant regions are preferably heavy chain constant regions, more preferably IgG1, IgG2, IgG3 and IgG4 type heavy chain constant regions, and even more preferably human IgG1, IgG2, IgG3 and IgG4 type heavy chain constant regions. The ami sequences Petition 870260047205, dated 05 / 18 / 2026, p. 58 / 2830 50 / 348 amino acid of the human IgG1 constant region, the human IgG2 constant region, the human IgG3 constant region, and the human IgG4 constant region are known. For the human IgG1, human IgG2, human IgG3, and human IgG4 constant regions, a plurality of allotype sequences with genetic polymorphism is described in Protein Sequences of Immunological Interest, NIH Publication No. 91-3242, and any of them can be used in the present invention. The amino acid-modified constant regions of the present invention may contain other amino acid mutations or modifications, provided they include an amino acid mutation of the present invention.

[00273] The term hinge region means a part of the antibody heavy chain polypeptide in a wild-type antibody heavy chain that joins the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system. It is known that in a native IgG antibody, the cysteine ​​residue at position 220 according to the EU numbering in the hinge region forms a disulfide bond with the cysteine ​​residue at position 214 in the antibody light chain. It is also known that between the two antibody heavy chains, disulfide bonds are formed between cysteine ​​residues at position 226 and between cysteine ​​residues at position 229 according to the EU numbering in the hinge region.A hinge region here includes wild-type hinge regions, as well as variants in which amino acid residues in a wild-type hinge region are altered through substitution, addition, or deletion.

[00274] The term Fc region in this invention is used to define a C-terminal region of an immunoglobulin heavy chain that Petition 870260047205, dated 05 / 18 / 2026, page 59 / 2830 51 / 348 contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446447) of the Fc region may or may not be present. Unless otherwise specified in this invention, the numbering of amino acid residues in the Fc region or constant region conforms to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[00275] Effector functions refer to the biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[00276] The term Fc receptor or FcR refers to a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subclasses, including allelic variants and alternatively linked forms of these receptors. Fc gamma RII receptors include Fc gamma RIIA (an activating receptor) and Fc gamma RIIB (an inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Petition 870260047205, dated 05 / 18 / 2026, p. 60 / 2830 52 / 348 The Fc gamma RIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The Fc gamma RIIB inhibitory receptor contains an immunoreceptor tyrosine-based inhibiting motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term FcR in this invention.

[00277] The term Fc receptor or FcR also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[00278] The term variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in antibody binding to the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved structural 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 can be isolated using a domain. Petition 870260047205, dated 05 / 18 / 2026, page 61 / 2830 53 / 348 VH or VL of an antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[00279] The term hypervariable region or HVR as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence (complementarity-determining regions or CDRs) and / or form structurally defined gauge loops (hypervariable gauge loops) and / or contain antigen-contact residues (antigen contacts). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

[00280] (a) hypervariable gauge loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 5355 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[00281] (b) CDRs that occur 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));

[00282] (c) antigen contacts that occur 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

[00283] (d) combinations 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).

[00284] Unless otherwise indicated, HVR residuals and other residuals in the variable domain (e.g., FR residuals) are numbered here in accordance with Kabat et al., supra. Petition 870260047205, dated 05 / 18 / 2026, p. 62 / 2830 54 / 348

[00285] Structure or FR refers to the residuals of the variable domain other than the residuals of the hypervariable region (HVR). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Consequently, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[00286] The terms full-length antibody, intact antibody, and whole antibody are used interchangeably in this invention to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains containing an Fc region as defined in this invention.

[00287] The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny derived independently of the number of passages. Progeny may not be completely identical in nucleic acid content to a mother cell, but may contain mutations. Mutant progeny that possesses the same biological function or activity as screened for or selected in the originally transformed cell is included here.

[00288] The term vector, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of operably directing the expression of nucleic acids to which they are attached. These vectors are referred to here as vectors. Petition 870260047205, dated 05 / 18 / 2026, page 63 / 2830 55 / 348 expression vectors.

[00289] A human antibody is one that has an amino acid sequence that corresponds to that of an antibody produced by a human being or human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-coding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[00290] A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A humanized form of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

[00291] An antibody fragment refers to a molecule other than an intact antibody comprising a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-stranded antibody molecules (e.g., scFv); single-stranded Fabs (scFabs); single-domain antibodies; and multispecific antibodies formed from antibody fragments. Petition 870260047205, dated 05 / 18 / 2026, p. 64 / 2830 56 / 348 Variable fragment (Fv)

[00292] Here, the term variable fragment (VF) refers to the smallest unit of an antibody-derived antigen-binding domain that is composed of a pair of antibody light chain variable region (VL) and antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies can be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing gene expression in E. coli (Science (1988) 240 (4855), 1038-1041). In the VF prepared from the periplasmic fraction, VH associates with VL in order to bind to an antigen. scFv, single-chain antibody, and sc(Fv)2

[00293] Here, the terms scFv, single-chain antibody, and sc(Fv)2 all refer to a single-chain polypeptide antibody fragment containing variable regions derived from the heavy and light chains, but not the constant region. In general, a single-chain antibody also contains a polypeptide linker between the VH and VL domains, which allows the formation of a desired structure believed to enable antigen binding. The single-chain antibody is examined in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994)”. See also International Patent Publication WO 1988 / 001649; U.S. Patents Nos. 4,946,778 and 5,260,203. In one particular embodiment, the single-chain antibody can be bispecific and / or humanized.

[00294] scFv is an antigen-binding domain in which VH and VL forming Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). VH and VL can be retained in close proximity by the peptide linker. Petition 870260047205, dated 05 / 18 / 2026, page 65 / 2830 57 / 348

[00295] sc(Fv)2 is a single-chain antibody in which four variable regions of two VL and two VH are linked by ligands, such as peptide ligands, to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VH and two VL may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes, for example, a bispecific sc(Fv)2 that recognizes two epitopes present on a single antigen, as reported in the Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFv with a ligand, such as a peptide ligand.

[00296] Here, the form of an antigen-binding domain that forms an sc(Fv)2 includes an antibody in which the two VH units and two VL units are arranged in the order VH, VL, VH, and VL ([VH]-ligand-[VL]-ligand-[VH]-ligand-[VL]) starting from the N-terminal of a single-chain polypeptide. The order of the two VH units and two VL units is not limited to the above form and they can be arranged in any order. An example order of the form is listed below.

[00297] [VL]-ligand-[VH]-ligand-[VH]-ligand-[VL]

[00298] [VH]-ligand-[VL]-ligand-[VL]-ligand-[VH]

[00299] [VH]-ligand-[VH]-ligand-[VL]-ligand-[VL]

[00300] [VL]-ligand-[VL]-ligand-[VH]-ligand-[VH]

[00301] [VL]-ligand-[VH]-ligand-[VL]-ligand-[VH] Fab, F(ab')2 and Fab'

[00302] Fab consists of a single light chain and a CH1 region and a variable region of a single heavy chain. The heavy chain of a wild-type Fab molecule cannot form disulfide bonds with another heavy-chain molecule. Here, in addition to wild-type Fab molecules, variants of Petition 870260047205, dated 05 / 18 / 2026, page 66 / 2830 58 / 348 Fab variants in which amino acid residues in a wild-type Fab molecule are altered by substitution, addition, or deletion. In one specific embodiment, mutated amino acid residues comprised in Fab variants (e.g., cysteine ​​residues or lysine residues after substitution, addition, or insertion) can form disulfide bonds with another heavy chain molecule or a part thereof (e.g., Fab molecule).

[00303] scFab is an antigen-binding domain in which a single light chain and CH1 region and a variable region of a single heavy chain that forms Fab are linked together by a peptide linker. The light chain and CH1 region and the variable region of the heavy chain can be retained in close proximity by the peptide linker.

[00304] F(ab')2 or Fab is produced by treating an immunoglobulin (monoclonal antibody) with a protease, such as pepsin and papain, and refers to an antibody fragment generated by the digestion of an immunoglobulin (monoclonal antibody) near the disulfide bonds present between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds present between the hinge regions in each of the two H chains to generate two homologous antibody fragments, in which an L chain comprising VL (variable region of L chain) and CL (constant region of L chain) is linked to an H chain fragment comprising VH (variable region of H chain) and CHyI (γ1 region in a constant region of H chain) via a disulfide bond in their C-terminal regions. Each of these two homologous antibody fragments is called Fab'.

[00305] F(ab')2 consists of two light chains and two heavy chains comprising the constant region of a CH1 domain and Petition 870260047205, dated 05 / 18 / 2026, page 67 / 2830 59 / 348 a portion of the CH2 domains, such that disulfide bonds are formed between the two heavy chains. OF(ab')2 disclosed in this invention can preferably be produced as follows. A complete monoclonal antibody or such comprising a domain binding to the desired antigen is partially digested with a protease, such as pepsin; and the Fc fragments are removed by adsorption onto a Protein A column. The protease is not particularly limited, provided it can cleave the entire antibody selectively to produce F(ab')2 under an appropriate enzymatic reaction configuration such as pH. Such proteases include, for example, pepsin and ficin. Single-domain antibodies

[00306] In this invention, those referred to by the term single-domain antibodies are not particularly limited in their structure, provided that the domain can exert antigen-binding activity by itself. Common antibodies exemplified by IgG antibodies exert antigen-binding activity in a state where a variable region is formed by the pairing of VH and VL. In contrast, a single-domain antibody is known to be capable of exerting antigen-binding activity by its own domain structure without pairing with another domain. Single-domain antibodies generally have a relatively low molecular weight and exist in the form of a monomer.

[00307] Examples of a single-domain antibody include, but are not limited to, antigen-binding molecules that naturally lack light chains, such as VHH from Camelidae animals and VNAR from sharks, and antibody fragments comprising all or part of a VH antibody domain or all or part of a VL antibody domain. Examples of a single-domain antibody that is an antibody fragment comprising the Petition 870260047205, dated 05 / 18 / 2026, p. 68 / 2830 60 / 348 all or part of a VH / VL antibody domain includes, but is not limited to, artificially prepared single-domain antibodies originating from a human VH antibody or a human VL antibody as described, for example, in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[00308] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which genes for the ability to produce a single-domain antibody have been introduced. Camelids include camel, llama, alpaca, dromedary, guanaco, and the like.Examples of a transgenic animal in which genes for the ability to produce a single-domain antibody have been introduced include, but are not limited to, transgenic animals described in International Publication No. WO2015 / 143414 or US Patent Publication No. US2011 / 0123527 A1. Humanized single-chain antibodies can also be obtained by replacing the structural sequences of a single-domain antibody obtained from an animal with human germline sequences or sequences similar to them. A humanized single-domain antibody (e.g., humanized VHH) is an embodiment of the single-domain antibody of the present invention.

[00309] Alternatively, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by ELISA, extraction, and similar methods. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, passive antibody libraries. Petition 870260047205, dated 05 / 18 / 2026, page 69 / 2830 61 / 348 vesicles obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78) and Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), and antibody libraries obtained through immunization of various animals (e.g., Journal of Applied Microbiology 2014 117: 2 (528-536)) and synthetic antibody libraries prepared from antibody genes of various animals and humans (e.g., Journal of Biomolecular Screening 2016 21: 1 (35-43), Journal of Biological Chemistry 2016 291: 24 (12641-12657), and AIDS 2016 30: 11 (1691-1701)).

[00310] Binding activity refers to the strength of the total sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Here, binding activity is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). For example, when the members of a binding pair reflect a monovalent 1:1 interaction, the binding activity refers to the intrinsic binding affinity (affinity). When a member of a binding pair is capable of both monovalent and multivalent binding, the binding activity is the sum of each binding strength. The binding activity of a molecule X to its partner Y can generally be represented by the dissociation constant (KD) or the amount of analyte bound per unit amount of ligand.Binding activity can be measured by common methods known in the art, including those described herein.

[00311] An antigen-binding agonist molecule or agonist antibody, as used herein, is an antigen-binding molecule or antibody that significantly enhances a biological activity of the antigen to which it binds.

[00312] A blocking antigen-binding molecule or blocking antibody or an antigen-binding molecule Petition 870260047205, dated 05 / 18 / 2026, p. 70 / 2830 62 / 348 An agonist or antagonist antibody, as used herein, is an antigen-binding molecule or antibody that significantly (partially or completely) inhibits a biological activity of the antigen to which it binds.

[00313] The phrase substantially reduced or substantially different, as used herein, refers to a sufficiently high degree of difference between two numerical values ​​(usually one associated with a molecule and the other associated with a reference / comparator molecule), such that a person skilled in the art would consider the difference between the two values ​​to be of statistical significance within the context of the biological characteristic measured by the said values ​​(e.g., KD values).

[00314] The term substantially similar or substantially the same, as used herein, refers to a sufficiently high degree of similarity between two numerical values ​​(for example, one associated with an antibody of the invention and the other associated with a reference / comparator antibody), such that a person skilled in the art would consider the difference between the two values ​​to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values ​​(for example, KD values).

[00315] The terms pharmaceutical formulation and pharmaceutical composition refer to a preparation that is in a form that allows the biological activity of an active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to an individual to whom the formulation would be administered.

[00316] A pharmaceutically acceptable vehicle refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is not toxic to an individual. A pharmaceutical vehicle Petition 870260047205, dated 05 / 18 / 2026, p. 71 / 2830 63 / 348 acceptable mind includes, but is not limited to, a buffer, excipient, stabilizer or preservative.

[00317] A person or individual is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain modalities, the person or individual is a human being. II. Antigen-binding molecule

[00318] In one aspect, the present invention is partly based on the discovery that various activities of an antigen-binding molecule containing a first antigen-binding domain and a second antigen-binding domain in which the antigen-binding domains are linked together through one or more bonds are increased or decreased compared with a control antigen-binding molecule containing antigen-binding domains without the linkage or linked by fewer bonds. In certain embodiments, an antigen-binding molecule possessing the activity of retaining two or more antigen molecules in spatially close positions is provided. The antigen-binding molecule of the present invention is useful, for example, insofar as it can regulate the activation of two antigen molecules that are activated through association with each other.In certain other embodiments, an antigen-binding molecule that has acquired resistance to protease digestion through linkage between antigen-binding domains is provided. A. Exemplary antigen-binding molecules Structures of antigen-binding molecules

[00319] In one aspect, the present invention provides an antigen-binding molecule comprising a first binding domain. Petition 870260047205, dated 05 / 18 / 2026, page 72 / 2830 64 / 348 antigen binding and a second antigen-binding domain, and the antigen-binding domains are linked together by one or more bonds.

[00320] In one embodiment of the above aspects, at least one of the one or more bonds linking the two antigen-binding domains is a covalent bond. In certain embodiments, the covalent bond is formed by direct cross-linking of an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain. The cross-linked amino acid residues are, for example, cysteine, and the covalent bond formed is, for example, a disulfide bond.

[00321] In certain other embodiments, the 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 by means of a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the crosslinked amino acid residues are, for example, lysine.

[00322] In one embodiment of the above aspects, at least one of the one or more bonds linking the antigen-binding domains is a non-covalent bond. In certain embodiments, the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond. The ionic bond is formed, for example, between an acidic amino acid and a basic amino acid. The acidic amino acid is, for example, aspartic acid (Asp) or glutamic acid (Glu). The basic amino acid is, for example, histidine (His), lysine (Lys), or arginine (Arg).

[00323] The amino acid residues from which the linkages between antigen-binding domains (the linkages that connect two antigen-binding domains) originate are respectively present in the first and second antigen-binding domains, Petition 870260047205, dated 05 / 18 / 2026, page 73 / 2830 65 / 348 and the linkages between antigen-binding domains are formed through the linkage of these amino acid residues. In one embodiment of the above aspects, at least one of the amino acid residues from which the linkage between antigen-binding domains originates is an artificially introduced mutated amino acid residue, and, for example, is an artificially introduced cysteine ​​residue. This mutated amino acid residue can be introduced into a wild-type antigen-binding domain, for example, through an amino acid substitution method.This descriptive report discloses the amino acid residue sites from which the binding between antigen-binding domains can originate for each of the CH1, CL, and hinge regions as constant regions and the VH, VL, and VHH regions as variable regions when the antigen-binding domains comprise, for example, an antibody fragment and, for example, cysteine ​​residues can be introduced into such sites.

[00324] In one embodiment of the above aspects, at least one of the first and second antigen-binding domains independently possesses antigen-binding activity (that is, a single antigen-binding domain independently possesses antigen-binding activity). In certain embodiments, each of the first and second antigen-binding domains independently possesses antigen-binding activity.

[00325] In one embodiment of the above aspects, the first and second antigen-binding domains are both antigen-binding domains of the same type. As mentioned below, examples of proteins that constitute antigen-binding domains include polypeptides derived from an antibody or a non-antibody protein and its fragments (e.g., Fab, Fab', scFab, Fv, scFv, and single-domain antibody). From the point of view of Petition 870260047205, dated 05 / 18 / 2026, page 74 / 2830 66 / 348 such molecular forms, when the structures of the proteins that constitute the first and second antigen-binding domains are identical, the antigen-binding domains are determined to be of the same type.

[00326] In one embodiment of the above aspects, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain can be formed by linking amino acid residues present at the same position in the first antigen-binding domain and the second antigen-binding domain to each other, or it can be formed by linking amino acid residues present at a position that is respectively different from each other.

[00327] The positions of amino acid residues in the antigen-binding domain can be shown according to the Kabat numbering or EU numbering system (also called 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 from which the linkages between the first and second antigen-binding domains originate are present in an identical corresponding position in the antigen-binding domains, the position of these amino acid residues can be indicated as the same number according to the Kabat numbering or EU numbering system.Alternatively, if the amino acid residues from which the linkages between the first and second antigen-binding domains originate are present in different, non-corresponding positions in the antigen-binding domains, the positions of these amino acid residues may be indicated by different numbers according to the Kabat numbering or EU numbering system. Petition 870260047205, dated 05 / 18 / 2026, page 75 / 2830 67 / 348

[00328] In one embodiment of the above aspects, at least one of the first and second antigen-binding domains comprises an antibody fragment that binds to a specific antigen. In certain embodiments, the antibody fragment is a Fab, Fab', scFab, Fv, scFv, or single-domain antibody. In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present in an antibody fragment.

[00329] In one embodiment of the above aspects, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a constant region. In certain embodiments, the amino acid residue is present in a CH1 region and, for example, is present in any of the positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214 and 218 to 219 according to the EU numbering in the CH1 region. In certain embodiments, the amino acid residue is present at a selected position in the group consisting of positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218 and 219 according to the EU numbering in the CH1 region. In certain embodiments, the amino acid residue is present at positions 134, 135, 136, 137, 191, 192, 193, 194, 195 or 196 according to the EU numbering in the CH1 region. In certain embodiments, the amino acid residue is present at positions 135, 136 or 191 according to the EU numbering in the CH1 region.

[00330] In one embodiment of the above aspects, the constant region is derived from human. In certain embodiments, the heavy chain constant region subclass is any one of IgG1, IgG2, Petition 870260047205, dated 05 / 18 / 2026, page 76 / 2830 68 / 348 IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In certain embodiments, the subclass of the CH1 region is any one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[00331] In one embodiment of the above aspects, 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 and an amino acid residue in the CH1 region of the second antigen-binding domain. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 119, 120, 121, 122, and 123 according to EU numbering.In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 131, 132, 133, 134, 135, 136, 137, 138, 139 and 140 according to the EU numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 148, 149 and 150 according to the EU numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and 167 according to EU numbering.In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 174, 175, 176, 177 and 178 according to EU numbering. In certain embodiments, the residues... Petition 870260047205, dated 05 / 18 / 2026, page 77 / 2830 69 / 348 amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196 and 197 according to EU numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 and 214 according to EU numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 218 and 219 according to EU numbering.

[00332] In one embodiment of the above aspects, the difference in the positions of the amino acid residues from which the bonds originate in each of the first antigen-binding domain and the second antigen-binding domain is three amino acids or less. This means that when the position of the amino acid residue from which a bond originates in the CH1 region of the first antigen-binding domain and the position of the amino acid residue from which the bond originates in the CH1 region of the second antigen-binding domain are compared respectively according to EU numbering, the difference is three amino acids or less.In certain embodiments, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 135 according to EU numbering in the CH1 region of the first antigen-binding domain and an amino acid residue at either position 132 to 138 according to EU numbering in the CH1 region of the second antigen-binding domain. Petition 870260047205, dated 05 / 18 / 2026, page 78 / 2830 70 / 348 In certain embodiments, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain and an amino acid residue at any of EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain.

[00333] In certain embodiments, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and an amino acid residue at any of EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain.In an exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking amino acid residues at position 135 according to EU numbering in the CH1 regions of the two antigen-binding domains to each other. In an exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking amino acid residues at position 136 according to EU numbering in the CH1 regions of the two antigen-binding domains to each other. In an exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking amino acid residues at position 191 according to EU numbering in the CH1 regions of the two domains to each other.

[00334] In one of the above aspects, at least one Petition 870260047205, dated 05 / 18 / 2026, p. 79 / 2830 71 / 348 of the amino acid residues from which the bonds between the antigen-binding domains originate are present within a CL region and, for example, are present at any of the positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203 and 208 to 213 according to Kabat numbering in the CL region. In certain embodiments, the amino acid residue is present at a position selected from the group consisting of positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213 according to the Kabat numbering in the CL region. In certain embodiments, the amino acid residue is present at position 126 according to the Kabat numbering in the CL region.

[00335] In one embodiment of the above aspects, the constant region is derived from human. In certain embodiments, the subclass of the CL region is κ or λ.

[00336] In one embodiment of the above aspects, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue in the CL region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 108, 109, 110, 111, and 112 according to Kabat numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 according to Kabat numbering. In certain embodiments, the amino acid residues in Petition 870260047205, dated 05 / 18 / 2026, p. 80 / 2830 72 / 348 The first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of positions 151, 152, 153, 154, 155, and 156 according to Kabat numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of positions 184, 185, 186, 187, 188, 189, and 190 according to Kabat numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of positions 195 and 196 according to Kabat numbering.In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 200, 201, 202, and 203 according to the Kabat numbering. In certain embodiments, the amino acid residues in the first antigen-binding domain and in the second antigen-binding domain are each independently selected from the group consisting of positions 208, 209, 210, 211, 212, and 213 according to the Kabat numbering.

[00337] In one embodiment of the above aspects, the difference in the positions of the amino acid residues from which the bonds originate in each of the first antigen-binding domain and the second antigen-binding domain is three amino acids or less. This means that when the position of the amino acid residue from which a bond originates in the CL region of the first antigen-binding domain and the position of the amino acid residue from which the bond originates in the CL region of the second antigen-binding domain are compared respectively according to Petition 870260047205, dated 05 / 18 / 2026, page 81 / 2830 73 / 348 according to the EU numbering, the difference is three amino acids or less. In an exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking amino acid residues at position 126 according to the Kabat numbering in the CL regions of the two antigen-binding domains to each other.

[00338] In one embodiment of the above aspects, 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 and an amino acid residue in the CL region of the second antigen-binding domain. In certain embodiments, the amino acid residues in the CH1 region of the first antigen-binding domain are selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to the EU numbering, and the amino acid residues in the CL region of the second antigen-binding domain are selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 according to the Kabat numbering.In an exemplary embodiment, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 191 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at position 126 according to Kabat numbering in the CL region of the second antigen-binding domain.

[00339] In one embodiment of the above aspects, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a variable region. In certain embodiments, the amino acid residue Petition 870260047205, dated 05 / 18 / 2026, page 82 / 2830 74 / 348 is present in a VH region and, for example, is present at a selected position of the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b according to the Kabat numbering in the VH region. In certain embodiments, the amino acid residue is present in a VL region and, for example, is present at a selected position of the group consisting of positions 21, 27, 58, 77, 100, 105, and 107 according to the Kabat numbering in the VL region (κ subclass) and positions 6, 19, 33, and 34 according to the Kabat numbering in the VL region (λ subclass). In certain embodiments, the amino acid residue is present in a VHH region and, for example, is present in a selected position of the group consisting of positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94 and 107 according to the Kabat numbering in the VHH region.

[00340] In one embodiment of the above aspects, at least one of the first and second antigen-binding domains comprises a non-antibody protein binding to a particular antigen, or a fragment thereof. In certain embodiments, the non-antibody protein is a pair of a ligand and a receptor that bind specifically to each other. Such receptors include, for example, receptors belonging to cytokine receptor superfamilies, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[00341] In one embodiment of the above aspects, the first and / or second antigen-binding domains comprise a hinge region. In certain embodiments, at least one of the cysteine ​​residues present in a wild-type hinge region is replaced by another amino acid residue. These cysteine ​​residues Petition 870260047205, dated 05 / 18 / 2026, page 83 / 2830 75 / 348 are present, for example, at positions 226 and / or 229 according to the EU numbering in the wild-type hinge region. In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a hinge region and, for example, is present at a position selected from the group consisting of positions 216, 218, and 219 according to the EU numbering in the hinge region.

[00342] In one embodiment of the above aspects, the first antigen-binding domain and the second antigen-binding domain are linked together by means of two or more linkages.

[00343] In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is an amino acid residue present in a wild-type sequence and, for example, is a cysteine ​​residue in a wild-type hinge region. In certain embodiments, at least one linkage connecting the first antigen-binding domain and the second antigen-binding domain is a disulfide bond formed by cross-linking cysteine ​​residues present in wild-type hinge regions with each other. Such cysteine ​​residues are present, for example, at positions 226 and / or 229 according to the EU numbering of a wild-type hinge region.

[00344] In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within an antibody fragment, and at least one is present within a hinge region. In an exemplary embodiment, the antigen-binding molecule of the present invention is F(ab')2, wherein both the first and second antigen-binding domains comprise a Fab and a re Petition 870260047205, dated 05 / 18 / 2026, page 84 / 2830 76 / 348 hinge region.

[00345] In one embodiment of the above aspects, the antigen-binding molecule of the present invention further comprises an Fc region and, for example, is a full-length antibody. In certain embodiments, one or more amino acid mutations that promote the multimerization of Fc regions are introduced into the Fc region of the antigen-binding molecule of the present invention. Such amino acid mutations include, for example, amino acid mutations at at least one position selected from the group consisting of positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440 and 447 according to EU numbering (see, for example, WO 2016 / 164480). In certain forms, multimerization is hexamerization. Antigens bound by antigen-binding molecules

[00346] In one embodiment of the above aspects, both the first and second antigen-binding domains bind to the same antigen. In certain embodiments, both the first and second antigen-binding domains bind to the same epitope on the same antigen. In certain other embodiments, each of the first and second antigen-binding domains binds to a different epitope on the same antigen. In certain embodiments, the antigen-binding molecule of the present invention is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets a specific antigen.

[00347] In another embodiment of the above aspects, each of the first and second antigen-binding domains binds to a different antigen.

[00348] In another embodiment of the above aspects, the antigen-binding molecule of the present invention is a fixation antigen-binding molecule (for example, a fixation antibody). A Petition 870260047205, dated 05 / 18 / 2026, p. 85 / 2830 77 / 348 antigen-binding molecule in this descriptive report means an antigen-binding molecule that specifically binds to an antigen / antigen-binding molecule complex formed between a given antigen A and an antigen-binding molecule that binds to antigen A, and that thereby increases the antigen-binding activity of the antigen-binding molecule that binds to antigen A (or alternatively, stabilizes the antigen / antigen-binding molecule complex formed by antigen A and the antigen-binding molecule that binds to antigen A).For example, a CD3-binding antibody specifically binds to the antigen-antibody complex formed between CD3 and an antibody with reduced binding capacity for CD3 (binding-attenuated CD3 antibody) and can thus increase the binding activity of the binding-attenuated CD3 antibody to CD3 (or alternatively, stabilize the antigen-antibody complex formed by CD3 and the binding-attenuated CD3 antibody). In certain embodiments, the first and / or second antigen-binding domains in the antigen-binding molecule of the present invention may be antigen-binding domains (fixation antigen-binding domains) from fixation antigen-binding molecules.

[00349] In one embodiment of the above aspects, both the first and second antigen-binding domains have the same amino acid sequence. In another embodiment, each of the first and second antigen-binding domains has a different amino acid sequence.

[00350] In one embodiment of the above aspects, 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. Functions of antigen-binding molecules Petition 870260047205, dated 05 / 18 / 2026, page 86 / 2830 78 / 348

[00351] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has the activity of retaining two antigen molecules in spatially close positions. In certain embodiments, the antigen-binding molecule of the present invention is capable of maintaining two antigen molecules in positions closer than a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00352] In another embodiment of the above aspects, the antigen-binding molecule of the present invention has interaction-regulating activity between two antigen molecules. Without being limited by any particular theory, the interaction-regulating activity is considered to be the result of the antigen-binding molecule of the present invention retaining two antigen molecules in spatially closer positions. In certain embodiments, the antigen-binding molecule of the present invention is able to increase or decrease the interaction between two antigen molecules compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller bond between the Petition 870260047205, dated 05 / 18 / 2026, page 87 / 2830 79 / 348 two antigen-binding domains. In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00353] In certain embodiments, the two antigen molecules linked by the antigen-binding molecule of the present invention are a ligand and a receptor thereof, respectively, and the antigen-binding molecule of the present invention has activity promoting the activation of the receptor by the ligand. In another embodiment, the two antigen molecules linked by the antigen-binding molecule of the present invention are an enzyme and a substrate thereof, respectively, and the antigen-binding molecule of the present invention has activity promoting the catalytic reaction of the enzyme with the substrate.

[00354] Furthermore, in certain other embodiments, both antigen molecules bound by the antigen-binding molecule of the present invention are antigens (e.g., proteins) present on cell surfaces, and the antigen-binding molecule of the present invention has activity that promotes interaction between a cell expressing the first antigen and a cell expressing the second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen are, respectively, a cell with cytotoxic activity and a target cell thereof, and the antigen-binding molecule of the present invention promotes damage to the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, a T cell, a cell Petition 870260047205, dated 05 / 18 / 2026, page 88 / 2830 80 / 348 NK cell, monocyte, or macrophage.

[00355] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has activation-regulating activity on two antigen molecules that are activated by association with each other. Without being limited by any particular theory, the activation-regulating activity is believed to result from the retention of two antigen molecules in spatially closer positions by the antigen-binding molecule of the present invention. In certain embodiments, the antigen-binding molecule of the present invention may increase or decrease the activation of two antigen molecules compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only insofar as the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). For example, such antigen molecules are selected from the group consisting of receptors belonging to the cytokine receptor superfamilies, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[00356] In one embodiment of the above aspects, in the antigen-binding molecule of the present invention, two antigen-binding domains Petition 870260047205, dated 05 / 18 / 2026, p. 89 / 2830 81 / 348 antigens are present in spatially close positions and / or the mobility of the two antigen-binding domains is reduced. In certain embodiments, compared to a control antigen-binding molecule, the antigen-binding molecule of the present invention has two antigen-binding domains that are present in closer positions and / or the mobility of the two antigen-binding domains is more reduced, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only by having a smaller linkage between the two antigen-binding domains.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00357] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has resistance to protease cleavage. In certain embodiments, the antigen-binding molecule of the present invention has increased resistance to protease cleavage compared with a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains. In another embodiment, the smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., residues Petition 870260047205, dated 05 / 18 / 2026, page 90 / 2830 82 / 348 cysteine ​​duos that are not present in the Fab or wild-type hinge region). In certain embodiments, in the antigen-binding molecule of the present invention, the proportion of the full-length molecule (e.g., full-length IgG molecule) remaining after protease treatment is increased compared to the control antigen-binding molecule. In certain embodiments, in the antigen-binding molecule of the present invention, the proportion of a particular fragment (e.g., Fab monomer) produced after protease treatment is reduced compared to the control antigen-binding molecule.

[00358] In one embodiment of the above aspects, when the antigen-binding molecule of the present invention is treated with a protease, a dimer of the antigen-binding domains or fragments thereof (e.g., cross-linked Fab dimer) is removed. In certain embodiments, when the control antigen-binding molecule, which differs from the antigen-binding molecule of the present invention only in that it has a smaller linkage between the two antigen-binding domains, is treated with the protease, monomers of the antigen-binding domains or fragments thereof are removed.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are 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.

[00359] In another embodiment, the control antigen-binding molecule differs from the antigen-binding molecule of the present Petition 870260047205, dated 05 / 18 / 2026, page 91 / 2830 83 / 348 the invention is based solely on the fact that it has a smaller linkage between the two antigen-binding domains, and a smaller linkage is one that is formed from mutated amino acid residues. The mutated amino acid residues are, for example, artificially introduced cysteine ​​residues. Pharmaceutical compositions

[00360] In one aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. Use of antigen-binding molecules

[00361] In one aspect, the present invention provides a method for maintaining two antigen molecules in spatially close positions, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together, and (c) contact the antigen-binding molecule produced in (b) with the two antigen molecules. In certain embodiments, the two antigen-binding domains in the antigen-binding molecule mentioned in (a) above may be linked together by one or more linkages, and in this case, some or all of the linkages are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region).In another embodiment, the at least one link mentioned in (b) above is a link in which the amino acid residues from which the link originates. Petition 870260047205, dated 05 / 18 / 2026, page 92 / 2830 84 / 348 between the antigen-binding domains are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). The present invention also provides a method for holding two antigen molecules in spatially close positions comprising contacting two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in retaining two antigen molecules in spatially close positions.

[00362] In another aspect, the present invention provides a method for regulating the interaction between two antigen molecules, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together, and (c) contact the antigen-binding molecule produced in (b) with the two antigen molecules. In certain embodiments, the two antigen-binding domains in the antigen-binding molecule mentioned in (a) above may be linked together by one or more linkages, and in this case, some or all of the linkages are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment- Petition 870260047205, dated 05 / 18 / 2026, page 93 / 2830 85 / 348 of, the aforementioned at least one linkage cited in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). The present invention also provides a method for regulating the interaction between two antigen molecules comprising contacting two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in regulating the interaction between two antigen molecules.

[00363] Furthermore, in another aspect, the present invention provides a method for regulating the activity of two antigen molecules that are activated through association with each other, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together, and (c) bring the antigen-binding molecule produced in (b) into contact with the two antigen molecules. In certain embodiments, the two antigen-binding domains in the antigen-binding molecule referred to in (a) above may be linked together by one or more linkages, and in this case, some or all of the linkages are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., Petition 870260047205, dated 05 / 18 / 2026, p. 94 / 2830 86 / 348 cysteine ​​residues in the hinge region). In another embodiment, said at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). The present invention also provides a method for regulating the activity of two antigen molecules that are activated through association with each other, comprising contacting two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention.The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in regulating the activity of two antigen molecules that are activated through association with each other.

[00364] In addition, in another aspect, the present invention provides a method for placing two antigen-binding domains in spatially close positions and / or reducing the mobility of two antigen-binding domains, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, and (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together. In certain embodiments, the two antigen-binding domains in the antigen-binding molecule referred to in (a) above may be linked together by one or more linkages, and in this case, some or all of the linkages are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are Petition 870260047205, dated 05 / 18 / 2026, p. 95 / 2830 87 / 348 so present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, said at least one linkage cited in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00365] In addition, in another aspect, the present invention provides a method for increasing the resistance of an antigen-binding molecule to cleavage by protease, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, and (b) add to the antigen-binding molecule at least one linkage that links the two antigen-binding domains together. In certain embodiments, the two antigen-binding domains in the antigen-binding molecule referred to in (a) above may be linked together by one or more linkages, and in this case, some or all of the linkages are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region).In another embodiment, the at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the Fab or hinge region). Petition 870260047205, dated 05 / 18 / 2026, p. 96 / 2830 88 / 348 wild type bradiça).

[00366] The antigen-binding molecule used in these various methods may have the characteristics of the antigen-binding molecules described here. Methods for producing antigen-binding molecules

[00367] In one aspect, the present invention provides a method for producing an antigen-binding molecule that has the activity of retaining two antigen molecules in spatially close positions, comprising: (a) 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, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) grow the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.In certain embodiments, each of the two antigen-binding domains mentioned in (a) above may comprise one or more amino acid residues from which the linkages to link the two antigen-binding domains originate and, in this case, some or all of the one or more amino acid residues from which the linkage between the domains originates. Petition 870260047205, dated 05 / 18 / 2026, p. 97 / 2830 89 / 348 antigen-binding domains are amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, said at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00368] In another aspect, the present invention provides a method for producing an antigen-binding molecule that has regulatory activity on the interaction between two antigen molecules, comprising: (a) 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, (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) cultivate the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages. In certain mo Petition 870260047205, dated 05 / 18 / 2026, page 98 / 2830 90 / 348 embodiments, each of the two antigen-binding domains mentioned in (a) above may comprise one or more amino acid residues from which the linkages to link the two antigen-binding domains originate and, in this case, some or all of the one or more amino acid residues from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, said at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00369] In addition, in another aspect, the present invention provides a method for producing an antigen-binding molecule that has activation-regulating activity between two antigen molecules that are activated through association with each other, comprising: (a) 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, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, Petition 870260047205, dated 05 / 18 / 2026, page 99 / 2830 91 / 348 (d) cultivate the host cell in such a way that both polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages. In certain embodiments, each of the two antigen-binding domains mentioned in (a) above may comprise one or more amino acid residues from which the linkages to link the two antigen-binding domains originate and, in this case, some or all of the one or more amino acid residues from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region).In another embodiment, the at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00370] In addition, in another aspect, the present invention provides a method for producing an antigen-binding molecule in which two antigen-binding domains are present in spatially close positions and / or the mobility of two antigen-binding domains is reduced, comprising: (a) 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, Petition 870260047205, dated 05 / 18 / 2026, p. 100 / 2830 92 / 348 (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) grow the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.In certain embodiments, each of the two antigen-binding domains mentioned in (a) above may comprise one or more amino acid residues from which the linkages to connect the two antigen-binding domains originate, and in this case, some or all of the one or more amino acid residues from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, the referred at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00371] In addition, in another aspect, the present invention provides a method for producing an antigen-binding molecule that has increased resistance to protease cleavage, comprising: Petition 870260047205, dated 05 / 18 / 2026, page 101 / 2830 93 / 348 (a) 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, (b) introduce a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) grow the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more linkages.In certain embodiments, each of the two antigen-binding domains mentioned in (a) above may comprise one or more amino acid residues from which the linkages to connect the two antigen-binding domains originate, and in this case, some or all of the one or more amino acid residues from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, said at least one linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the Fab or hinge region of the wild-type). Petition 870260047205, dated 05 / 18 / 2026, p. 102 / 2830 94 / 348 wild type).

[00372] The antigen-binding molecule produced in these various aspects may have the characteristics of the antigen-binding molecules described here. Methods for tracking antigen-binding molecules

[00373] In another aspect, the present invention provides a method for identifying a novel pair of protein molecules that are activated through association with each other, comprising: (a) provide two arbitrary protein molecules, (b) produce, by the production method of the present invention, an antigen-binding molecule comprising two antigen-binding domains that bind respectively to the two protein molecules, (c) bring the antigen-binding molecule produced in (b) into contact with the two protein molecules, and (d) evaluate whether the two protein molecules are activated or not.

[00374] In certain embodiments, at least one of the two protein molecules is selected from the group consisting of receptors belonging to superfamilies of cytokine receptors, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules. A. Exemplary antigen-binding molecules Structures of antigen-binding molecules

[00375] In one aspect, the present invention provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, and the antigen-binding domains are linked together by means of Petition 870260047205, dated 05 / 18 / 2026, page 103 / 2830 95 / 348 two or more linkages. In one embodiment, at least one of the first and second antigen-binding domains independently possesses antigen-binding activity (i.e., a single antigen-binding domain independently possesses antigen-binding activity). In certain embodiments, each of the first and second antigen-binding domains independently possesses antigen-binding activity.

[00376] In one embodiment of the above aspects, at least one of the first and second antigen-binding domains comprises an antibody fragment that binds to a particular antigen. In certain embodiments, the first and / or second antigen-binding domains comprise a hinge region. The amino acid residues from which the linkages between the antigen-binding domains originate are respectively present in the first and second antigen-binding domains, and the linkages between the antigen-binding domains are formed by the linkage of these amino acid residues. In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present in the antibody fragment. In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a hinge region.In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within the antibody fragment, and at least one of the amino acid residues is present within a hinge region.

[00377] In one embodiment of the above aspects, in at least one of the first and second antigen-binding domains, multiple amino acid residues from which the links between the domains Petition 870260047205, dated 05 / 18 / 2026, page 104 / 2830 96 / 348 Antigen-binding domains originate from positions seven or more amino acids apart in the primary structure. This means that between any two amino acid residues of the multiple amino acid residues mentioned above, six or more amino acid residues that are not the aforementioned amino acid residues are present. In certain embodiments, the combinations of multiple amino acid residues from which the linkages between antigen-binding domains originate include a pair of amino acid residues that are present at positions less than seven amino acid apart in the primary structure.In certain embodiments, if the first and second antigen-binding domains are linked together by three or more linkages, the linkages between the antigen-binding domains may originate from three or more amino acid residues, including a pair of amino acid residues that are present at positions seven or more amino acids apart in the primary structure.

[00378] In certain embodiments, amino acid residues present at the same position in the first antigen-binding domain and in the second antigen-binding domain are linked together to form a bond. In certain embodiments, amino acid residues present at a different position in the first antigen-binding domain and in the second antigen-binding domain are linked together to form a bond.

[00379] The positions of amino acid residues in the antigen-binding domain can be shown according to the Kabat numbering or EU numbering system (also called 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 from which the bonds between the first and second domains of Petition 870260047205, dated 05 / 18 / 2026, page 105 / 2830 97 / 348 If the amino acid residues from which the linkages between the first and second antigen-binding domains originate are present in identical corresponding positions in the antigen-binding domains, the position of these amino acid residues can be indicated by the same number according to the Kabat numbering or EU numbering system. Alternatively, if the amino acid residues from which the linkages between the first and second antigen-binding domains originate are present in different, non-corresponding positions in the antigen-binding domains, the positions of these amino acid residues can be indicated by different numbers according to the Kabat numbering or EU numbering system.

[00380] In one embodiment of the above aspects, at least one of the two or more bonds linking the antigen-binding domains is a covalent bond. In certain embodiments, the covalent bond is formed by direct crosslinking of an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain. The crosslinked amino acid residues are, for example, cysteine, and the covalent bond formed is, for example, a disulfide bond. At least one of the crosslinked cysteine ​​residues may be present within a hinge region.

[00381] In certain other embodiments, the 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 by means of a crosslinking agent. The crosslinking agent is, for example, an amine-reactive crosslinking agent, and the crosslinked amino acid residues are, for example, lysine.

[00382] In one embodiment of the above aspects, at least one of the two or more links connecting the linking domains to the anti- Petition 870260047205, dated 05 / 18 / 2026, page 106 / 2830 98 / 348 geno is a non-covalent bond. In certain embodiments, the non-covalent bond is an ionic bond, hydrogen bond, or hydrophobic bond.

[00383] In one embodiment of the above aspects, the antibody fragment is a Fab, Fab', scFab, Fv, scFv, or single-domain antibody.

[00384] In one embodiment of the above aspects, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a constant region. In certain embodiments, the amino acid residue is present in a CH1 region and, for example, is present at a selected position of the group consisting of positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213 and 214 according to the numbering of EU in the CH1 region. In an exemplary embodiment, the amino acid residue is present at position 191 according to the EU numbering in the CH1 region, and the amino acid residues at position 191 according to the EU numbering in the CH1 region of the two antigen-binding domains are linked together to form a bond.

[00385] In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a hinge region and, for example, is present at a selected position of the group consisting of positions 216, 218 and 219 according to the EU numbering in the hinge region.

[00386] In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a CL region and, for example, is present at a selected position of the group that Petition 870260047205, dated 05 / 18 / 2026, page 107 / 2830 99 / 348 consists of positions 109, 112, 121, 126, 128, 151, 152, 153, 156, 184, 186, 188, 190, 200, 201, 202, 203, 208, 210, 211, 212, and 213 according to EU numbering in the CL region. In an exemplary embodiment, the amino acid residue is present at position 126 according to EU numbering in the CL region, and the amino acid residues at position 126 according to EU numbering in the CL region of the two antigen-binding domains are linked together to form a bond.

[00387] In certain embodiments, 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.

[00388] In one embodiment of the above aspects, the constant region is derived from human. In certain embodiments, the subclass of the heavy chain constant region is any one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In certain embodiments, the subclass of the CH1 region is any one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε. In certain embodiments, the subclass of the CL region is κ or λ.

[00389] In one embodiment of the above aspects, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is present within a variable region. In certain embodiments, the amino acid residue is present in a VH region and, for example, is present at a selected position of the group consisting of positions 8, 16, 28, 74, and 82b according to Kabat numbering in the VH region. In Petition 870260047205, dated 05 / 18 / 2026, page 108 / 2830 100 / 348 In certain embodiments, the amino acid residue is present in a VL region and, for example, is present in a position selected from the group consisting of positions 100, 105 and 107 according to Kabat numbering in the VL region.

[00390] In one embodiment of the above aspects, the first and second antigen-binding domains comprise a Fab and a hinge region.

[00391] In certain embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is an amino acid residue present in a wild-type Fab or hinge region and, for example, is a cysteine ​​residue in the hinge region. Examples of such cysteine ​​residues include the cysteine ​​residues at positions 226 and 229 according to EU numbering.

[00392] In certain other embodiments, at least one of the amino acid residues from which the linkages between the antigen-binding domains originate is a mutated amino acid residue that is not present in a wild-type Fab or hinge region and, for example, is a cysteine ​​residue that is not present in a wild-type Fab or hinge region. Such a mutated amino acid residue can be introduced into a wild-type Fab or hinge region, for example, through an amino acid substitution method. This descriptive report discloses the amino acid residue sites from which the linkages between the antigen-binding domains can originate for each of the CH1, hinge, CL, VH, and VL regions and, for example, cysteine ​​residues can be introduced into such sites.

[00393] Alternatively, in another embodiment, an amino acid residue that is present in a wild-type Fab or hinge region and that is involved in a linkage between domains Petition 870260047205, dated 05 / 18 / 2026, page 109 / 2830 The 101 / 348 antigen-binding residue (e.g., a cysteine ​​residue) can be replaced by another amino acid or deleted. Examples of such cysteine ​​residues include the cysteine ​​residues at positions 220, 226, and 229 according to the EU numbering in the hinge region and the cysteine ​​residue at position 214 in the CL region.

[00394] In certain embodiments, the antigen-binding molecule of the present invention is F(ab')2, wherein both the first and second antigen-binding domains comprise a Fab and a hinge region.

[00395] In one embodiment of the above aspects, at least one of the first and second antigen-binding domains comprises a non-antibody protein binding to a particular antigen, or a fragment thereof. In certain embodiments, the non-antibody protein is a ligand-receptor pair that binds specifically to each other. Such receptors include, for example, receptors belonging to cytokine receptor superfamilies, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[00396] In one embodiment of the above aspects, the antigen-binding molecule of the present invention further comprises an Fc region and, for example, is a full-length antibody. In certain embodiments, one or more amino acid mutations that promote the multimerization of Fc regions are introduced into the Fc region of the antigen-binding molecule of the present invention. Such amino acid mutations include, for example, amino acid mutations at at least one position selected from the group consisting of positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440 and 447 according to the numbering. Petition 870260047205, dated 05 / 18 / 2026, p. 110 / 2830 102 / 348 EU regulation (see, for example, WO 2016 / 164480). In certain embodiments, multimerization is hexamerization. Antigens bound by antigen-binding molecules

[00397] In one embodiment of the above aspects, both the first and second antigen-binding domains bind to the same antigen. In certain embodiments, the first and second antigen-binding domains bind to the same epitope on the same antigen. In certain other embodiments, each of the first and second antigen-binding domains binds to a different epitope on the same antigen. In certain embodiments, the antigen-binding molecule of the present invention is a biparatopic antigen-binding molecule (e.g., biparatopic antibody) that targets a specific antigen.

[00398] In one embodiment of the above aspects, each of the first and second antigen-binding domains binds to a different antigen.

[00399] In another embodiment of the above aspects, the antigen-binding molecule of the present invention is a fixation antigen-binding molecule (e.g., fixation antibody). Here, a fixation antigen-binding molecule refers to an antigen-binding molecule that specifically binds to an antigen / antigen-binding molecule complex formed by a given antigen A and an antigen-binding molecule that binds to antigen A, and thus enhances the activity of the antigen-binding molecule that binds to antigen A to bind antigen A (or stabilize the antigen / antigen-binding molecule complex formed by antigen A and the antigen-binding molecule that binds to antigen A). For example, a CD3 fixation antibody is capable of binding to an antigen-antibody complex formed by CD3 and an antibody with attenuated CD3-binding capacity (CD3 antibody). Petition 870260047205, dated 05 / 18 / 2026, p. 111 / 2830 103 / 348 attenuated by binding) and thus increase the CD3-binding activity of the CD3-attenuated antibody (or stabilize the antigen-antibody complex formed by CD3 and the CD3-attenuated antibody). In certain embodiments, the first and / or second antigen-binding domains in the antigen-binding molecule of the present invention may be antigen-binding domains derived from fixation antigen-binding molecules (fixation antigen-binding domains).

[00400] In one embodiment of the above aspects, both the first and second antigen-binding domains have the same amino acid sequence. In another embodiment, each of the first and second antigen-binding domains has a different amino acid sequence.

[00401] In one embodiment of the above aspects, 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. Functions of antigen-binding molecules

[00402] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has the activity of retaining two antigen molecules in spatially close positions. In certain embodiments, the antigen-binding molecule of the present invention is capable of maintaining two antigen molecules in positions closer than a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains. In another embodiment, a smaller linkage can be selected from linkages in which the amino acid residues from which the linkages between the Petition 870260047205, dated 05 / 18 / 2026, page 112 / 2830 104 / 348 antigen-binding domains originate from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00403] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has interaction-regulating activity between two antigen molecules. Without being limited by any particular theory, the interaction-regulating activity is considered to result from the retention of two antigen molecules in spatially closer positions by the antigen-binding molecule of the present invention. In certain embodiments, the antigen-binding molecule of the present invention is able to increase or decrease the interaction between two antigen molecules compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00404] In certain embodiments, the two antigen molecules linked by the antigen-binding molecule of the present invention are a ligand and a receptor thereof, respectively, and the antigen-binding molecule of the present invention has ligand-promoting receptor activation activity. In another embodiment... Petition 870260047205, dated 05 / 18 / 2026, p. 113 / 2830 105 / 348 In fact, the two antigen molecules linked by the antigen-binding molecule of the present invention are an enzyme and a substrate thereof, respectively, and the antigen-binding molecule of the present invention has activity in promoting the catalytic reaction of the enzyme with the substrate.

[00405] Furthermore, in certain other embodiments, both antigen molecules bound by the antigen-binding molecule of the present invention are antigens (e.g., proteins) present on cell surfaces, and the antigen-binding molecule of the present invention has activity that promotes interaction between a cell expressing the first antigen and a cell expressing the second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen are, respectively, a cell with cytotoxic activity and a target cell thereof, and the antigen-binding molecule of the present invention promotes damage to the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, a T cell, NK cell, monocyte, or macrophage.

[00406] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has activation-regulating activity on two antigen molecules that are activated through association with each other. Without being limited by any particular theory, it is believed that the activation-regulating activity results from the retention of two antigen molecules in spatially closer positions by the antigen-binding molecule of the present invention. In certain embodiments, the antigen-binding molecule of the present invention may increase or decrease the activation of two antigen molecules compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention by Petition 870260047205, dated 05 / 18 / 2026, page 114 / 2830 106 / 348 insofar as the control antigen-binding molecule has a minor linkage between the two antigen-binding domains. In another embodiment, a minor linkage may be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). For example, such antigen molecules are selected from the group consisting of receptors belonging to the cytokine receptor superfamilies, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[00407] In one embodiment of the above aspects, the antigen-binding molecule of the present invention has resistance to protease cleavage. In certain embodiments, the antigen-binding molecule of the present invention has increased protease cleavage resistance compared to a control antigen-binding molecule, and the control antigen-binding molecule differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule has a smaller linkage between the two antigen-binding domains. In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the Fab or fold region). Petition 870260047205, dated 05 / 18 / 2026, page 115 / 2830 107 / 348 wild-type diça). In certain embodiments, in the antigen-binding molecule of the present invention, the proportion of the full-length molecule (e.g., full-length IgG molecule) remaining after protease treatment is increased compared to the control antigen-binding molecule. In certain embodiments, in the antigen-binding molecule of the present invention, the proportion of a particular fragment (e.g., Fab monomer) produced after protease treatment is reduced compared to the control antigen-binding molecule.

[00408] In one embodiment of the above aspects, when the antigen-binding molecule of the present invention is treated with a protease, a dimer of the antigen-binding domains or fragments thereof (e.g., cross-linked Fab dimer) is removed. In certain embodiments, when the control antigen-binding molecule, which differs from the antigen-binding molecule of the present invention only by having a smaller linkage between the two antigen-binding domains, is treated with the protease, monomers of the antigen-binding domains or fragments thereof are removed.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are 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. Pharmaceutical compositions.

[00409] In one aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. Petition 870260047205, dated 05 / 18 / 2026, page 116 / 2830 108 / 348 Use of antigen-binding molecules

[00410] In one aspect, the present invention provides a method for maintaining two antigen molecules in spatially close positions, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more bonds, (b) adding to the antigen-binding molecule another bond that links the two antigen-binding domains together, and (c) bringing the antigen-binding molecule produced in (b) into contact with the two antigen-binding molecules.

[00411] In certain embodiments, some or all of the linkages mentioned in (a) above are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, said linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).The present invention also provides a method for maintaining two antigen molecules in spatially close positions comprising contacting the two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or pharmaceutical composition of the present invention. Petition 870260047205, dated 05 / 18 / 2026, page 117 / 2830 109 / 348 invention for use in retaining two antigen molecules in spatially close positions.

[00412] In another aspect, the present invention provides a method for regulating 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 together by one or more bonds, (b) adding to the antigen-binding molecule another bond that links the two antigen-binding domains together, and (c) bringing the antigen-binding molecule produced in (b) into contact with the two antigen-binding molecules.

[00413] In certain embodiments, some or all of the linkages mentioned in (a) above are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, the other linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).The present invention also provides a method for regulating the interaction between two antigen molecules comprising contacting two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. The present invention further provides an antigen-binding molecule or... Petition 870260047205, dated 05 / 18 / 2026, page 118 / 2830 110 / 348 pharmaceutical composition of the present invention for use in regulating the interaction between two antigen molecules.

[00414] Furthermore, in another aspect, the present invention provides a method for regulating the activity of two antigen molecules that are activated through association with each other, comprising: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more bonds, (b) adding to the antigen-binding molecule another bond that links the two antigen-binding domains together, and (c) bringing the antigen-binding molecule produced in (b) into contact with the two antigen-binding molecules.

[00415] In certain embodiments, some or all of the linkages mentioned in (a) above are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, the other linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).The present invention also provides a method for regulating the activity of two antigen molecules that are activated through association with each other, comprising contact of two antigen molecules with the antigen-binding molecule or pharmaceutical composition of the present invention. Petition 870260047205, dated 05 / 18 / 2026, page 119 / 2830 111 / 348 further provides an antigen-binding molecule or pharmaceutical composition of the present invention for use in regulating the activity of two antigen molecules that are activated through association with each other.

[00416] In addition, in another aspect, the present invention provides a method for increasing the resistance of an antigen-binding molecule to cleavage by protease, comprising: (a) provide an antigen-binding molecule comprising two antigen-binding domains, wherein the two antigen-binding domains are linked together by one or more bonds, and (b) add to the antigen-binding molecule another bond that links the two antigen-binding domains together.

[00417] In certain embodiments, some or all of the linkages mentioned in (a) above are linkages in which the amino acid residues from which the linkages between the antigen-binding domains originate are derived from amino acid residues that are present in a wild-type Fab or hinge region (e.g., cysteine ​​residues in the hinge region). In another embodiment, the other linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00418] The antigen-binding molecule used in these various methods may have the characteristics of the antigen-binding molecules described here. Methods for producing antigen-binding molecules Petition 870260047205, dated 05 / 18 / 2026, page 120 / 2830 112 / 348

[00419] In one aspect, the present invention provides a method for producing an antigen-binding molecule that has the activity of retaining two antigen molecules in spatially close positions, comprising: (a) provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains comprises one or more amino acid residues from which a linkage to connect the two antigen-binding domains originates, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that another linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) cultivate the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains,in which the two antigen-binding domains are linked together by two or more bonds.

[00420] In certain embodiments, some or all of the one or more amino acid residues mentioned in (a) above from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (for example, cysteine ​​residues in the hinge region). In another embodiment, the linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage originates are present in a wild-type Fab or hinge region (for example, cysteine ​​residues in the hinge region). Petition 870260047205, dated 05 / 18 / 2026, page 121 / 2830 113 / 348 originates the linkage between antigen-binding domains derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00421] In another aspect, the present invention provides a method for producing an antigen-binding molecule that has regulatory activity on the interaction between two antigen molecules, comprising: (a) provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains comprises one or more amino acid residues from which a linkage to connect the two antigen-binding domains originates, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that another linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) cultivate the host cell such that the two polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains,in which the two antigen-binding domains are linked together by two or more bonds.

[00422] In certain embodiments, some or all of the one or more amino acid residues mentioned in (a) above from which the originates Petition 870260047205, dated 05 / 18 / 2026, page 122 / 2830 114 / 348 The linkage between antigen-binding domains is an amino acid residue that is present in a wild-type Fab or hinge region (for example, cysteine ​​residues in the hinge region). In another embodiment, the aforementioned linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (for example, cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00423] In addition, in another aspect, the present invention provides a method for producing an antigen-binding molecule that has activation-regulating activity between two antigen molecules that are activated through association with each other, comprising: (a) provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains comprises one or more amino acid residues from which a linkage to connect the two antigen-binding domains originates, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that another linkage connecting the two antigen-binding domains is added, (c) introduce the nucleic acids produced in (b) into a host cell, (d) cultivate the host cell such that the two polypeptides are expressed, and Petition 870260047205, dated 05 / 18 / 2026, page 123 / 2830 115 / 348 (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by means of two or more linkages.

[00424] In certain embodiments, some or all of the one or more amino acid residues mentioned in (a) above from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (for example, cysteine ​​residues in the hinge region). In another embodiment, the linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (for example, cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00425] In addition, in another aspect, the present invention provides a method for producing an antigen-binding molecule that has increased resistance to protease cleavage, comprising: (a) provide a nucleic acid encoding a polypeptide comprising a first antigen-binding domain and a nucleic acid encoding a polypeptide comprising a second antigen-binding domain, wherein each of the two antigen-binding domains comprises one or more amino acid residues from which a linkage to connect the two antigen-binding domains originates, (b) introduce a mutation into the nucleic acids encoding the two antigen-binding domains such that another linkage connecting the two antigen-binding domains is added, Petition 870260047205, dated 05 / 18 / 2026, page 124 / 2830 116 / 348 (c) introduce the nucleic acids produced in (b) into a host cell, (d) cultivate the host cell in such a way that both polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by means of two or more linkages.

[00426] In certain embodiments, some or all of the one or more amino acid residues mentioned in (a) above from which the linkage between the antigen-binding domains originates are amino acid residues that are present in a wild-type Fab or hinge region (for example, cysteine ​​residues in the hinge region). In another embodiment, the linkage mentioned in (b) above is a linkage in which the amino acid residues from which the linkage between the antigen-binding domains originates are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (for example, cysteine ​​residues that are not present in the wild-type Fab or hinge region).

[00427] The antigen-binding molecule produced in these various aspects may have the characteristics of the antigen-binding molecules described here. Methods for tracking antigen-binding molecules

[00428] In another aspect, the present invention provides a method for identifying a novel pair of protein molecules that are activated through association with each other, comprising: (a) provide two arbitrary protein molecules, (b) produce, by the production method of the present invention, an antigen-binding molecule comprising two domains Petition 870260047205, dated 05 / 18 / 2026, page 125 / 2830 117 / 348 antigen-binding molecules that bind respectively to the two protein molecules, wherein the antigen-binding molecule has the activity of keeping the two protein molecules in close positions, (c) bringing the antigen-binding molecule produced in (b) into contact with the two protein molecules, and (d) evaluating whether the two protein molecules are activated or not.

[00429] In certain embodiments, at least one of the protein molecules is selected from the group consisting of receptors belonging to the cytokine receptor superfamilies, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules. Binding of antigen-binding domains

[00430] In a non-limiting embodiment, two or more antigen-binding domains contained in an antigen-binding molecule of the present invention are linked together by one or more bonds. In a preferred embodiment, an antigen-binding domain contained in an antigen-binding molecule of the present invention has, individually, activity to bind to an antigen. In such an embodiment, the antigen-binding molecule of the present invention containing two antigen-binding domains can bind to two or more antigen molecules; the antigen-binding molecule of the present invention containing three antigen-binding domains can bind to three or more antigen molecules; the antigen-binding molecule of the present invention containing four antigen-binding domains can bind to four or more antigen molecules; and the antigen-binding molecule of the present invention... Petition 870260047205, dated 05 / 18 / 2026, page 126 / 2830 118 / 348 invention containing N antigen-binding domains can bind to N or more antigen molecules.

[00431] In certain embodiments, at least one of the linkages between the antigen-binding domains contained in an antigen-binding molecule of the present invention is different from a linkage found in a naturally occurring antibody (e.g., in a Fab or wild-type hinge region). Examples of the linkages found between the antigen-binding domains of a naturally occurring antibody (e.g., naturally occurring IgG antibody) include disulfide linkages in the hinge region. The linkages between amino acid residues positioned in a region other than the hinge region may be linkages between amino acid residues within an antibody fragment (e.g., Fab) and include linkages between heavy chains (HH), linkages between light chains (LL), and linkages between heavy and light chains (HL or LH) (see Figure 1).Examples of amino acid residues in the heavy or light chain from which the linkages between antigen-binding domains originate include amino acid residues in the aforementioned positions within the variable region (VH region or VL region) or within the constant region (CH1 region, hinge region, or CL region).

[00432] In a non-limiting embodiment, the linkages between antigen-binding domains may originate from several amino acid residues present in positions separated from each other in the primary structure in at least one of two or more antigen-binding domains contained in an antigen-binding molecule of the present invention. The distance between the multiple amino acid residues is a distance that allows obtaining the structures of two or more antigen-binding domains sufficiently close as a result of the linkage between the antigen-binding domains. Petition 870260047205, dated 05 / 18 / 2026, p. 127 / 2830 119 / 348 antigen by the bonds that originate from amino acid residues.The distance between multiple amino acid residues can be, for example, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 7 or more amino acids, 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 11 or more amino acids, 12 or more amino acids, 13 or more amino acids, 14 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 30 or more amino acids, 35 or more amino acids, 40 or more amino acids, 45 or more amino acids, 50 or more amino acids, 60 or more amino acids, 70 or more amino acids, 80 or more amino acids, 90 or more amino acids, 100 or more amino acids, 110 or more amino acids, 120 or more amino acids, 130 or more amino acids, 140 or more amino acids, 150 or more amino acids, 160 or more amino acids, 170 or more amino acids, 180 or more amino acids. 190 amino acids or more, 200 amino acids or more, 210 amino acids or more, or 220 amino acids or more.

[00433] Furthermore, the number of linkages between antigen-binding domains and the number of amino acid residues from which the linkages originate is a number that allows the formation of structures of two or more antigen-binding domains sufficiently close together as a result of the linkage between the antigen-binding domains by the linkages. The number can be, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more.

[00434] In certain embodiments, provided that the structures of two or more sufficiently close antigen-binding domains are achieved as a result of the linkage between the antigen-binding domains by three or more linkages that respectively originate from three or more amino acid residues in the antigen-binding domains, the distance in the primary structure between any two Petition 870260047205, dated 05 / 18 / 2026, p. 128 / 2830 120 / 348 amino acid residues selected from the three amino acid residues may be seven or more amino acids in at least one pair of amino acid residues and may be less than seven amino acids in the remaining pairs of amino acid residues.

[00435] In connection with the antigen-binding domains contained in the antigen-binding molecules of the present invention, sufficiently close means that two or more antigen-binding domains are close enough to achieve the desired functions (activities) of the antigen-binding molecule of the present invention.Examples of desired functions (activities) include the activity of maintaining two antigen molecules in spatially close positions; the activity of regulating 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 with a substrate; the activity of promoting the interaction between a cell expressing a first antigen and a cell expressing a second antigen; the activity of promoting damage to a target cell by a cell with cytotoxic activity (such as a T cell, NK cell, monocyte, macrophage); the activity of regulating the activation of two antigen molecules that are activated through association with each other; and resistance to protease cleavage of antigen-binding molecules.

[00436] In a non-limiting embodiment, the linkage between the antigen-binding domains contained in an antigen-binding molecule of the present invention may be a covalent bond or a non-covalent bond. The covalent bond may be a covalent bond formed by the direct crosslinking of an amino acid residue in a first antigen-binding domain and an amino acid residue of a second antigen-binding domain, for example, a disulfide bond between cysteine ​​residues. The residue Petition 870260047205, dated 05 / 18 / 2026, p. 129 / 2830 The 121 / 348 amino acid duo directly cross-linked may be present in an antibody fragment such as Fab, or within a hinge region.

[00437] In another embodiment, a covalent bond is formed by crosslinking an amino acid residue in a first antigen-binding domain and an amino acid residue in a second antigen-binding domain by means of a crosslinking agent. For example, when an amine-reactive crosslinking agent is used for crosslinking, the crosslinking can be done through a free amino group of the N-terminal amino acid of the antigen-binding domain, or a primary amine of the side chain of a lysine residue in the antigen-binding domain. Amine-reactive crosslinking agents include a functional group that forms a chemical bond with a primary amine, such as isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluoroester.Representative examples include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl souberate), BS3 (bis(sulfosuccinimidyl) souberate), DSP (dithiobis(succinimidyl propionate)), DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), BSOCOES (bis(2(succinimidooxycarbonyloxy)ethyl)sulfone), EGS (ethylene glycol bis(succinimidyl succinate)), Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), and DFDNB (1,5-difluoro-2,4-dinitrobenzene). Examples of other crosslinking agents include carboxyl / amine reactive, sulfhydryl reactive, aldehyde reactive, and light-reactive crosslinking agents.

[00438] The non-covalent link to connect the antigen-binding domains can be an ionic bond, a hydrogen bond, or a li Petition 870260047205, dated 05 / 18 / 2026, page 130 / 2830 122 / 348 hydrophobic coating.

[00439] If the number of linkages between antigen-binding domains is greater than that of a control antigen-binding molecule (e.g., an antigen-binding molecule having a structure substantially similar to a naturally occurring antibody structure), it can be assessed, 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 cleaves the antigen-binding domain (e.g., a protease that cleaves the N-terminal side of the crosslinking site of hinge regions such as papain and Lys-C) and then subjected to non-reducing electrophoresis. Then, an antibody that recognizes a portion of the antigen-binding domain (e.g., HRP-labeled anti-cap chain antibody) is used to detect fragments that are present after protease treatment.When only one antigen-binding domain monomer (e.g., Fab monomer) is detected for the control antigen-binding molecule and one antigen-binding domain multimer (e.g., Fab dimer) is detected for the antigen-binding molecule of interest, then it can be assessed that the number of linkages between the antigen-binding domains of the antigen-binding molecule of interest is greater than that of the control antigen-binding molecule.

[00440] The formation of a disulfide bond between cysteines in a modified antigen-binding molecule produced by introducing cysteines 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 then the mass of peptides expected to be generated is measured. Petition 870260047205, dated 05 / 18 / 2026, page 131 / 2830 123 / 348 from the amino acid sequence of each antibody is detected by LC / MS. If a component corresponding to the theoretical mass of a peptide that should be generated when the newly introduced cysteines form a disulfide bond is detected, the introduced cysteines can be assessed as having formed a disulfide bond. Furthermore, if this component becomes undetectable when the sample containing the aforementioned antigen-binding molecule is analyzed after the addition of an agent to reduce disulfide bonds (e.g., tris(2-carboxyethyl)phosphine) to the sample, the accuracy of the above assessment will still be strongly verified. Resistance to protease cleavage

[00441] In a non-limiting embodiment, the antigen-binding molecule of the present invention has resistance to protease cleavage. In certain embodiments, the resistance to protease cleavage of the antigen-binding molecule of the present invention is increased compared to a control antigen-binding molecule (e.g., an antigen-binding molecule having a structure substantially similar to a naturally occurring antibody structure) where the number of linkages between the antigen-binding domains is lower by one or more compared to the antigen-binding molecule.In another embodiment, a smaller linkage can be selected from linkages where the amino acid residues from which the linkages between the antigen-binding domains originate are derived from mutated amino acid residues that are not present in a wild-type Fab or hinge region (e.g., cysteine ​​residues that are not present in the wild-type Fab or hinge region). If the proportion of the full-length molecule (e.g., full-length IgG molecule) remaining after protease treatment is increased, or the proportion of a particulate fragment... Petition 870260047205, dated 05 / 18 / 2026, page 132 / 2830 124 / 348 lar (e.g., Fab monomer) produced after protease treatment is reduced to an antigen-binding molecule compared to a control antigen-binding molecule, so it can be assessed that resistance to protease cleavage is increased (protease resistance is improved).

[00442] In certain embodiments, the proportion of the remaining total length molecule after protease treatment may be, in relation to all antigen-binding molecules, for example, 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 7.5% or more, 10% or more, 12.5% ​​or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more or 50% or more. In certain other embodiments, the proportion of a monomer of an antigen-binding domain (e.g., Fab) produced after protease treatment may be, relative to all antigen-binding molecules, 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.In certain other embodiments, the proportion of a dimer of an antigen-binding domain (e.g., Fab) produced after protease treatment may be, relative to all antigen-binding molecules, 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.

[00443] Examples of proteases include, but are not limited to Petition 870260047205, dated 05 / 18 / 2026, p. 133 / 2830 125 / 348 these, Lys-C, plasmin, human neutrophil elastase (HNE) and papain.

[00444] In another aspect, an antigen-binding molecule according to any of the above embodiments may incorporate any of the features, individually or in combination, as described in Sections 1 to 7 below: 1. Affinity of the Binding Molecule to the Antigen

[00445] In certain embodiments, an antigen-binding molecule provided herein has a dissociation constant (KD) of 1 micro M or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less or 0.001 nM or less (e.g., 10 to 8 M or less, e.g., from 10 to 8 M to 10 to 13 M, e.g., from 10 to 9 M to 10 to 13 M). 2. Antibody Fragments

[00446] In certain embodiments, an antigen-binding molecule provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments and other fragments described herein. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For the argument regarding the Fab and F(ab')2 fragments comprising epitope-binding residues for the recovery receptor and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[00447] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med. Petition 870260047205, dated 05 / 18 / 2026, p. 134 / 2830 126 / 348 9:129-134 (2003); and 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). 3. Chimeric and Humanized Antibodies

[00448] In certain embodiments, an antigen-binding molecule provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a class-switched antibody in which the class or subclass has been altered from that of the precursor antibody. Chimeric antibodies include their antigen-binding fragments.

[00449] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the non-human precursor antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, for example, CDRs, (or parts thereof) are derived from a non-human antibody, and FRs (or parts thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve the antibody's specificity or affinity. Petition 870260047205, dated 05 / 18 / 2026, p. 135 / 2830 127 / 348 4. Human Antibodies

[00450] In certain embodiments, an antigen-binding molecule provided here is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described in general in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). 5. Library-Derived Antigen-Binding Molecules

[00451] The antigen-binding molecules of the invention can be isolated by combinatorial library screening for antigen-binding molecules with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antigen-binding molecules possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Academic. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004). 6. Multispecific Antigen-Binding Molecules

[00452] In certain embodiments, an antigen-binding molecule provided here is a multispecific antigen-binding molecule, for example, a bispecific antigen-binding molecule. Multispecific antigen-binding molecules are monoclonal antigen-binding molecules that possess specificities of Petition 870260047205, dated 05 / 18 / 2026, page 136 / 2830 128 / 348 binding to at least two different sites. In certain embodiments, one of the binding specificities is for a particular antigen (e.g., CD3) and the other is for any other antigen (e.g., CD28 or cancer antigen). In certain embodiments, bispecific antigen-binding molecules can bind to two different epitopes on a single antigen. Bispecific antigen-binding molecules can be prepared as full-length antibodies or antibody fragments.

[00453] Techniques for constructing multispecific antigen-binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole engineering (see, for example, US Patent No. 5,731,168). Multispecific antigen-binding molecules can also be constructed by engineering electrostatic steering effects for the production of Fc heterodimeric antibody molecules (WO 2009 / 089004A1); Crosslinking of two or more antibodies or fragments (see, for example, US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-stranded Fv dimers (scFv) (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[00454] Engineered antibodies with three or more functional antigen-binding sites, including Octopus antibodies, are also Petition 870260047205, dated 05 / 18 / 2026, p. 137 / 2830 129 / 348 included in this invention (see, for example, US 2006 / 0025576A1). 7. Variants of Antigen-Binding Molecules

[00455] In certain embodiments, variants of the amino acid sequence of the antigen-binding molecules provided in this invention are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecule. Variants of the amino acid sequence of an antigen-binding molecule can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antigen-binding molecule or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequences of the antigen-binding molecule. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, for example, antigen binding. a) Replacement, Insertion and Deletion Variants

[00456] In certain embodiments, variants of the antigen-binding molecule having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in the table below under the heading of preferred substitutions. More substantial changes are provided in the table under the heading of exemplary substitutions, and as further described below, with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antigen-binding molecule of interest and the products screened for a desired activity, e.g., retained / enhanced antigen binding, decreased immunogenicity, or enhanced ADCC or CDC. Petition 870260047205, dated 05 / 18 / 2026, p. 138 / 2830 130 / 348 Original Residue Exemplary Substitutions Preferred Substitutions Wing (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Only Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; But; No; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; But; No; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; No; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; But; Phe; No; Norleucine Leu

[00457] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[00458] Non-conservative substitutions will involve the exchange of Petition 870260047205, dated 05 / 18 / 2026, page 139 / 2830 131 / 348 a member of one of these classes by another class.

[00459] One type of substitution variant involves the replacement of one or more residues in the hypervariable region of a precursor antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have modifications (e.g., enhancements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the precursor antigen-binding molecule and / or will have certain biological properties substantially retained from the precursor antigen-binding molecule. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein.In short, one or more HVR residues are modified and the variant antibodies displayed on the phage are screened for particular biological activity (e.g., binding affinity).

[00460] Alterations (e.g., substitutions) can be made to HVRs, for example, to improve the affinity of the binding molecule to the antigen. Such alterations can be made at HVR access points, i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that bring the antigen into contact with the resulting VH or VL variant being tested for binding affinity. Affinity maturation by constructing and reselecting secondary libraries has been 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 affinity maturation modalities, diversity is introduced into the selected variable genes. Petition 870260047205, dated 05 / 18 / 2026, p. 140 / 2830 132 / 348 for maturation through any of a variety of methods (e.g., error-prone PCR, chain rearrangement, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antigen-binding molecule variants with the desired affinity. Another method for introducing diversity involves HVR-targeted approaches, in which multiple HVR residues (e.g., 4 to 6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using mutagenesis or alanine scan modeling. CDR-H3 and CDR-L3 in particular are frequently targeted.

[00461] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the antigen-binding molecule's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside the antigen-contact residues in HVRs. In certain embodiments of the VH and VL variant sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[00462] A useful method for identifying residues or regions of an antigen-binding molecule that can be targeted for mutagenesis is termed alanine scavenging mutagenesis, as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a target residue or group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or charged amino acid. Petition 870260047205, dated 05 / 18 / 2026, page 141 / 2830 133 / 348 of negatively (e.g., alanine or polyalanine) to determine if the interaction of the antigen-binding molecule with the antigen is affected. Additional substitutions can be introduced at amino acid sites demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antigen-binding molecule complex can be analyzed to identify contact points between the antigen-binding molecule and the antigen. These contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired properties.

[00463] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions that vary in length by one residue with polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antigen-binding molecule with an N-terminal methionyl residue. Other variants of antigen-binding molecule insertion include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antigen-binding molecule to the N- or C-terminal of the antigen-binding molecule. b) Glycosylation variants

[00464] In certain embodiments, an antigen-binding molecule provided in this invention is altered to increase or decrease the extent to which the antigen-binding molecule is glycosylated. The addition or deletion of glycosylation sites to an antigen-binding molecule can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[00465] When the antigen-binding molecule comprises Petition 870260047205, dated 05 / 18 / 2026, p. 142 / 2830 134 / 348 an Fc region, the carbohydrate attached to it can be altered. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide that is usually linked by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose linked to a GlcNAc in the stem of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antigen-binding molecule of the invention can be made in order to create variants of the antigen-binding molecule with certain enhanced properties.

[00466] In one embodiment, variants of the antigen-binding molecule are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antigen-binding molecule may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain in Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example.Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antigen-binding molecules. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publications Nos. Petition 870260047205, dated 05 / 18 / 2026, page 143 / 2830 135 / 348 US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to variants of defucosylated or fucose-deficient antigen-binding molecules include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; 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 CHO Lec13 cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially in Example 11), and neutralized cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, CHO-neutralized 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).

[00467] Variants of the antigen-binding molecule are further provided with two-part oligosaccharides, for example, where a biantennary oligosaccharide linked to the Fc region of the antigen-binding molecule is split in two by GlcNAc. Such variants of the antigen-binding molecule may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants of the antigen-binding molecule with at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such variants of the molecule Petition 870260047205, dated 05 / 18 / 2026, page 144 / 2830 136 / 348 antigen-binding molecules may have enhanced CDC function. Such variants of the antigen-binding molecule are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Variants of the Fc region

[00468] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antigen-binding molecule provided in this invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[00469] In certain embodiments, the invention contemplates a variant of the antigen-binding molecule that possesses some, but not all, effector functions, making it a desirable candidate for applications where the half-life of the antigen-binding molecule in vivo is important, although certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antigen-binding molecule lacks Fc gamma R binding (therefore, probably no ADCC activity), but retains the ability to bind to FcRn. The primary cells for mediating ADCC, NK cells, express only Fc gamma RIII, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII.The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457492 (1991). Non-limiting examples of in vitro assays to evaluate the... Petition 870260047205, dated 05 / 18 / 2026, p. 145 / 2830 137 / 348 ADCC activity of a molecule of interest are described in US Patent No. 5,500,362 (see, for example, 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96 (trademarked) non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al.C1q binding assays can also be performed to confirm that the antigen-binding molecule is unable to bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (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)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[00470] Antigen-binding molecules with reduced effector function include those with substitution of one or more of the residues in the Fc region 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions. Petition 870260047205, dated 05 / 18 / 2026, p. 146 / 2830 138 / 348 tions in two or more positions of amino acids 265, 269, 270, 297 and 327, including the so-called Fc DANA mutant with substitution of residues 265 and 297 in alanine (US Patent No. 7,332,581).

[00471] Certain variants of the antigen-binding molecule with increased or decreased binding to FcRs are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

[00472] In certain embodiments, a variant of the antigen-binding molecule comprises an Fc region with one or more amino acid substitutions that enhance ADCC, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (EU residue numbering).

[00473] In some embodiments, alterations are made to the Fc region that result in altered (i.e., increased or decreased) C1q binding and / or Complement-Dependent Cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[00474] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. Such Fc variants include those with substitutions in one or more of the residues of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, for example, replacement of the residue in the Fc 434 region (US Patent No. 7,371,826).

[00475] See also Duncan & Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO Petition 870260047205, dated 05 / 18 / 2026, page 147 / 2830 139 / 348 94 / 29351 in relation to other examples of variants of the Fc region. d) Cysteine-designed antigen-binding molecule variants

[00476] In certain embodiments, it may be desirable to create cysteine-engineered antigen-binding molecules, for example, thioMAbs, in which one or more residues of an antigen-binding molecule are replaced by cysteine ​​residues. In particular embodiments, the substituted residues occur at accessible sites of the antigen-binding molecule. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antigen-binding molecule and can be used to conjugate the antigen-binding molecule with other components, such as drug components or drug-linking components, to create an immunoconjugate, as described further below. In certain embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain.Antigen-binding molecules engineered with cysteine ​​can be generated as described, for example, in U.S. Patent No. 7,521,541. e) Derivatives of Antigen-Binding Molecules

[00477] In certain embodiments, an antigen-binding molecule provided in this invention may be further modified to contain additional non-protein components that are known in the art and readily available. Suitable components for deriving the antigen-binding molecule 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 Petition 870260047205, dated 05 / 18 / 2026, page 148 / 2830 140 / 348 1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (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 may offer manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antigen-binding molecule may vary, and if more than one polymer is attached, they may be the same or different molecules.In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antigen-binding molecule to be improved, whether the antigen-binding molecule derivative will be used in a therapy under defined conditions, etc.

[00478] In connection with an antigen-binding molecule in the present invention, examples of the desired property (activity) may include, but are not particularly limited to, binding activity, neutralizing activity, cytotoxic activity, agonist activity, antagonist activity, and enzymatic activity. Agonist activity is an intracellular signal transduction activity, for example, through the binding of an antibody to an antigen such as a receptor, to induce a change in some physiological activity. Examples of physiological activity may include, but are not limited to, proliferative 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, induction activity. Petition 870260047205, dated 05 / 18 / 2026, p. 149 / 2830 141 / 348 of cell death and apoptosis-inducing activity.

[00479] In another embodiment, conjugates of an antigen-binding molecule and a non-protein component that can be selectively heated through radiation exposure are provided. In one embodiment, the non-protein component is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells but heat the non-protein component to a temperature at which cells proximal to the antigen-binding molecule-non-protein component are neutralized. B. Recombinant Methods and Compositions

[00480] Antigen-binding molecules can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, the isolated nucleic acid encoding an antigen-binding molecule in the present invention (a polypeptide comprising an antigen-binding domain described herein) is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antigen-binding molecule (e.g., the light and / or heavy chains of the antigen-binding molecule). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In yet another embodiment, a host cell comprising such nucleic acid is provided.In such an embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and an amino acid sequence comprising the VH of the antigen-binding molecule. Petition 870260047205, dated 05 / 18 / 2026, page 150 / 2830 142 / 348 or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antigen-binding molecule and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antigen-binding molecule. In one embodiment, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of producing an antigen-binding molecule is provided in the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antigen-binding molecule, as provided above, under conditions suitable for expression of the antigen-binding molecule and, optionally, recovery of the antigen-binding molecule from the host cell (or host cell culture medium).

[00481] For the recombinant production of an antigen-binding molecule in the present invention, the nucleic acid encoding an antigen-binding molecule, for example, as described above, is isolated and inserted into one or more vectors for subsequent cloning and / or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (for example, through the use of oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antigen-binding molecule).

[00482] Suitable host cells for cloning or expression of vectors encoding antigen-binding molecules include the prokaryotic or eukaryotic cells described herein. For example, antigen-binding molecules can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of anti-antigen fragments Petition 870260047205, dated 05 / 18 / 2026, p. 151 / 2830 143 / 348 bodies and polypeptides in bacteria, see, for example, US Patents Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.). After expression, the antigen-binding molecule can be isolated from the bacterial cell paste in a soluble fraction and can be subsequently purified.

[00483] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antigen-binding molecules, including fungal and yeast strains whose glycosylation pathways have been humanized, resulting in the production of an antigen-binding molecule with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[00484] Suitable host cells for the expression of glycosylated antigen-binding molecules are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified, which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[00485] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing the PLANTIBODIES™ technology for the production of antigen-binding molecules in transgenic plants).

[00486] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Or Petition 870260047205, dated 05 / 18 / 2026, page 152 / 2830 144 / 348 Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described, for example, in Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine renal cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for the production of antigen-binding molecules, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). C. Essays

[00487] The antigen-binding molecules provided in this invention can be identified, tracked, or characterized by their physical / chemical properties and / or biological activities by various assays known in the art. 1. Bond tests and other tests

[00488] In one aspect, an antigen-binding molecule in the present invention is tested for its antigen-binding activity. Petition 870260047205, dated 05 / 18 / 2026, page 153 / 2830 145 / 348 tygen, for example, by known methods such as ELISA, Western blot, etc. 2. Activity trials

[00489] In one aspect, assays are provided to identify antigen-binding molecules that have biological activity. Biological activity may include, for example, activity of maintaining two antigen molecules in spatially close positions, activity of regulating the interaction between two antigen molecules, activity of promoting the activation of a receptor by a ligand, activity of promoting the catalytic reaction of an enzyme with a substrate, promotion of the interaction between a cell expressing a first antigen and a cell expressing a second antigen, activity of promoting damage to a target cell by a cell with cytotoxic activity (e.g., a T cell, NK cell, monocyte, or macrophage), activity of regulating the activation of two antigen molecules that are activated through association with each other, and resistance to cleavage by protease.Antigen-binding molecules possessing such biological activity in vivo and / or in vitro are also provided.

[00490] Furthermore, an antigen-binding molecule in the present invention may exert various biological activities, depending on the type of antigen molecule to which the antigen-binding molecule binds. Examples of such antigen-binding molecules include an antigen-binding molecule that binds to a T cell receptor (TCR) complex (e.g., CD3) and possesses T cell activation-inducing activity (agonist activity); and an antigen-binding molecule that binds to a molecule of the TNF receptor superfamily (e.g., OX40 or 4-1BB) or other co-stimulatory molecules (e.g., CD28 or ICOS) and possesses the aforementioned activation-promoting activity (agonist activity). Petition 870260047205, dated 05 / 18 / 2026, p. 154 / 2830 146 / 348 of agonist). In certain embodiments, this biological activity exerted through binding to an antigen molecule is increased or decreased by the binding of two or more antigen-binding domains comprised in the antigen-binding molecule in the present invention. Without being limited by theory, in certain embodiments, such increase or decrease can be achieved because the interaction between two or more antigen molecules is regulated through binding to the antigen-binding molecule in the present invention (for example, the association between two or more antigen molecules is promoted).

[00491] In certain embodiments, an antigen-binding molecule of the invention is tested with respect to this biological activity. If two antigen molecules are held spatially close, it can be evaluated using techniques such as crystal structure analysis, electron microscopy, and electron tomography-based structural analysis of a complex composed of antigens and an antigen-binding molecule. If two antigen-binding domains are spatially close to each other or if the mobility of two antigen-binding domains is reduced, it can also be evaluated by the aforementioned techniques. In particular, regarding techniques for analyzing the three-dimensional structure of IgG molecules using electron tomography, see, for example, Zhang et al., Sci. Rep. 5:9803 (2015).In electron tomography, the frequency of occurrence of structures that a target molecule can form can be shown by histograms, allowing the distributional evaluation of structural changes, such as reduced domain mobility. For example, when the connection between values ​​that can be obtained from structure-related parameters, such as distance and angle between two domains, and their frequency of occurrence is shown by histograms, it can be determined that the mobility of the two domains is decreased if their distribution areas are reduced. The activity exerted by means of... Petition 870260047205, dated 05 / 18 / 2026, page 155 / 2830 147 / 348 The interaction of two antigen molecules can be evaluated by selecting and using an appropriate activity measurement system from those known according to the type of target antigen molecules. The effect on protease cleavage can be evaluated using methods known to those skilled in the art or methods described in the Examples below. D. Pharmaceutical Formulations (Pharmaceutical Compositions)

[00492] Pharmaceutical formulations of an antigen-binding molecule, as described in this invention, are prepared by mixing said antigen-binding molecule having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);Low molecular weight polypeptides (less than about 10 residues); 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants; Petition 870260047205, dated 05 / 18 / 2026, page 156 / 2830 148 / 348 cos, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable vehicles also include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, soluble human PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, an sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[00493] Exemplary lyophilized formulations of antigen-binding molecule are described in U.S. Patent No. 6,267,958. Aqueous formulations of antigen-binding molecule include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine acetate buffer.

[00494] The formulation herein may also contain more than one active ingredient as needed for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are appropriately present in combination in amounts that are effective for the intended purpose.

[00495] Active ingredients can be captured in microcapsules prepared, for example, by coacervation techniques or through interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Petition 870260047205, dated 05 / 18 / 2026, page 157 / 2830 149 / 348

[00496] Extended-release preparations may be prepared. Suitable examples of extended-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antigen-binding molecule, the matrices of which are in the form of molded articles, for example, films or microcapsules.

[00497] Formulations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes. Examples

[00498] The following are examples of antigen-binding molecules and methods of the present invention. It will be understood that various other embodiments may be practiced, given the general description provided above. Example 1: Concept of a Fab-crosslinked antibody

[00499] Agonist antibodies are superior in properties such as stability, pharmacokinetics, and production methods compared to natural ligands and their fusion proteins, and their pharmaceutical development is ongoing. However, in general, agonist antibodies with strong activity are more difficult to obtain than simple binding or neutralizing antibodies. A solution to this problem is therefore being sought.

[00500] The properties required for an agonist antibody may depend on the type of ligand. For agonist antibodies against the TNF receptor superfamily, typified by death receptor (DR), OX40, 4-1BB, CD40 and the like, it has been reported that antibody or ligand multimerization contributes to activation. Techniques to enhance this effect include the use of natural ligands, crosslinking with anti-Fc antibodies, crosslinking via FcyRs, multimerization of antibody-binding domains, and multimerization via antibodies. Petition 870260047205, dated 05 / 18 / 2026, p. 158 / 2830 150 / 348 po Fc, and similar, have been reported to increase agonist activity. It is also known that, for certain types of antigens, adjusting the distance of the antigen-binding sites using the antibody's Fab or scFv structure leads to increased agonist activity independently of multimerization.

[00501] As another technique, an agonist antibody against a cytokine receptor that is a bispecific antibody capable of binding to different epitopes within the same antigen has been reported. Furthermore, a method for improving agonist activity using chemical conjugation to crosslink two different Fabs in a similar manner has been reported.

[00502] Further methods beyond those mentioned above to improve the activity of agonist antibodies are desired. However, no simple method to achieve this has been reported. Thus, the inventors developed a method to crosslink Fabs with each other through the introduction of minimal mutations and demonstrated that this actually increased agonist activity, thus completing the invention. An exemplary embodiment is shown in Fig. 1. Example 2: Production of expression vectors for modified antibodies and expression and purification of modified antibodies.

[00503] An antibody gene inserted into an expression vector for animal cells was subjected to amino acid sequence substitution by a method known to the skilled in the art using PCR, the In-Fusion Advantage PCR cloning kit (TAKARA), or similar, to construct an expression vector for a modified antibody. The nucleotide sequence of the resulting expression vector was determined by a method known to the skilled in the art. The produced expression vector was transiently introduced into FreeStyle293® or Expi293® (Invitrogen) cells, and the cells were allowed to express the modified antibody. Petition 870260047205, dated 05 / 18 / 2026, page 159 / 2830 151 / 348 in the culture supernatant. The modified antibody was purified from the culture supernatant obtained by a method known to a person skilled in the art using rProtein A Sepharose® Fast Flow (GE Healthcare). The absorbance at 280 nm was measured using a spectrophotometer. An absorption coefficient was calculated from the value measured using the PACE method and used to calculate the antibody concentration (Protein Science 1995;4:2411-2423).

[00504] The amount of aggregates of the modified antibody was analyzed by a method known to a person skilled in the art using an Agilent 1260 Infinity® (Agilent Technologies) for HPLC and a G3000SWXL (TOSOH) as a gel filtration chromatography column. The concentration of the purified antibody was 0.1 mg / ml, and 10 μI of the antibody were injected.

[00505] The antibodies prepared by this method (anti-CD3ε antibodies, anti-CD28 antibodies and bispecific anti-CD3ε x anti-CD28 antibodies) are shown in Table 1. Table 1 Antibody names, SEQ ID NOS Antibody Name SEQ ID NO: Heavy Chain 1 Light Chain 1 Heavy Chain 2 Light Chain 2 CD3-G4s 1 10 - - CD3-G4sHH 2 10 - - CD3-G4sLL 1 11 - - CD3-G1s 4 10 - - CD3-G1s 5 12 - - CD28-G1 6 13 - - CD3-G1sLL 4 11 - - CD3-G1sHH 7 10 - - CD3 / / CD28-G1s 4 10 6 13 CD3 / / CD28-G1sLL 4 11 6 14 Petition 870260047205, dated 05 / 18 / 2026, page 160 / 2830 152 / 348 Antibody Name SEQ ID NO: Heavy Chain 1 Light Chain 1 Heavy Chain 2 Light Chain 2 CD3 / / CD28-G1sHH 7 10 9 13 CD3 / / CD28-G1sLH 4 11 9 13 CD3 / / CD28-G1sHL 7 10 6 14 OKT3 / / CD28-G1s 5 12 6 13 OKT3 / / CD28-G1sHH 8 12 9 13 OKT3 / / CD28-G1sHL 8 12 6 14 HH: position 191 (EU numbering) was changed to Cys in the two constant regions of the H chain. LL: position 126 (EU numbering) was changed to Cys in the two constant L-string regions. HL, LH: position 191 (EU numbering) was changed to Cys in a constant region of the H chain, and position 126 (EU numbering) was changed to Cys in a constant region of the L chain. Example 3: Preparation of bispecific antibodies.

[00506] The purified antibody was dialyzed in TBS buffer (WAKO) and its concentration was adjusted to 1 mg / ml. As a 10x reaction buffer, 2-MEA (SIGMA) 250 mM was prepared. Two different homodimeric antibodies prepared in Example 2 were mixed in equal amounts. To this mixture, a volume of 1 / 10 of the 10x reaction buffer was added and mixed. The mixture was left to stand at 37°C for 90 minutes. After the reaction, the mixture was dialyzed in TBS to obtain a solution of a bispecific antibody in which the two different antibodies above were heterodimerized. The antibody concentration was measured by the method mentioned above and the antibody was subjected to subsequent experiments. Example 4: Evaluation of agonist activity Example 4-1 Preparation of Jurkat cell suspension

[00507] Jurkat cells (TCR / CD3 effector cells (NFAT), Prome Petition 870260047205, dated 05 / 18 / 2026, page 161 / 2830 Cells (153 / 348 ga) were collected from flasks. The cells were washed with assay buffer (RPMI 1640 medium (Gibco), 10% FBS (HyClone), 1% non-essential amino acids MEM (Invitrogen) and 1 mM sodium pyruvate (Invitrogen)) and then suspended at 3 x 10⁶ cells / ml in assay buffer. This Jurkat cell suspension was subjected to subsequent experiments. Example 4-2 Preparation of luminescence reagent solution

[00508] 100 ml of Bio-Glo Luciferase Assay Buffer (Promega) were added to the flask of Bio-Glo Luciferase Assay Substrate (Promega) and mixed by inversion. The flask was protected from light and frozen at -20 °C. This luminescence reagent solution was subjected to subsequent experiments. Example 4-3 T-cell activation assay

[00509] T cell activation via agonist signaling was assessed based on the change in luciferase luminescence fold. The aforementioned Jurkat cells are transformed cells with a luciferase reporter gene having an NFAT-responsive sequence. When the cells are stimulated by an anti-TCR / CD3 antibody, the NFAT pathway is activated via intracellular signaling, thus inducing luciferase expression. The Jurkat cell suspension prepared as described above was added to a 384-well flat-bottom white plate at 10 μL per well (3 x 10⁴ cells / well). Then, the antibody solution prepared at each concentration (150, 15, 1.5, 0.15, 0.015, 0.0015, 0.00015, 0.000015 nM) was added at 20 μL per well. This plate was left to stand in a 5% CO2 incubator at 37 °C for 24 hours.After incubation, the luminescence reagent solution was thawed and 30 µl of the solution were added to each well. The plate was then left to stand at room temperature for 10 minutes. Luciferase luminescence was observed in each well of the plate. Petition 870260047205, dated 05 / 18 / 2026, page 162 / 2830 154 / 348 was measured using a luminometer.

[00510] As a result, molecules modified with an additional Fab-Fab linking disulfide bond of the anti-CD3ε antibody showed varied CD3-mediated signaling compared to the wild-type molecule (unmodified molecule) as shown in Figs. 2 and 3. Furthermore, as shown in Figs. 4 and 5, modified molecules of a bispecific antibody composed of an anti-CD3ε antibody and an anti-CD28 antibody with an additional Fab-Fab linking disulfide bond also showed widely varied CD3 and / or CD28-mediated signaling compared to the wild-type molecule.

[00511] These results suggest that introducing modifications of the present invention can increase or decrease the agonist activity possessed by antigen-binding molecules, such as antibodies. Example 5: Evaluation of antibodies having cysteine ​​substitution at various positions in the heavy chain. Example 5-1 Evaluation of antibodies having cysteine ​​substitution at various positions in the heavy chain

[00512] The variable region and the constant region of the heavy chain of an anti-human IL6R neutralizing antibody, MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)) were subjected to a study in which an arbitrary amino acid residue structurally exposed on the surface was replaced with cysteine.

[00513] The amino acid residues within the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) were replaced with cysteine ​​to produce variants of the MRA heavy chain variable region shown in Table 2. These variants of the MRA heavy chain variable region were each linked to the constant region. Petition 870260047205, dated 05 / 18 / 2026, page 163 / 2830 155 / 348 MRA heavy chain (G1T4, SEQ ID NO: 18) to produce MRA heavy chain variants, and the expression vectors encoding the corresponding genes were produced by a method known to a person skilled in the art.

[00514] In addition, amino acid residues within the MRA heavy chain constant region (G1T4, SEQ ID NO: 18) were replaced with cysteine ​​to produce variants of the MRA heavy chain constant region shown in Table 3. These variants of the MRA heavy chain constant region were linked to the MRA heavy chain variable region (MRAH, SEQ ID NO: 17) to produce MRA heavy chain variants, and the expression vectors encoding the corresponding genes were produced by a method known to a person skilled in the art.

[00515] The heavy chain variants of MRA produced above were combined with the light chain of MRA. The resulting MRA variants shown in Table 4 were expressed via transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life technologies) by a method known to those skilled in the art and purified with Protein A by a method known to those skilled in the art. Table 2 Variants of the MRA heavy chain variable region and cysteine ​​substitution position Variant of the variable region of MRA heavy chain Cysteine ​​substitution position (Kabat numbering) SEQ ID NO: MRAH.Q5C 5 21 MRAH.E6C 6 22 MRAH.S7C 7 23 MRAH.G8C 8 24 MRAH.P9C 9 25 MRAH.G10C 10 26 Petition 870260047205, dated 05 / 18 / 2026, page 164 / 2830 156 / 348 Variante da região variável de cadeia pesada de MRA Posição da substituição de cisteína (numeração de Kabat) SEQ ID NO: MRAH.L11C 11 27 MRAH.V12C 12 28 MRAH.R13C 13 29 MRAH.P14C 14 30 MRAH.S15C 15 31 MRAH.Q16C 16 32 MRAH.T17C 17 33 MRAH.L18C 18 34 MRAH.S19C 19 35 MRAH.L20C 20 36 MRAH.T21C 21 37 MRAH.T23C 23 38 MRAH.S25C 25 39 MRAH.G26C 26 40 MRAH.S28C 28 41 MRAH.T30C 30 42 MRAH.R66C 66 43 MRAH.V67C 67 44 MRAH.T68C 68 45 MRAH.L70C 70 46 MRAH.D72C 72 47 MRAH.T73C 73 48 MRAH.S74C 74 49 MRAH.K75C 75 50 MRAH.N76C 76 51 MRAH.Q77C 77 52 MRAH.S79C 79 53 MRAH.L80C 80 54 MRAH.R81C 81 55 MRAH.L82C 82 56 MRAH.S82aC 82a 57 Petição 870260047205, de 18 / 05 / 2026, pág. 165 / 2830 157 / 348 Variant of the variable region of the heavy chain of MRA. Cysteine ​​substitution position (Kabat numbering). SEQ ID NO: MRAH.S82bC 82b 58 MRAH.V82cC 82c 59 MRAH.S112C 112 60 MRAH.S113C 113 61 MRAH.S31C 31 62 MRAH.W35C 35 63 MRAH.S35aC 35a 64 MRAH.Y50C 50 65 MRAH.I51C 51 66 MRAH.S52C 52 67 MRAH.S62C 62 68 MRAH.L63C 63 69 MRAH.K64C 64 70 MRAH.S65C 65 71 MRAH.D101C 101 72 MRAH.Y102C 102 73 Table 3 Variants of the MRA heavy chain constant region and cysteine ​​substitution position Variant of the MRA heavy chain constant region Cysteine ​​substitution position (EU numbering) SEQ ID NO: G1T4.A118C 118 74 G1T4.S119C 119 75 G1T4.T120C 120 76 G1T4.K121C 121 77 G1T4.G122C 122 78 G1T4.P123C 123 79 G1T4.S124C 124 80 G1T4.V125C 125 81 G1T4.F126C 126 82 G1T4.P127C 127 83 Petition 870260047205, dated 05 / 18 / 2026, page 166 / 2830 158 / 348 Variant of the MRA heavy chain constant region Cysteine ​​substitution position (EU numbering) SEQ ID NO: G1T4.S131C 131 84 G1T4.S132C 132 85 G1T4.K133C 133 86 G1T4.S134C 134 87 G1T4.T135C 135 88 G1T4.S136C 136 89 G1T4.G137C 137 90 G1T4.G138C 138 91 G1T4.T139C 139 92 G1T4.A140C 140 93 G1T4.A141C 141 94 G1T4.D148C 148 95 G1T4.Y149C 149 96 G1T4.F150C 150 97 G1T4.P151C 151 98 G1T4.E152C 152 99 G1T4.P153C 153 100 G1T4.V154C 154 101 G1T4.T155C 155 102 G1T4.V156C 156 103 G1T4.S157C 157 104 G1T4.W158C 158 105 G1T4.N159C 159 106 G1T4.S160C 160 107 G1T4.G161C 161 108 G1T4.A162C 162 109 G1T4.L163C 163 110 G1T4.T164C 164 111 G1T4.S165C 165 112 G1T4.G166C 166 113 G1T4.V167C 167 114 Petition 870260047205, dated 05 / 18 / 2026, page 167 / 2830 159 / 348 Variant of the MRA heavy chain constant region Cysteine ​​substitution position (EU numbering) SEQ ID NO: G1T4.V173C 173 115 G1T4.L174C 174 116 G1T4.Q175C 175 117 G1T4.S176C 176 118 G1T4.S177C 177 119 G1T4.G178C 178 120 G1T4.L179C 179 121 G1T4.Y180C 180 122 G1T4.V186C 186 123 G1T4.T187C 187 124 G1T4.V188C 188 125 G1T4.P189C 189 126 G1T4.S190C 190 127 G1T4.S191C 191 128 G1T4.S192C 192 129 G1T4.L193C 193 130 G1T4.G194C 194 131 G1T4.T195C 195 132 G1T4.Q196C 196 133 G1T4.T197C 197 134 G1T4.Y198C 198 135 G1T4.I199C 199 136 G1T4.N201C 201 137 G1T4.V202C 202 138 G1T4.N203C 203 139 G1T4.H204C 204 140 G1T4.K205C 205 141 G1T4.P206C 206 142 G1T4.S207C 207 143 G1T4.N208C 208 144 G1T4.T209C 209 145 Petition 870260047205, dated 05 / 18 / 2026, page 168 / 2830 160 / 348 Variant of the MRA heavy chain constant region Cysteine ​​substitution position (EU numbering) SEQ ID NO: G1T4.K210C 210 146 G1T4.V211C 211 147 G1T4.D212C 212 148 G1T4.K213C 213 149 G1T4.R214C 214 150 G1T4.V215C 215 151 G1T4.E216C 216 152 G1T4.P217C 217 153 G1T4.K218C 218 154 G1T4.S219C 219 155 Table 4 MRA variants SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH.Q5C-G1T4 21 18 19 20 MRAH.E6C-G1T4 22 18 19 20 MRAH.S7C-G1T4 23 18 19 20 MRAH.G8C-G1T4 24 18 19 20 MRAH.P9C-G1T4 25 18 19 20 MRAH.G10C-G1T4 26 18 19 20 MRAH.L11C-G1T4 27 18 19 20 MRAH.V12C-G1T4 28 18 19 20 MRAH.R13C-G1T4 29 18 19 20 MRAH.P14C-G1T4 30 18 19 20 MRAH.S15C-G1T4 31 18 19 20 MRAH.Q16C-G1T4 32 18 19 20 MRAH.T17C-G1T4 33 18 19 20 MRAH.L18C-G1T4 34 18 19 20 Petition 870260047205, dated 05 / 18 / 2026, page 169 / 2830 161 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH.S19C-G1T4 35 18 19 20 MRAH.L20C-G1T4 36 18 19 20 MRAH.T21C-G1T4 37 18 19 20 MRAH.T23C-G1T4 38 18 19 20 MRAH.S25C-G1T4 39 18 19 20 MRAH.G26C-G1T4 40 18 19 20 MRAH.S28C-G1T4 41 18 19 20 MRAH.T30C-G1T4 42 18 19 20 MRAH.R66C-G1T4 43 18 19 20 MRAH.V67C-G1T4 44 18 19 20 MRAH.T68C-G1T4 45 18 19 20 MRAH.L70C-G1T4 46 18 19 20 MRAH.D72C-G1T4 47 18 19 20 MRAH.T73C-G1T4 48 18 19 20 MRAH.S74C-G1T4 49 18 19 20 MRAH.K75C-G1T4 50 18 19 20 MRAH.N76C-G1T4 51 18 19 20 MRAH.Q77C-G1T4 52 18 19 20 MRAH.S79C-G1T4 53 18 19 20 MRAH.L80C-G1T4 54 18 19 20 MRAH.R81C-G1T4 55 18 19 20 MRAH.L82C-G1T4 56 18 19 20 MRAH.S82aC-G1T4 57 18 19 20 MRAH.S82bC-G1T4 58 18 19 20 MRAH.V82cC-G1T4 59 18 19 20 MRAH.S112C-G1T4 60 18 19 20 MRAH.S113C-G1T4 61 18 19 20 MRAH.S31C-G1T4 62 18 19 20 Petition 870260047205, dated 05 / 18 / 2026, page 170 / 2830 162 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH.W35C-G1T4 63 18 19 20 MRAH.S35aC-G1T4 64 18 19 20 MRAH.Y50C-G1T4 65 18 19 20 MRAH.I51C-G1T4 66 18 19 20 MRAH.S52C-G1T4 67 18 19 20 MRAH.S62C-G1T4 68 18 19 20 MRAH.L63C-G1T4 69 18 19 20 MRAH.K64C-G1T4 70 18 19 20 MRAH.S65C-G1T4 71 18 19 20 MRAH.D101C-G1T4 72 18 19 20 MRAH.Y102C-G1T4 73 18 19 20 MRAH-G1T4.A118C 17 74 19 20 MRAH-G1T4.S119C 17 75 19 20 MRAH-G1T4.T120C 17 76 19 20 MRAH-G1T4.K121C 17 77 19 20 MRAH-G1T4.G122C 17 78 19 20 MRAH-G1T4.P123C 17 79 19 20 MRAH-G1T4.S124C 17 80 19 20 MRAH-G1T4.V125C 17 81 19 20 MRAH-G1T4.F126C 17 82 19 20 MRAH-G1T4.S132C 17 85 19 20 MRAH-G1T4.K133C 17 86 19 20 MRAH-G1T4.S134C 17 87 19 20 MRAH-G1T4.T135C 17 88 19 20 MRAH-G1T4.S136C 17 89 19 20 MRAH-G1T4.G137C 17 90 19 20 Petition 870260047205, dated 05 / 18 / 2026, p. 171 / 2830 163 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH-G1T4.G138C 17 91 19 20 MRAH-G1T4.T139C 17 92 19 20 MRAH-G1T4.A140C 17 93 19 20 MRAH-G1T4.A141C 17 94 19 20 MRAH-G1T4.D148C 17 95 19 20 MRAH-G1T4.Y149C 17 96 19 20 MRAH-G1T4.F150C 17 97 19 20 MRAH-G1T4.P151C 17 98 19 20 MRAH-G1T4.E152C 17 99 19 20 MRAH-G1T4.P153C 17 100 19 20 MRAH-G1T4.V154C 17 101 19 20 MRAH-G1T4.T155C 17 102 19 20 MRAH-G1T4.V156C 17 103 19 20 MRAH-G1T4.S157C 17 104 19 20 MRAH-G1T4.S160C 17 107 19 20 MRAH-G1T4.G161C 17 108 19 20 MRAH-G1T4.A162C 17 109 19 20 MRAH-G1T4.L163C 17 110 19 20 MRAH-G1T4.T164C 17 111 19 20 MRAH-G1T4.S165C 17 112 19 20 MRAH-G1T4.G166C 17 113 19 20 MRAH-G1T4.L174C 17 116 19 20 MRAH-G1T4.Q175C 17 117 19 20 MRAH-G1T4.S176C 17 118 19 20 Petition 870260047205, dated 05 / 18 / 2026, p. 172 / 2830 164 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH-G1T4.S177C 17 119 19 20 MRAH-G1T4.G178C 17 120 19 20 MRAH-G1T4.L179C 17 121 19 20 MRAH-G1T4.Y180C 17 122 19 20 MRAH-G1T4.V186C 17 123 19 20 MRAH-G1T4.T187C 17 124 19 20 MRAH-G1T4.V188C 17 125 19 20 MRAH-G1T4.P189C 17 126 19 20 MRAH-G1T4.S190C 17 127 19 20 MRAH-G1T4.S191C 17 128 19 20 MRAH-G1T4.G194C 17 131 19 20 MRAH-G1T4.T195C 17 132 19 20 MRAH-G1T4.Y198C 17 135 19 20 MRAH-G1T4.I199C 17 136 19 20 MRAH-G1T4.H204C 17 140 19 20 MRAH-G1T4.K205C 17 141 19 20 MRAH-G1T4.N208C 17 144 19 20 MRAH-G1T4.T209C 17 145 19 20 MRAH-G1T4.K210C 17 146 19 20 Petition 870260047205, dated 05 / 18 / 2026, page 173 / 2830 165 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH-G1T4.V211C 17 147 19 20 MRAH-G1T4.D212C 17 148 19 20 MRAH-G1T4.K213C 17 149 19 20 MRAH-G1T4.R214C 17 150 19 20 MRAH-G1T4.V215C 17 151 19 20 MRAH-G1T4.E216C 17 152 19 20 MRAH-G1T4.P217C 17 153 19 20 MRAH-G1T4.K218C 17 154 19 20 MRAH-G1T4.S219C 17 155 19 20 Example 5-2. Evaluation of protease-mediated Fab fragmentation of antibodies having cysteine ​​substitution at multiple positions in the heavy chain.

[00516] Using a protease that cleaves the heavy chain hinge region of the antibody to induce Fab fragmentation, the MRA variants produced in Example 5-1 were examined to verify whether they had acquired protease resistance such that their fragmentation was inhibited. The protease used was Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001). The reaction was performed under the conditions of 2 ng / μL protease, 100 pg / ml antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS and 35 °C for two hours, or under the conditions of 2 ng / μL protease, 20 pg / ml antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS and 35 °C for one hour. The sample was then subjected to non-reducing capillary electrophoresis. Wes (Protein Simple) was used for capillary electrophoresis, and an HRP-labeled anti-capa chain antibody (abcam; ab46527) was used for detection. The results are shown in Figs. 6 to 13. Treatment with MRA Lys-C caused cleavage of Petition 870260047205, dated 05 / 18 / 2026, page 174 / 2830 166 / 348 heavy chain hinge region, resulting in the disappearance of the IgG band at approximately 150 kDa and the appearance of the Fab band at approximately 50 kDa. For the MRA variants produced in Example 5-1, some showed the Fab dimer band appearing at approximately 96 kDa and some showed the undigested IgG band detected at approximately 150 kDa after protease treatment. The area of ​​each band obtained after protease treatment was submitted using proprietary software for Wes (Compass for SW; Protein Simple) to calculate the percentage of areas of undigested IgG band, Fab dimer, etc. The calculated percentage of each band is shown in Table 5. Table 5 IgG Antibody Name (%) FabFab (%) Fab (%) Heavy Chain SEQ ID NO: Light Chain SEQ ID NO: MRAH.Q5C-G1T4 0.2 1.5 97.6 21 16 MRAH.E6C-G1T4 0 0.3 80.7 22 16 MRAH.S7C-G1T4 0.4 1.9 96.9 23 16 MRAH.G8C-G1T4 16.6 1.1 76.7 24 16 MRAH.P9C-G1T4 0.2 1.5 97.2 25 16 MRAH.G10C-G1T4 0.6 1.9 96.9 26 16 MRAH.L11C-G1T4 0 1.2 98.3 27 16 MRAH.V12C-G1T4 0.2 1 97.6 28 16 MRAH.R13C-G1T4 0.6 1.9 96.6 29 16 MRAH.P14C-G1T4 0.3 1.7 97.7 30 16 MRAH.S15C-G1T4 0.9 1.3 81.4 31 16 MRAH.Q16C-G1T4 92.5 0 2 32 16 MRAH.T17C-G1T4 0.4 1.4 97.8 33 16 MRAH.L18C-G1T4 0.3 0.6 96.1 34 16 MRAH.S19C-G1T4 0.3 1.2 98.1 35 16 MRAH.L20C-G1T4 1 0.3 93.3 36 16 MRAH.T21C-G1T4 0.5 1 98.3 37 16 Petition 870260047205, dated 05 / 18 / 2026, page 175 / 2830 167 / 348 Nome do anticopo IgG (%) FabFab (%) Fab (%) Cadeia pesada SEQ ID NO: Cadeia leve SEQ ID NO: MRAH.T23C-G1T4 sem dados sem dados sem dados 38 16 MRAH.S25C-G1T4 0,3 2,8 87 39 16 MRAH.G26C-G1T4 0,4 ​​1,7 85,5 40 16 MRAH.S28C-G1T4 98,6 0 0,2 41 16 MRAH.T30C-G1T4 0,5 0,7 97,8 42 16 MRAH.R66C-G1T4 0,2 1,2 97,9 43 16 MRAH.V67C-G1T4 0,3 0,4 97,8 44 16 MRAH.T68C-G1T4 0.2 1.4 97.7 45 16 MRAH.L70C-G1T4 0.2 0.9 98 46 16 MRAH.D72C-G1T4 0.3 0.8 97.6 47 16 MRAH.T73C-G1T4 0.5 0.9 97.7 48 16 MRAH.S74C-G1T4 97.1 0 0.3 49 16 MRAH.K75C-G1T4 0.1 1.5 97 50 16 MRAH.N76C-G1T4 0.4 0.4 93.1 51 16 MRAH.Q77C-G1T4 0.1 0.2 99.6 52 16 MRAH.S79C-G1T4 0.1 1.6 96.7 53 16 MRAH.L80C-G1T4 0.2 0 96.5 54 16 MRAH.R81C-G1T4 0 1.4 98 55 16 MRAH.L82C-G1T4 0 0 96.8 56 16 MRAH.S82aC-G1T4 0.6 1 96.7 57 16 MRAH.S82bC-G1T4 97.5 0 0.3 58 16 MRAH.V82cC-G1T4 0.1 0.3 95.6 59 16 MRAH.S112C-G1T4 0.1 1.1 97.6 60 16 MRAH.S113C-G1T4 0.1 2.8 95.9 61 16 MRAH.S31C-G1T4 0.5 2 75.7 62 16 MRAH.W35C-G1T4 0.1 0.3 91.1 63 16 MRAH.S35aC-G1T4 0 0.6 90.7 64 16 MRAH.Y50C-G1T4 0.2 1.5 95.8 65 16 MRAH.I51C-G1T4 0.2 0.8 94.4 66 16. Petition 870260047205, dated 05 / 18 / 2026, page 176 / 2830 168 / 348 Nome do anticopo IgG (%) FabFab (%) Fab (%) Cadeia pesada SEQ ID NO: Cadeia leve SEQ ID NO: MRAH.S52C-G1T4 0,3 1,7 96,4 67 16 MRAH.S62C-G1T4 0,2 1,1 97,6 68 16 MRAH.L63C-G1T4 0,4 ​​1,4 94,2 69 16 MRAH.K64C-G1T4 0 1,6 91,7 70 16 MRAH.S65C-G1T4 0,3 1,7 95,6 71 16 MRAH.D101C-G1T4 0 1,2 97 72 16 MRAH.Y102C-G1T4 0,2 1,3 96,8 73 16 MRAH-G1T4.A118C 1.2 1 89 74 16 MRAH-G1T4.S119C 2.3 14 77.7 75 16 MRAH-G1T4.T120C 0 0.1 0.1 76 16 MRAH-G1T4.K121C 2.4 1.1 82.2 77 16 MRAH-G1T4.G122C 8 1.4 79.8 78 16 MRAH-G1T4.P123C 7.1 0 45.7 79 16 MRAH-G1T4.S124C 0.8 1.7 94.5 80 16 MRAH-G1T4.V125C 2.3 0 62 81 16 MRAH-G1T4.F126C 2.1 1 85.5 82 16 MRAH-G1T4.P127C 2.9 1.4 77.4 83 16 MRAH-G1T4.S131C 68.4 0 0 84 16 MRAH-G1T4.S132C 13.9 0.8 54.6 85 16 MRAH-G1T4.K133C 66.8 0 0 86 16 MRAH-G1T4.S134C 63.5 0 21.9 87 16 MRAH-G1T4.T135C 44.7 13.2 23.6 88 16 MRAH-G1T4.S136C 22.9 27.3 35.1 89 16 MRAH-G1T4.G137C 8,4 18,1 62,1 90 16 MRAH-G1T4.G138C sem dados sem dados sem dados 91 16 MRAH-G1T4.T139C 7,4 1,4 82,1 92 16 MRAH-G1T4.A140C 20.2 0 47.2 93 16 MRAH-G1T4.A141C 0.3 0 31.9 94 16 MRAH-G1T4.D148C 21 0 64.8 95 16. Petition 870260047205, dated 05 / 18 / 2026, page 177 / 2830 169 / 348 Nome do anticopo IgG (%) FabFab (%) Fab (%) Cadeia pesada SEQ ID NO: Cadeia leve SEQ ID NO: MRAH-G1T4.Y149C 0,5 0 58,1 96 16 MRAH-G1T4.F150C 79,2 0 0,4 97 16 MRAH-G1T4.P151C 2 0 56,1 98 16 MRAH-G1T4.E152C 0,9 ​​0,3 84,8 99 16 MRAH-G1T4.P153C 4,4 0,8 86,6 100 16 MRAH-G1T4.V154C 4 0 45,7 101 16 MRAH-G1T4.T155C 20,2 1,4 67.6 102 16 MRAH-G1T4.V156C 7 0 39.2 103 16 MRAH-G1T4.S157C 13.5 3.2 75.9 104 16 MRAH-G1T4.W158C 4.2 0 66.1 105 16 MRAH-G1T4.N159C 13.9 1.9 76.1 106 16 MRAH-G1T4.S160C 7.7 20.9 66.2 107 16 MRAH-G1T4.G161C 14.1 12 68.6 108 16 MRAH-G1T4.A162C 9.6 17.9 65.8 109 16 MRAH-G1T4.L163C 10.2 6.1 75.9 110 16 MRAH-G1T4.T164C 3.8 3.2 88.7 111 16 MRAH-G1T4.S165C 7.8 4.1 81.5 112 16 MRAH-G1T4.G166C 4.5 2.2 89.4 113 16 MRAH-G1T4.V167C 5.5 2.5 81.2 114 16 MRAH-G1T4.V173C 2.1 1.6 92.2 115 16 MRAH-G1T4.L174C 19.8 0 67.1 116 16 MRAH-G1T4.Q175C 4.4 1.1 86.6 117 16 MRAH-G1T4.S176C 2.3 7.7 85.5 118 16 MRAH-G1T4.S177C 7.1 12.4 71.6 119 16 MRAH-G1T4.G178C 6.2 2.4 85.5 120 16 MRAH-G1T4.L179C 0.2 0 0 121 16 MRAH-G1T4.Y180C 0 0 72.7 122 16 MRAH-G1T4.V186C 0 0 73.3 123 16 MRAH-G1T4.T187C 0.8 2.5 90.3 124 16 MRAH-G1T4.V188C 0.3 4 82.7 125 16. Petition 870260047205, dated 05 / 18 / 2026, page 178 / 2830 170 / 348 Nome do anticopo IgG (%) FabFab (%) Fab (%) Cadeia pesada SEQ ID NO: Cadeia leve SEQ ID NO: MRAH-G1T4.P189C 0,9 ​​4,7 89,6 126 16 MRAH-G1T4.S190C 10,9 0 74,4 127 16 MRAH-G1T4.S191C 2,3 46,4 45,1 128 16 MRAH-G1T4.S192C 1,3 11 83 129 16 MRAH-G1T4.L193C 3,6 0 70,5 130 16 MRAH-G1T4.G194C 13,8 0 0 131 16 MRAH-G1T4.T195C 29.6 0 57.3 132 16 MRAH-G1T4.Q196C 1.5 0 92.6 133 16 MRAH-G1T4.T197C 81.5 0 4.5 134 16 MRAH-G1T4.Y198C 0.1 0.3 17.1 135 16 MRAH-G1T4.I199C 1 1.7 91.6 136 16 MRAH-G1T4.N201C 0.7 4 90.3 137 16 MRAH-G1T4.V202C 0 0.1 6.6 138 16 MRAH-G1T4.N203C 0.6 2.4 89.8 139 16 MRAH-G1T4.H204C 0.4 2.2 77.7 140 16 MRAH-G1T4.K205C 0.2 2.3 85.5 141 16 MRAH-G1T4.P206C 0.4 2.1 86.9 142 16 MRAH-G1T4.S207C no data no data no data 143 16 MRAH-G1T4.N208C 0.4 0 86.2 144 16 MRAH-G1T4.T209C 0.7 0 83.1 145 16 MRAH-G1T4.K210C 0.6 0 81.7 146 16 MRAH-G1T4.V211C 0.3 1 67.6 147 16 MRAH-G1T4.D212C 1.1 1.8 80.9 148 16 MRAH-G1T4.K213C 6.5 0 41.9 149 16 MRAH-G1T4.R214C 18.6 0 42.7 150 16 MRAH-G1T4.V215C 0 0 11.8 151 16 MRAH-G1T4.E216C 7.4 0 64.8 152 16 MRAH-G1T4.P217C 4.5 0.2 43.3 153 16 MRAH-G1T4.K218C 30.8 0 29.5 154 16. Petition 870260047205, dated 05 / 18 / 2026, page 179 / 2830 171 / 348 IgG Antibody Name (%) Fab- Fab (%) Fab (%) Heavy Chain SEQ ID NO: Light Chain SEQ ID NO: MRAH-G1T4.S219C 46.9 0.1 18 155 16

[00517] From this result, it was observed that cysteine ​​substitution in the variable region of the heavy chain or the constant region of the heavy chain improved protease resistance in the hinge region of the heavy chain in the MRA variants shown in Table 6. Alternatively, the result suggested that a Fab dimer was formed by a covalent bond between Fab-Fab. Table 6 MRA variants SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH.G8C-G1T4 24 18 19 20 MRAH.Q16C-G1T4 32 18 19 20 MRAH.S28C-G1T4 41 18 19 20 MRAH.S74C-G1T4 49 18 19 20 MRAH.S82bC-G1T4 58 18 19 20 MRAH-G1T4.S119C 17 75 19 20 MRAH-G1T4.G122C 17 78 19 20 MRAH-G1T4.P123C 17 79 19 20 MRAH-G1T4.S131C 17 84 19 20 MRAH-G1T4.S132C 17 85 19 20 MRAH-G1T4.K133C 17 86 19 20 MRAH-G1T4.S134C 17 87 19 20 MRAH-G1T4.T135C 17 88 19 20 MRAH-G1T4.S136C 17 89 19 20 MRAH-G1T4.G137C 17 90 19 20 MRAH-G1T4.T139C 17 92 19 20 Petition 870260047205, dated 05 / 18 / 2026, pages 180 / 2830 172 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAH-G1T4.A140C 17 93 19 20 MRAH-G1T4.D148C 17 95 19 20 MRAH-G1T4.F150C 17 97 19 20 MRAH-G1T4.T155C 17 102 19 20 MRAH-G1T4.V156C 17 103 19 20 MRAH-G1T4.S157C 17 104 19 20 MRAH-G1T4.N159C 17 106 19 20 MRAH-G1T4.S160C 17 107 19 20 MRAH-G1T4.G161C 17 108 19 20 MRAH-G1T4.A162C 17 109 19 20 MRAH-G1T4.V167C 17 114 19 20 MRAH-G1T4.L174C 17 116 19 20 MRAH-G1T4.G178C 17 120 19 20 MRAH-G1T4.S190C 17 127 19 20 MRAH-G1T4.S191C 17 128 19 20 MRAH-G1T4.T195C 17 132 19 20 MRAH-G1T4.E216C 17 152 19 20 MRAH-G1T4.K218C 17 154 19 20 MRAH-G1T4.S219C 17 155 19 20 Petition 870260047205, dated 05 / 18 / 2026, pp. 181 / 2830 173 / 348 Example 6: Evaluation of antibodies having cysteine ​​substitution at various positions in the light chain. Example 6-1. Evaluation of antibodies with cysteine ​​substitution at various positions in the light chain.

[00518] The variable region of the light chain and the constant region of a human anti-IL6R neutralizing antibody, MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 15), light chain: MRAL-k0 (SEQ ID NO: 16)) were subjected to a study in which an arbitrary amino acid residue structurally exposed on the surface was replaced with cysteine.

[00519] The amino acid residues in the variable light chain region of MRA (MRAL, SEQ ID NO: 19) were replaced with cysteine ​​to produce variants of the variable light chain region of MRA shown in Table 7. These variants of the variable light chain region of MRA were each linked to the constant light chain region of MRA (k0, SEQ ID NO: 20) to produce MRA light chain variants, and the expression vectors encoding the corresponding genes were produced by a method known to a person skilled in the art.

[00520] In addition, amino acid residues within the constant light chain region of MRA (k0, SEQ ID NO: 20) were replaced with cysteine ​​to produce variants of the constant light chain region of MRA shown in Table 8. These variants of the constant light chain region of MRA were each linked to the variable light chain region of MRA (MRAL, SEQ ID NO: 19) to produce MRA light chain variants, and expression vectors encoding the corresponding genes were produced by a method known to a person skilled in the art.

[00521] The light chain variants of MRA produced above were combined with the heavy chain of MRA. The MRA variants Petition 870260047205, dated 05 / 18 / 2026, pp. 182 / 2830 174 / 348 results shown in Table 9 were expressed via transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by a method known to those skilled in the art, and purified with Protein A by a method known to those skilled in the art. Table 7 Variants of the variable region of the MRA light chain and cysteine ​​substitution position. Variant of the variable region of MRA light chain Cysteine ​​substitution position (Kabat numbering) SEQ ID NO: MRAL.T5C 5 156 MRAL.Q6C 6 157 MRAL.S7C 7 158 MRAL.P8C 8 159 MRAL.S9C 9 160 MRAL.S10C 10 161 MRAL.L11C 11 162 MRAL.S12C 12 163 MRAL.A13C 13 164 MRAL.S14C 14 165 MRAL.V15C 15 166 MRAL.G16C 16 167 MRAL.D17C 17 168 MRAL.R18C 18 169 MRAL.V19C 19 170 MRAL.T20C 20 171 MRAL.I21C 21 172 MRAL.T22C 22 173 MRAL.G57C 57 174 MRAL.V58C 58 175 MRAL.P59C 59 176 MRAL.S60C 60 177 MRAL.R61C 61 178 Petition 870260047205, de 18 / 05 / 2026, pág. 183 / 2830 175 / 348 Variante da região variácil de cáeda leve de MRA Posição da attitução de cisteína (numeração de Kabat) SEQ ID NO: MRAL.F62C 62 179 MRAL.S63C 63 180 MRAL.S65C 65 181 MRAL.S67C 67 182 MRAL.G68C 68 183 MRAL.T69C 69 184 MRAL.D70C 70 185 MRAL.T72C 72 186 MRAL.F73C 73 187 MRAL.T74C 74 188 MRAL.I75C 75 189 MRAL.S76C 76 190 MRAL.S77C 77 191 MRAL.L78C 78 192 MRAL.Q79C 79,193 MRAL.F98C 98 194 MRAL.G99C 99 195 MRAL.Q100C 100 196 MRAL.G101C 101 197 MRAL.T102C 102 198 MRAL.K103C 103 199 MRAL.V104C 104 200 MRAL.E105C 105 201 MRAL.I106C 106 202 MRAL.K107C 107 203 MRAL.A25C 25 204 MRAL.S26C 26 205 MRAL.Q27C 27 206 MRAL.Y32C 32 207 MRAL.L33C 33 208 MRAL.N34C 34 209 MRAL.Y50C 50 210 Petition 870260047205, de 18 / 05 / 2026, pág. 184 / 2830 176 / 348 Variant of the variable region of MRA light chain Cysteine ​​substitution position (Kabat numbering) SEQ ID NO: MRAL.T51C 51 211 MRAL.H55C 55 212 MRAL.S56C 56 213 MRAL.Y96C 96 214 MRAL.T97C 97 215 Table 8 Variants of the MRA light chain constant region and cysteine ​​substitution position Variant of the constant region of MRA light chain Cysteine ​​substitution position (EU numbering) SEQ ID NO: k0.R108C 108 216 k0.T109C 109 217 k0.V110C 110 218 k0.A111C 111 219 k0.A112C 112 220 k0.P113C 113 221 k0.S114C 114 222 k0.V115C 115 223 k0.F116C 116 224 k0.P120C 120 225 k0.S121C 121 226 k0.D122C 122 227 k0.E123C 123 228 k0.Q124C 124 229 k0.L125C 125 230 k0.K126C 126 231 k0.S127C 127 232 k0.G128C 128 233 k0.T129C 129 234 k0.A130C 130 235 Petition 870260047205, dated 05 / 18 / 2026, pages 185 / 2830 177 / 348 Variant of the constant region of MRA light chain Cysteine ​​substitution position (EU numbering) SEQ ID NO: k0.S131C 131 236 k0.L136C 136 237 k0.N137C 137 238 k0.N138C 138 239 k0.F139C 139 240 k0.Y140C 140 241 k0.P141C 141 242 k0.R142C 142 243 k0.E143C 143 244 k0.A144C 144 245 k0.K145C 145 246 k0.V146C 146 247 k0.Q147C 147 248 k0.W148C 148 249 k0.K149C 149 250 k0.V150C 150 251 k0.D151C 151 252 k0.N152C 152 253 k0.A153C 153 254 k0.L154C 154 255 k0.Q155C 155 256 k0.S156C 156 257 k0.G157C 157 258 k0.N158C 158 259 k0.S159C 159 260 k0.Q160C 160 261 k0.E161C 161 262 k0.S162C 162 263 k0.V163C 163 264 k0.T164C 164 265 Petition 870260047205, dated 05 / 18 / 2026, pages 186 / 2830 178 / 348 Variant of the constant region of MRA light chain Cysteine ​​substitution position (EU numbering) SEQ ID NO: k0.E165C 165 266 k0.Q166C 166 267 k0.D167C 167 268 k0.S168C 168 269 k0.K169C 169 270 k0.D170C 170 271 k0.S171C 171 272 k0.T172C 172 273 k0.Y173C 173 274 k0.S174C 174 275 k0.L175C 175 276 k0.T180C 180 277 k0.L181C 181 278 k0.S182C 182 279 k0.K183C 183 280 k0.A184C 184 281 k0.D185C 185 282 k0.Y186C 186 283 k0.E187C 187 284 k0.K188C 188 285 k0.H189C 189 286 k0.K190C 190 287 k0.V191C 191 288 k0.Y192C 192 289 k0.A193C 193 290 k0.E195C 195 291 k0.V196C 196 292 k0.T197C 197 293 k0.H198C 198 294 k0.Q199C 199 295 Petition 870260047205, dated 05 / 18 / 2026, pp. 187 / 2830 179 / 348 Variant of the constant region of MRA light chain Cysteine ​​substitution position (EU numbering) SEQ ID NO: k0.G200C 200 296 k0.L201C 201 297 k0.S202C 202 298 k0.S203C 203 299 k0.P204C 204 300 k0.V205C 205 301 k0.T206C 206 302 k0.K207C 207 303 k0.S208C 208 304 k0.F209C 209 305 k0.N210C 210 306 k0.R211C 211 307 k0.G212C 212 308 k0.E213C 213 309 Table 9 MRA variants SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAL.T5C-k0 17 18 156 20 MRAL.Q6C-k0 17 18 157 20 MRAL.S7C-k0 17 18 158 20 MRAL.P8C-k0 17 18 159 20 MRAL.S9C-k0 17 18 160 20 MRAL.S10C-k0 17 18 161 20 MRAL.L11C-k0 17 18 162 20 MRAL.S12C-k0 17 18 163 20 MRAL.A13C-k0 17 18 164 20 MRAL.S14C-k0 17 18 165 20 Petition 870260047205, dated 05 / 18 / 2026, pages 188 / 2830 180 / 348 SEQ ID NO: Antibody Name Heavy Chain Variable Region Heavy Chain Constant Region Light Chain Variable Region Light Chain Constant Region MRAL.V15C-k0 17 18 166 20 MRAL.G16C-k0 17 18 187 MRAL. 17 18 168 20 MRAL.R18C-k0 17 18 169 20 MRAL.V19C-k0 17 18 170 20 MRAL.T20C-k0 17 18 171 20 MRAL.I21C-k0 180 172 172 MRAL.T22C-k0 17 18 173 20 MRAL.G57C-k0 17 18 174 20 MRAL.V58C-k0 17 18 175 20 MRAL.P59C-k0 17 18 176 20 MRAL. 20 MRAL.R61C-k0 17 18 178 20 MRAL.F62C-k0 17 18 179 20 MRAL.S63C-k0 17 18 180 20 MRAL.S65C-k0 17 18 181 MRAL 2077C-187. 182 20 MRAL.G68C-k0 17 18 183 20 MRAL.T69C-k0 17 18 184 20 MRAL.D70C-k0 17 18 185 20 MRAL.T72C-k0 17 18 20 MRAL. 18 187 20 MRAL.T74C-k0 17 18 188 20 MRAL.I75C-k0 17 18 189 20 MRAL.S76C-k0 17 18 190 20 MRAL.S77C-k0 17 17 108 MRAL-208C-L. 17 18 192 20 MRAL.Q79C-k0 17 18 193 20 MRAL.F98C-k0 17 18 194 20 Petition 870260047205, of 18 / 05 / 2026, p. 189 / 2830 181 / 348 SEQ ID NO: Nome do anticopo Região de caiade pesada Região constante de caiade pesada Região caiade leve Região constante de caiade leve MRAL.G99C-k0 17 18 195 20 MRAL.Q100C-k0 17 18 196 20 MRAL.G101C-k0 17 18 197 20 MRAL.T102C-k0 17 18 198 20 MRAL.K103C-k0 17 18 199 20 MRAL.V104C-k0 17 18 200 20 MRAL.E105C-k0 17 18 201 20 MRAL.I106C-k0 17 18 202 20 MRAL.K107C-k0 17 18 203 20 MRAL.A25C-k0 17 18 204 20 MRAL.S26C-k0 17 18 205 20 MRAL.Q27C-k0 17 18 206 20 MRAL.Y32C-k0 17 18 207 20 MRAL.L33C-k0 17 18 208 20 MRAL.N34C-k0 17 18 209 20 MRAL.Y50C-k0 17 18 210 20 MRAL.T51C-k0 17 18 211 20 MRAL.H55C-k0 17 18 212 20 MRAL.S56C-k0 17 18 213 20 MRAL.Y96C-k0 17 18 214 20 MRAL.T97C-k0 17 18 215 20 MRAL-k0.R108C 17 18 19 216 MRAL-k0.T109C 17 18 19 217 MRAL-k0.V110C 17 18 19 218 MRAL-k0.A111C 17 18 19 219 MRAL-k0.A112C 17 18 19 220 MRAL-k0.P113C 17 18 19 221 MRAL-k0.S114C 17 18 19 222 MRAL-k0.V115C 17 18 19 223 Petition 870260047205, dated 05 / 18 / 2026, pp. 190 / 2830 182 / 348 SEQ ID NO: Nome do antibody Heavy chain variable region Heavy chain variable region Light chain variable region MRAL-k0.F116C 17 18 19 224 MRAL-k0.P120C 17 18 19 225 MRAL-k0.S121C 17 18 19 226 MRAL-k0.D122C 17 18 19 227 MRAL-k0.E123C 17 18 19 228 MRAL-k0.Q124C 17 18 19 229 MRAL-k0.L125C 17 18 19 230 MRAL-k0.K126C 17 18 19 231 MRAL-k0.S127C 17 18 19 232 MRAL-k0.G128C 17 18 19 233 MRAL-k0.T129C 17 18 19 234 MRAL-k0.A130C 17 18 19 235 MRAL-k0.S131C 17 18 19 236 MRAL-k0.L136C 17 18 19 237 MRAL-k0.N137C 17 18 19 238 MRAL-k0.N138C 17 18 19 239 MRAL-k0.F139C 17 18 19 240 MRAL-k0.Y140C 17 18 19 241 MRAL-k0.P141C 17 18 19 242 MRAL-k0.R142C 17 18 19 243 MRAL-k0.E143C 17 18 19 244 MRAL-k0.A144C 17 18 19 245 MRAL-k0.K145C 17 18 19 246 MRAL-k0.V146C 17 18 19 247 MRAL-k0.Q147C 17 18 19 248 MRAL-k0.W148C 17 18 19 249 MRAL-k0.K149C 17 18 19 250 MRAL-k0.V150C 17 18 19 251 MRAL-k0.D151C 17 18 19 252 Petition 870260047205, dated 05 / 18 / 2026, pp. 191 / 2830 183 / 348 SEQ ID NO: Nome do antibody Heavy chain variable region Heavy chain variable region Light chain variable region MRAL-k0.N152C 17 18 19 253 MRAL-k0.A153C 17 18 19 254 MRAL-k0.L154C 17 18 19 255 MRAL-k0.Q155C 17 18 19 256 MRAL-k0.S156C 17 18 19 257 MRAL-k0.G157C 17 18 19 258 MRAL-k0.N158C 17 18 19 259 MRAL-k0.S159C 17 18 19 260 MRAL-k0.Q160C 17 18 19 261 MRAL-k0.E161C 17 18 19 262 MRAL-k0.S162C 17 18 19 263 MRAL-k0.V163C 17 18 19 264 MRAL-k0.T164C 17 18 19 265 MRAL-k0.E165C 17 18 19 266 MRAL-k0.Q166C 17 18 19 267 MRAL-k0.D167C 17 18 19 268 MRAL-k0.S168C 17 18 19 269 MRAL-k0.K169C 17 18 19 270 MRAL-k0.D170C 17 18 19 271 MRAL-k0.S171C 17 18 19 272 MRAL-k0.T172C 17 18 19 273 MRAL-k0.Y173C 17 18 19 274 MRAL-k0.S174C 17 18 19 275 MRAL-k0.L175C 17 18 19 276 MRAL-k0.T180C 17 18 19 277 MRAL-k0.L181C 17 18 19 278 MRAL-k0.S182C 17 18 19 279 MRAL-k0.K183C 17 18 19 280 MRAL-k0.A184C 17 18 19 281 Petition 870260047205, dated 05 / 18 / 2026, page 192 / 2830 184 / 348 SEQ ID NO: Antibody Name Variable Heavy Chain Region Constant Heavy Chain Region Variable Light Chain Region Constant Light Chain Region MRAL-k0.D185C 17 18 19 282 MRAL-k0.Y186C 17 18 19 283 MRAL-k0.E187C 17 18 19 284 MRAL-k0.K188C 17 18 19 285 MRAL-k0.H189C 17 18 19 286 MRAL-k0.K190C 17 18 19 287 MRAL-k0.V191C 17 18 19 288 MRAL-k0.Y192C 17 18 19 289 MRAL-k0.A193C 17 18 19 290 MRAL-k0.E195C 17 18 19 291 MRAL-k0.V196C 17 18 19 292 MRAL-k0.T197C 17 18 19 293 MRAL-k0.H198C 17 18 19 294 MRAL-k0.Q199C 17 18 19 295 MRAL-k0.G200C 17 18 19 296 MRAL-k0.L201C 17 18 19 297 MRAL-k0.S202C 17 18 19 298 MRAL-k0.S203C 17 18 19 299 MRAL-k0.P204C 17 18 19 300 MRAL-k0.V205C 17 18 19 301 MRAL-k0.T206C 17 18 19 302 MRAL-k0.K207C 17 18 19 303 MRAL-k0.S208C 17 18 19 304 MRAL-k0.F209C 17 18 19 305 MRAL-k0.N210C 17 18 19 306 MRAL-k0.R211C 17 18 19 307 MRAL-k0.G212C 17 18 19 308 MRAL-k0.E213C 17 18 19 309 Petition 870260047205, dated 05 / 18 / 2026, page 193 / 2830 185 / 348 Example 6-2. Evaluation of Fab fragmentation mediated by protease antibodies having cysteine ​​substitution at multiple positions in the light chain.

[00522] Using a protease that cleaves the heavy chain hinge region of the antibody to induce Fab fragmentation, the MRA variants produced in Example 6-1 were examined to see if they had acquired protease resistance so that their fragmentation was inhibited. The protease used was Lys-C (Endoproteinase Lys-C Sequencing Grade) (SIGMA; 11047825001). The reaction was performed under the conditions of 2 ng / µg protease, 100 pg / µl antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS and 35 °C for two hours, or under the conditions of 2 ng / µg protease, 20 pg / µl antibody, 80% 25 mM Tris-HCl pH 8.0, 20% PBS and 35 °C for one hour. The sample was then subjected to non-reducing capillary electrophoresis. Wes (Protein Simple) was used for capillary electrophoresis, and an HRP-labeled anti-cap chain antibody (abcam; ab46527) was used for detection. The results are shown in Figs. 14 to 23.Lys-C treatment of MRA caused cleavage of the heavy chain hinge region, resulting in the disappearance of the IgG band at approximately 150 kDa and the appearance of the Fab band at approximately 50 kDa. For the MRA variants produced in Example 6-1, some showed the Fab dimer band appearing at approximately 96 kDa and some showed the undigested IgG band detected at approximately 150 kDa after protease treatment. The area of ​​each band obtained after protease treatment was submitted using proprietary software for Wes (Compass for SW; Protein Simple) to calculate the percentage of areas of the undigested IgG band, Fab dimer, etc. The c...

Claims

1. Antigen-binding molecule, characterized in that it comprises a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains comprise a hinge region; wherein the first and second antigen-binding domains comprise an antibody fragment that binds to a particular antigen; wherein the first antigen-binding domain and the second antigen-binding domain are linked together by means of two or more disulfide bonds; wherein 1) at least one of the disulfide bonds linking the first antigen-binding domain and the second antigen-binding domain is formed by the linkage of cysteine ​​residues in a variable region of the antibody fragment of the first and second antigen-binding domains, and 2) at least one disulfide bond is present in the hinge region, and wherein said antigen-binding molecule exhibits greater resistance to protease cleavage compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from said antigen-binding molecule only by having one less bond between the two antigen-binding domains.

2. Antigen-binding molecule according to claim 1, characterized in that at least one of the bonds linking the first and second antigen-binding domains is formed by the linkage of an amino acid residue in a VH region of the first antigen-binding domain with an amino acid residue in a VH region of the second antigen-binding domain.

3. Antigen-binding molecule according to claim 2,characterized in that at least one of the linkages joining the first and second antigen-binding domains is formed by the linkage of any two amino acid residues selected from the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74 and 82b, according to Kabat numbering, in the VH region of the first antigen-binding domain and in the VH region of the second antigen-binding domain.

4. Antigen-binding molecule according to claim 3, characterized in that at least one linkage joining the first and second antigen-binding domains is formed by the linkage of amino acid residues to each other at any of the selected positions of the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74 and 82b, according to Kabat numbering, in the VH region of the first and second antigen-binding domains.

5. Antigen-binding molecule according to claim 1,characterized by the fact that at least one of the linkages connecting the first and second antigen-binding domains is formed by the linkage of an amino acid residue in a VL region of the first antigen-binding domain with an amino acid residue in a VL region of the second antigen-binding domain.

6. Antigen-binding molecule according to claim 5, characterized in that at least one of the linkages joining the first and second antigen-binding domains is formed by the linkage of any two amino acid residues selected from the group consisting of positions 21, 27, 58, 77, 100, 105 and 107, according to Kabat numbering, in the VL region (subclass κ) of the first antigen-binding domain and in the VL region (subclass κ) of the second antigen-binding domain.

7. Antigen-binding molecule according to claim 6,characterized in that at least one linkage joining the first and second antigen-binding domains is formed by linking amino acid residues to each other at any of the selected positions of the group consisting of positions 21, 27, 58, 77, 100, 105 and 107, according to Kabat numbering, in the VL region (κ subclass) of the first and second antigen-binding domains.

8. Antigen-binding molecule according to claim 5, characterized in that at least one of the linkages joining the first and second antigen-binding domains is formed by linking any two amino acid residues selected from the group consisting of positions 6, 19, 33 and 34, according to Kabat numbering, in the VL region (λ subclass) of the first antigen-binding domain and in the VL region (λ subclass) of the second antigen-binding domain.

9. Antigen-binding molecule according to claim 8,characterized in that at least one linkage joining the first and second antigen-binding domains is formed by the linkage of amino acid residues to each other at any of the selected positions of the group consisting of positions 6, 19, 33 and 34 according to Kabat numbering, in the VL region (λ subclass) of the first and second antigen-binding domains.

10. Antigen-binding molecule, according to any one of claims 1 to 9, characterized in that the antibody fragment is a Fab, Fab', or scFab.

11. Antigen-binding molecule, according to any one of claims 1 to 10, characterized in that the antigen-binding domain comprises an Fc region.

12. Antigen-binding molecule according to any one of claims 1 to 11,characterized by the fact that it has regulatory activity on the interaction between two antigen molecules.

13. Pharmaceutical composition, characterized by the fact that it comprises the antigen-binding molecule, as defined in any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. Method for regulating the interaction between two antigen molecules, characterized by the fact that it comprises: (a) providing an antigen-binding molecule comprising two antigen-binding domains, wherein each of the antigen-binding domains comprises a hinge region and an antibody fragment that binds to a particular antigen; (b) adding to the antigen-binding molecule at least one disulfide bond linking the two antigen-binding domains to each other, such that the antigen-binding domains are linked to each other by means of two or more disulfide bonds,wherein (1) at least one of the cysteine ​​residues from which disulfide bonds originate between the antigen-binding domains is present in a variable region of the antibody fragment of the first and second antigen-binding domains, and (2) at least one of the two or more disulfide bonds is present in the hinge region, and (c) contacting the antigen-binding molecule produced in (b) with the two antigen molecules, and wherein said antigen-binding molecule exhibits greater resistance to protease cleavage compared with a control antigen-binding molecule, wherein the antigen-binding molecule Petition 870260047205, dated 05 / 18 / 2026,p. 361 / 2830 5 / 6 to the control antigen differs from the said antigen-binding molecule only by having one less linkage between the two antigen-binding domains.

15. Method for producing an antigen-binding molecule that has regulatory activity of the interaction between two antigen molecules, characterized in that it comprises: (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 antigen-binding domains comprises a hinge region and an antibody fragment that binds to a particular antigen, (b) introducing a mutation in the nucleic acids encoding the two antigen-binding domains such that at least one linkage connecting the two antigen-binding domains is added,so that the antigen-binding domains are linked together by two or more disulfide bonds, wherein 1) at least one of the cysteine ​​residues from which the linkages between the antigen-binding domains originate is present in a variable region of the antibody fragment of the first and second antigen-binding domains, and 2) at least one of the two or more disulfide bonds is present in the hinge region, (c) introduce the nucleic acids produced in (b) into a host cell, (d) culture the host cell such that both polypeptides are expressed, and (e) obtain an antigen-binding molecule that is a polypeptide comprising the first and second antigen-binding domains, wherein the two antigen-binding domains are linked together by two or more disulfide bonds,and wherein said antigen-binding molecule exhibits greater resistance to protease cleavage compared to a control antigen-binding molecule, the control antigen-binding molecule differing from said antigen-binding molecule only by having one less linkage between the two antigen-binding domains.

16. Isolated nucleic acid, characterized in that it encodes the antigen-binding molecule, as defined in any one of claims 1 to 12.

17. Vector, characterized in that it comprises the isolated nucleic acid, as defined in claim 16.

18. Host cell, characterized in that it comprises the isolated nucleic acid, as defined in claim 16, or the vector, as defined in claim 17.

Images