Method for producing proteins

Abstract

In one embodiment, it was found that a preparation containing an antigen-binding molecule can be produced more efficiently and with high reproducibility by carrying out the following steps in chromatography, the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region; contacting a mixture containing the antigen-binding molecule and a misdisulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule with a solution containing a reducing agent, the antigen binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region; and subsequently, removing the reducing agent.

Description

Technical Field

[0001] The present disclosure relates to methods for producing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. The present disclosure also relates to methods for increasing or enriching such antigen-binding molecules, methods for eliminating the heterogeneity of disulfide bonds of antigen-binding molecules, and the like. Background Art

[0002] Antigen-binding molecules (LINC-Ig) having an artificial disulfide bond between amino acid residues in a region other than the hinge region (“LINC”) are known (PTL 1 to PTL 4). For example, LINC-Ig has a disulfide bond between the CH1 region of one of the two heavy chains constituting the antigen-binding molecule and the CH1 region of the other heavy chain in the heavy chain. However, when such an antigen-binding molecule is obtained by expressing it in a cell culture medium, two forms (a molecule having an appropriate disulfide bond between the heavy chains (LINC form) and a molecule having no appropriate disulfide bond, such as a mis-disulfide-bonded form and a non-disulfide-bonded form (unLINC form)) will coexist in the cell culture medium. When preparing LINC-Ig, it is necessary to convert molecules in the cell culture medium in which appropriate disulfide bonds are not formed between the heavy chains, such as open-type molecules having capped sulfur atoms, into molecules having appropriate disulfide bonds.

[0003] The following are known methods for obtaining LINC-Ig, which form appropriate disulfide bonds between the heavy chains via amino acid residues in a region other than the hinge region.

[0004] PTL 2 and PTL 4 disclose a method for effectively obtaining an antibody having LINC, which includes treating a preparation containing a molecule having a disulfide bond between the heavy chains and a molecule in which an appropriate disulfide bond is not formed with a reducing agent, and then performing reoxidation by buffer exchange or the like to form a disulfide bond.

[0005] PTL 5 discloses using a redox buffer (e.g., cysteine / cystine) so that a reduction-oxidation reaction can be carried out in a downstream process to prevent fragmentation of the antibody.

[0006] PTL 6 discloses a method for refolding a recombinant antibody by contacting the recombinant antibody with a reduction / oxidation coupling reagent.

[0007] NPL 1 discloses using a redox buffer to inhibit batch-to-batch non-uniformity caused by cysteinylation of a specific antibody (MAB007).

[0008] [Citation List]

[0009] [Patent Documents]

[0010] [PTL 1]WO2020 / 027330

[0011] [PTL 2]WO2021 / 157679

[0012] [PTL 3]WO2021 / 200898

[0013] [PTL 4]WO2021 / 201087

[0014] [PTL 5]WO2020 / 037016

[0015] [PTL 6]WO2006 / 047340

[0016] [Non - Patent Literature]

[0017] [NPL 1]Pharm Sci. Feb. 2008; 97(2):775 - 90,Removal of Cysteinylationfrom an Unpaired Sulfhydryl in the Variable Region of a RecombinantMonoclonal IgG1 Antibody Improves Homogeneity,Stability,and BiologicalActivity Summary of the Invention

[0018] [Technical Problem]

[0019] As described above, a method is known for effectively obtaining an antigen - binding molecule having LINC by treating a mixture containing an antigen - binding molecule having appropriate disulfide bonds between heavy chains and an antigen - binding molecule in which appropriate disulfide bonds are not formed (e.g., a capped molecule) with a reducing agent and then performing re - oxidation. However, from the viewpoints of productivity and ease of manufacture, this method has problems including low yield and long reaction time.

[0020] To solve the above problems, the present disclosure aims to provide a method for effectively and easily producing and purifying an antigen - binding molecule having appropriate disulfide bonds between antibody heavy chains. The present disclosure relates to a method for increasing the structural homogeneity and relative abundance of an antigen - binding molecule having one or more disulfide bonds formed between amino acid residues in a region other than the hinge region at each of two antibody heavy chains. In other words, the present disclosure relates to a method for reducing the relative abundance of an antigen - binding molecule in which appropriate disulfide bonds are not formed between amino acid residues in a region other than the hinge region.

[0021] [Solution to the Problem]

[0022] As a result of investigations to solve the above problems, the present inventors found that by contacting an antigen-binding molecule with a reducing agent and then removing the reducing agent, at least one disulfide bond can be formed more effectively and with high reproducibility between amino acid residues in regions other than the hinge region, where the antigen-binding molecule binds to an affinity column and has amino acid residues capable of forming at least one disulfide bond between amino acid residues in regions other than the hinge region (these antigen-binding molecules include antigen-binding molecules that form appropriate disulfide bonds between amino acid residues in regions other than the hinge region and antigen-binding molecules that do not form at least one appropriate disulfide bond between amino acid residues in regions other than the hinge region). Specifically, the present inventors developed a technique to stably obtain a high yield of an antigen-binding molecule (LINC-Ig form) having at least one disulfide bond between amino acid residues in regions other than the hinge region by inserting two steps (flowing a solution containing a reducing agent and removing the reducing agent) in the step of protein A affinity chromatography purification (a commonly used technique for primary purification from cell culture supernatants), the harvested cell culture fluid (HCCF).

[0023] This disclosure is based on these findings and specifically relates to the following:

[0024] [1] A method for producing a preparation containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises subjecting a mixture to chromatography in the presence of a reducing agent, the antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region.

[0025] [2] A method for producing a preparation containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises:

[0026] (a) contacting the antigen-binding molecule with a solution containing a reducing agent in chromatography, the antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region, and

[0027] (b) removing the reducing agent.

[0028] [3] A method for producing a preparation containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises subjecting a mixture to chromatography in the presence of a reducing agent, the mixture comprising:

[0029] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0030] a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule.

[0031] [4] A method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises:

[0032] (a) contacting a mixture in chromatography with a solution comprising a reducing agent, the mixture comprising:

[0033] an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0034] a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule;

[0035] and

[0036] (b) removing the reducing agent.

[0037] [5] The method according to any one of [1] to [4], wherein the antigen-binding molecule comprises two or more polypeptide chains, and the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the polypeptide chains.

[0038] [6] The method according to [5], wherein either or both of the polypeptide chains comprise a mutated, substituted or introduced cysteine residue in a region other than the hinge region.

[0039] [7] The method according to any one of [1] to [6], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the first antigen-binding domain and the second antigen-binding domain.

[0040] [8] The method according to any one of [1] to [7], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the heavy chain of the first antigen-binding domain and the heavy chain of the second antigen-binding domain.

[0041] [9] The method according to any one of [1] to [8], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain.

[0042]

[10] The method according to any one of [1] to [9], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the amino acid residue at EU numbering position 191 in the heavy chain of the first antigen-binding domain and the amino acid residue at EU numbering position 191 in the heavy chain of the second antigen-binding domain.

[0043]

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

[10] , wherein the chromatography comprises an affinity chromatography matrix, an ion exchange chromatography matrix, a hydrophobic interaction chromatography matrix, a multimodal chromatography matrix comprising both ion exchange chromatography and hydrophobic interaction chromatography, or a hydroxyapatite matrix.

[0044]

[12] The method according to

[11] , wherein the affinity chromatography matrix is selected from the group consisting of: a protein A matrix, a protein G matrix, a protein L matrix, a sequence-selective peptide matrix, and a matrix that selectively binds to the antigen-binding molecule.

[0045]

[13] The method according to

[11] , wherein the ion exchange chromatography matrix is a cation exchange ligand or an anion exchange ligand.

[0046]

[14] The method according to

[11] , wherein the hydrophobic interaction chromatography matrix is a hydrophobic ligand.

[0047]

[15] The method according to

[11] , wherein the multimodal chromatography matrix is a matrix having a combination of a cation exchange ligand and a hydrophobic ligand, or a matrix having a combination of an anion exchange ligand and a hydrophobic ligand.

[0048]

[16] The method according to

[11] , wherein the hydroxyapatite matrix is hydroxyapatite or a derivative thereof (such as fluorapatite).

[0049]

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

[16] , wherein the method comprises:

[0050] (a) contacting a mixture with a chromatography matrix, the mixture comprising:

[0051] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0052] a misdisulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule;

[0053] the chromatography matrix is packed into a column for column chromatography or coated on a membrane for membrane chromatography,

[0054]

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

[17] , wherein the reducing agent is selected from the group consisting of monothiols, dithiols, phosphines, and inorganic reagents, and combinations of two or more thereof.

[0055]

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

[18] , wherein the reducing agent is cysteine or TCEP.

[0056]

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

[18] , wherein the reducing agent is a monothiol and the concentration of the reducing agent is from about 0.01 mM to about 100 mM.

[0057]

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

[19] , wherein the reducing agent is cysteine and the concentration of the reducing agent is from about 0.01 mM to about 100 mM, from about 0.0001 mM to about 100.0 mM, from about 0.001 mM to about 100.0 mM, from about 0.005 mM to about 75.0 mM, from about 0.01 mM to about 50.0 mM, from about 0.05 mM to about 25.0 mM, or from about 0.1 mM to about 10 mM.

[0058]

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

[18] , wherein the reducing agent is a dithiol and the concentration of the reducing agent is from about 0.001 mM to about 10 mM.

[0059]

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

[18] , wherein the reducing agent is a phosphine and the concentration of the reducing agent is from about 0.0001 mM to about 1 mM or from about 0.001 mM to about 0.01 mM.

[0060]

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

[19] , wherein the reducing agent is TCEP and the concentration of the reducing agent is from about 0.00001 mM to about 10.0 mM, from about 0.00005 mM to about 5.0 mM, from about 0.0001 mM to about 1 mM, from about 0.0005 mM to about 0.5 mM, from about 0.001 mM to about 0.1 mM, or from about 0.001 mM to about 0.01 mM.

[0061]

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

[18] , wherein the reducing agent is an inorganic reagent and the concentration of the reducing agent is from about 0.0001 mM to about 10 mM or from about 0.001 mM to about 0.1 mM.

[0062]

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

[19] , wherein the reducing agent is cysteine and the concentration of the reducing agent is about 0.1 mM, about 0.15 mM, about 1.0 mM, about 10.0 mM or about 100 mM.

[0063]

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

[19] , wherein the reducing agent is TCEP and the concentration of the reducing agent is about 0.001 mM, about 0.01 mM, about 0.1 mM or about 1.0 mM.

[0064]

[28] The method according to [2] and any one of [4] to

[27] , wherein the pH of the solution containing the reducing agent is from about 4.5 to about 10.0, from about 5.0 to about 9.0, from about 6.5 to about 8.5, or from about 7.0 to about 8.0.

[0065]

[29] The method according to [2] and any one of [4] to

[28] , wherein the pH of the solution containing the reducing agent is about 7.0, about 7.5 or about 8.0.

[0066]

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

[29] , wherein the chromatography is column chromatography or membrane chromatography.

[0067]

[31] The method according to

[30] , wherein the solution containing the reducing agent is passed through a column for column chromatography or a device for membrane chromatography, and the residence time is from about 2 seconds to about 80 minutes or from about 3 seconds to about 24 minutes, or the flow is temporarily stopped when the column or device is filled with the reducing agent.

[0068]

[32] The method according to

[30] or

[31] , wherein the solution containing the reducing agent is passed through a column for column chromatography or a device for membrane chromatography, and the residence time is about 12 minutes or about 30 seconds, or the flow is temporarily stopped when the column or device is filled with the solution containing the reducing agent.

[0069]

[33] The method according to [2] and any one of [4] to

[32] , wherein the mixture is contacted with the solution containing the reducing agent for about 6 seconds to about 1440 minutes, or about 18 seconds to about 300 minutes.

[0070]

[34] The method according to [2] and any one of [4] to

[33] , wherein the mixture is contacted with the solution containing the reducing agent for about 120 minutes or about 7.5 minutes.

[0071]

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

[34] , wherein the removal of the reducing agent comprises contacting the antigen-binding molecule with a solution free of the reducing agent.

[0072]

[36] The method according to

[35] , wherein the chromatography is column chromatography or membrane chromatography, and wherein the contacting of the mixture with the solution free of the reducing agent comprises flowing the solution free of the reducing agent through a column for column chromatography or a device for membrane chromatography.

[0073]

[37] The method according to

[35] or

[36] , wherein the solution free of the reducing agent is flowed through a column for column chromatography or a device for membrane chromatography for a residence time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes, and optionally the flow is temporarily stopped when the column or device is filled with the solution.

[0074]

[38] The method according to any one of

[35] to

[37] , wherein the solution free of the reducing agent is flowed through a column for column chromatography or a device for membrane chromatography for a residence time of about 4 minutes or about 30 seconds, and optionally the flow is temporarily stopped when the column or device is filled with the solution.

[0075]

[39] The method according to any one of

[35] to

[38] , wherein the mixture is contacted with the solution free of the reducing agent for about 6 seconds to about 1440 minutes, or about 18 seconds to about 300 minutes.

[0076]

[40] The method according to any one of

[35] to

[39] , wherein the mixture is contacted with the solution free of the reducing agent for about 20 minutes or about 7.5 minutes.

[0077]

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

[40] , wherein the mixture is contacted with the solution containing the reducing agent at about 4°C to about 37°C, preferably at about 15°C to about 37°C.

[0078]

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

[41] , wherein the method further comprises: before step (a), contacting the mixture with a chromatography matrix, the mixture comprising:

[0079] an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0080] mis-disulfide-bonded forms and / or non-disulfide-bonded forms of the antigen-binding molecule;

[0081] to pack the antigen-binding molecule into a column for column chromatography or to immobilize the antigen-binding molecule on a membrane for membrane chromatography.

[0082]

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

[42] , wherein the method further comprises: removing impurities in chromatography before step (a).

[0083]

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

[43] , wherein the method further comprises: recovering an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, before or after step (b).

[0084]

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

[44] , wherein contacting the antigen-binding molecule with a solution containing a reducing agent results in the cleavage of disulfide bonds formed between amino acid residues capable of forming disulfide bonds, and / or the de-capping of sulfur atoms in amino acid residues capable of forming disulfide bonds.

[0085]

[46] The method according to [2] and any one of [4] to

[45] , wherein the removal of the reducing agent results in the formation of disulfide bonds between amino acid residues capable of forming disulfide bonds.

[0086]

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

[46] , wherein amino acid residues capable of forming disulfide bonds are introduced or engineered to be cysteine residues.

[0087]

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

[47] , wherein at least one disulfide bond in a region other than the hinge region is an inter-chain disulfide bond.

[0088]

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

[48] , wherein at least one disulfide bond formed between amino acid residues in a region other than the hinge region is one, two, three, four or more inter-chain disulfide bonds.

[0089]

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

[49] , wherein at least one disulfide bond formed between amino acid residues in a region other than the hinge region is an engineered disulfide bond that does not exist in wild-type IgG.

[0090]

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

[50] , the method for increasing the ratio (LINC ratio) of an antigen-binding molecule (LINC form) having at least one disulfide bond in a region other than the hinge region to the sum of:

[0091] (i) an antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0092] (ii) mis-disulfide-bonded forms and / or non-disulfide-bonded forms (unLINC forms) of the antigen-binding molecule.

[0093]

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

[51] , the method for producing an antigen-binding molecule (LINC form) having at least one disulfide bond in a region other than the hinge region in a molar ratio of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to the sum of the following:

[0094] (i) an antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and

[0095] (ii) mis-disulfide-bonded forms and / or non-disulfide-bonded forms (unLINC forms) of the antigen-binding molecule.

[0096] This disclosure also relates to the following inventions:

[0097] [A1] The method according to any one of [1] to

[52] , wherein the chromatography is column chromatography.

[0098] [A2] The method according to [A1], wherein the method comprises:

[0099] (a) contacting a mixture with a chromatography matrix, the mixture comprising:

[0100] an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0101] mis-disulfide-bonded forms and / or non-disulfide-bonded forms of the antigen-binding molecule;

[0102] filling the chromatography matrix into a column.

[0103] [A3] The method according to [A1] or [A2], wherein a solution containing a reducing agent is passed through the column for a residence time of about 2 minutes to about 80 minutes, and optionally the flow is temporarily stopped when the column is filled with the solution containing the reducing agent.

[0104] [A4] The method according to any one of [A1] to [A3], wherein a solution containing a reducing agent is passed through the column for a residence time of about 4 minutes to about 24 minutes, and optionally the flow is temporarily stopped when the column is filled with the solution containing the reducing agent.

[0105] [A5] The method according to any one of [A1] to [A4], wherein a solution containing a reducing agent is passed through the column for a residence time of about 12 minutes, and optionally the flow is temporarily stopped when the column is filled with the solution containing the reducing agent.

[0106] [A6]The method according to any one of [A1] to [A5], wherein a solution containing a reducing agent is passed through a column for column chromatography at a rate of about 25 cm / h to about 500 cm / h, or about 50 cm / h to about 400 cm / h, and optionally the flow is temporarily stopped when the column is filled with the solution containing the reducing agent.

[0107] [A7]The method according to any one of [A1] to [A6], wherein a solution containing a reducing agent is passed through a column for column chromatography at a rate of about 100 cm / h, and optionally the flow is temporarily stopped when the column is filled with the solution containing the reducing agent.

[0108] [A8]The method according to any one of [A1] to [A7], wherein the flow volume of the solution containing the reducing agent is about 0.1 times the column volume (about 0.1 CV (column volume)) to about 100 times the column volume (about 100 CV).

[0109] [A9]The method according to any one of [A1] to [A8], wherein the flow volume of the solution containing the reducing agent is about 1 times the column volume (1 CV) to about 20 times the column volume (about 20 CV).

[0110] [A10]The method according to any one of [A1] to [A9], wherein the flow volume of the solution containing the reducing agent is about 10.0 times the column volume (about 10.0 CV).

[0111] [A11]The method according to any one of [A1] to [A10], wherein the mixture is contacted with the solution containing the reducing agent for about 4 minutes to about 1440 minutes.

[0112] [A12]The method according to any one of [A1] to [A11], wherein the mixture is contacted with the solution containing the reducing agent for about 8 minutes to about 300 minutes.

[0113] [A13]The method according to any one of [A1] to [A12], wherein the mixture is contacted with the solution containing the reducing agent for about 120 minutes.

[0114] [A14]The method according to any one of [A1] to [A13], wherein the amount of antigen-binding molecules carried by the matrix is about 5 g to about 80 g, about 7 g to about 50 g, or about 10 g to about 40 g per 1 L of the matrix.

[0115] [A15]The method according to any one of [A1] to [A14], wherein a solution without a reducing agent is passed through the column, and the residence time is about 2 minutes to about 80 minutes, or the flow is temporarily stopped when the column is filled with the solution.

[0116] [A16]The method according to any one of [A1] to [A15], wherein a solution without a reducing agent is passed through the column, with a residence time of about 4 minutes to about 24 minutes, or the flow is temporarily stopped when the column is filled with the solution.

[0117] [A17]The method according to any one of [A1] to [A16], wherein a solution without a reducing agent is passed through the column, with a residence time of about 4 minutes, or the flow is temporarily stopped when the column is filled with the solution.

[0118] [A18]The method according to any one of [A1] to [A17], wherein a solution without a reducing agent is passed through the column for column chromatography at a rate of about 25 cm / h to about 500 cm / h, or about 50 cm / h to about 400 cm / h, or the flow is temporarily stopped when the column is filled with the solution.

[0119] [A19]The method according to any one of [A1] to [A18], wherein a solution without a reducing agent is passed through the column for column chromatography at a rate of about 300 cm / h, or the flow is temporarily stopped when the column is filled with the solution.

[0120] [A20]The method according to any one of [A1] to [A19], wherein the flow volume of the solution without a reducing agent is about 0.1 times (about 0.1 CV) to about 100 times (about 100 CV) of the column volume, or about 1 time (about 1 CV) to about 20 times (about 20 CV) of the column volume. [A21]The method according to any one of [A1] to [A20], wherein the flow volume of the solution without a reducing agent is about 5.0 times (about 5.0 CV) of the column volume.

[0121] [A22]The method according to any one of [A1] to [A21], wherein the mixture is contacted with the solution without the reducing agent for about 4 minutes to about 1440 minutes.

[0122] [A23]The method according to any one of [A1] to [A22], wherein the mixture is contacted with the solution without the reducing agent for about 8 minutes to about 300 minutes.

[0123] [A24]The method according to any one of [A1] to [A23], wherein the mixture is contacted with the solution without the reducing agent for about 20 minutes.

[0124] The present disclosure also relates to the following inventions:

[0125] [B1]The method according to any one of [1] to

[52] , wherein the chromatography is membrane chromatography.

[0126] [B2]The method according to [B1], wherein the method comprises:

[0127] (a) contacting a mixture with a chromatography matrix, the mixture comprising:

[0128] an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and

[0129] a misdisulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule;

[0130] the chromatography matrix being coated or immobilized on a membrane for membrane chromatography.

[0131] [B3]The method according to [B1] or [B2], wherein a solution containing a reducing agent is passed through the membrane device for a residence time of about 2 seconds to about 60 minutes, or the flow is temporarily stopped when the membrane device is filled with the solution containing the reducing agent.

[0132] [B4]The method according to any one of [B1] to [B3], wherein a solution containing a reducing agent is passed through the membrane device for a residence time of about 3 seconds to about 6 minutes, such as about 30 seconds, or the flow is temporarily stopped when the membrane device is filled with the solution containing the reducing agent.

[0133] [B5]The method according to any one of [B1] to [B4], wherein the flow volume of the solution containing the reducing agent is about 1 times (about 1 MV (membrane volume)) to about 500 times (about 500 MV) the volume of the membrane device.

[0134] [B6]The method according to any one of [B1] to [B5], wherein the flow volume of the solution containing the reducing agent is about 2 times (about 2 MV) to about 100 times (about 100 MV) the volume of the membrane device, such as about 15 times (about 15 MV) the volume of the membrane device.

[0135] [B7]The method according to any one of [B1] to [B6], wherein the mixture is contacted with the solution containing the reducing agent for about 6 seconds to about 600 minutes.

[0136] [B8]The method according to any one of [B1] to [B7], wherein the mixture is contacted with the solution containing the reducing agent for about 18 seconds to about 120 minutes, such as about 7.5 minutes.

[0137] [B9]The method according to any one of [B1] to [B8], wherein the amount of the antigen-binding molecule carried by the matrix is about 5 g to about 100 g or about 7 g to about 70 g, such as about 25 g, per 1 L of the volume of the membrane device.

[0138] [B10]The method according to any one of [B1] to [B9], wherein a solution without a reducing agent is passed through the membrane device with a residence time of about 2 seconds to about 60 minutes, or the flow is temporarily stopped when the membrane device is filled with the solution.

[0139] [B11]The method according to any one of [B1] to [B10], wherein a solution without a reducing agent is passed through the membrane device with a residence time of about 3 seconds to about 6 minutes, such as about 30 seconds, or the flow is temporarily stopped when the membrane device is filled with the solution.

[0140] [B12]The method according to any one of [B1] to [B11], wherein the flow volume of the solution without a reducing agent is about 1 times (about 1MV) the volume of the membrane device to about 500 times (about 500MV) the volume of the membrane device.

[0141] [B13]The method according to any one of [B1] to [B12], wherein the flow volume of the solution without a reducing agent is about 2 times (about 2MV) the volume of the membrane device to about 100 times (about 100MV) the volume of the membrane device, such as about 15 times (about 15MV) the volume of the membrane device.

[0142] [B14]The method according to any one of [B1] to [B13], wherein the mixture is contacted with the solution without the reducing agent for about 6 seconds to about 600 minutes.

[0143] [B15]The method according to any one of [B1] to [B14], wherein the mixture is contacted with the solution without the reducing agent for about 18 seconds to about 120 minutes, such as about 7.5 minutes.

[0144] The present disclosure also relates to the following inventions:

[0145]

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

[52] , [A1] to [A24] and [B1] to [B15], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain that can be connected to each other via at least one disulfide bond.

[0146]

[102] The method according to

[101] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a Fab, Fab', scFab, Fv, scFv or VHH structure.

[0147]

[103] The method according to

[101] or

[102] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises or does not comprise a hinge region.

[0148]

[104] The method according to any one of

[101] to

[103] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a Fab and a hinge region forming an F(ab')2 structure.

[0149]

[105] The method according to any one of

[101] to

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

[0150]

[106] The method according to any one of

[101] to

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

[0151]

[107] The method according to any one of

[101] to

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

[0152]

[108] The method according to any one of

[101] to

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

[0153]

[109] The method according to any one of

[101] to

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

[0154]

[110] The method according to any one of

[101] to

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

[0155]

[111] The method according to any one of

[101] to

[110] , wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds to a soluble protein.

[0156]

[112] The method according to any one of

[101] to

[111] , wherein at least one of the first antigen-binding domain and the second antigen-binding domain binds to a membrane protein.

[0157]

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

[52] , [A1] to [A24], [B1] to [B15] and

[101] to

[112] , wherein the antigen-binding molecule has the activity of regulating the interaction between two antigen molecules.

[0158]

[114] The method according to any one of

[101] to

[104] , wherein the first antigen-binding domain and the second antigen-binding domain bind to a ligand and its receptor, respectively, and wherein the antigen-binding molecule has an activity of promoting the activation of the receptor mediated by the ligand.

[0159]

[115] The method according to any one of

[101] to

[104] , wherein the first antigen-binding domain and the second antigen-binding domain bind to an enzyme and its substrate, respectively, and wherein the antigen-binding molecule has an activity of promoting the catalytic reaction between the enzyme and the substrate.

[0160]

[116] The method according to any one of

[101] to

[104] , wherein the first antigen-binding domain and the second antigen-binding domain both bind to proteins (a first antigen and a second antigen, respectively) present on the surface of a cell, and wherein the antigen-binding molecule has an activity of promoting the interaction between the cell expressing the first antigen and the cell expressing the second antigen.

[0161]

[117] The method according to

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

[0162]

[118] The method according to

[117] , wherein the cell having cytotoxic activity is a T cell, an NK cell, a monocyte or a macrophage.

[0163]

[119] The method according to any one of

[105] to

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

[0164]

[120] The method according to any one of

[101] to

[104] , wherein the first antigen-binding domain and the second antigen-binding domain are each capable of binding to CD3 and / or CD137.

[0165]

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

[52] , [A1] to [A24], [B1] to [B15] and

[101] to

[120] , wherein the antigen-binding molecule further comprises a third antigen-binding domain.

[0166]

[122] The method according to any one of

[101] to

[121] , wherein the third antigen-binding domain is fused to the first antigen-binding domain or the second antigen-binding domain.

[0167]

[123] The method according to

[121] or

[122] , wherein the third antigen-binding domain is a Fab or scFv.

[0168]

[124] The method according to any one of

[102] to

[123] , wherein the third antigen-binding domain is optionally fused at its C-terminus via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of the first antigen-binding domain or the second antigen-binding domain.

[0169]

[125] The method according to any one of

[121] to

[124] , wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each a Fab molecule, and wherein the third antigen-binding domain is optionally fused at the C-terminus of its Fab heavy chain (CH1 region) via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of the first antigen-binding domain or the second antigen-binding domain.

[0170]

[126] The method according to

[124] or

[125] , wherein the peptide linker comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0171]

[127] The method according to any one of

[121] to

[126] , wherein the third antigen-binding domain is a cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the first antigen-binding domain and the second antigen-binding domain are conventional Fab molecules.

[0172]

[128] The method according to any one of

[121] to

[127] , wherein the third antigen-binding domain is capable of binding to an antigen expressed on a cancer cell or cancer tissue.

[0173]

[129] The method according to any one of

[121] to

[128] , wherein the third antigen-binding domain is capable of binding to DLL3, preferably human DLL3.

[0174]

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

[52] , [A1] to [A24], [B1] to [B15], and

[101] to

[129] , wherein the antigen-binding molecule further comprises an Fc region.

[0175]

[131] The method according to

[130] , wherein the Fc region consists of a first Fc region subunit and a second Fc region subunit that can stably associate with each other.

[0176]

[132] The method according to

[131] , wherein the first antigen-binding domain and the second antigen-binding domain are Fab, wherein the first antigen-binding domain is fused to the N-terminus of either the first Fc region subunit or the second Fc region subunit of the Fc region at the C-terminus of the Fab heavy chain, and wherein the second antigen-binding domain is fused to the N-terminus of the other subunit of the Fc region at the C-terminus of the Fab heavy chain.

[0177]

[133] The method according to any one of

[130] to

[132] , wherein the Fc region is of human origin.

[0178]

[134] The method according to any one of

[130] to

[133] , wherein the Fc region is an IgG Fc region, preferably a human IgG Fc region, or more preferably a human IgG1 Fc region.

[0179]

[135] The method according to any one of

[130] to

[134] , wherein the Fc region shows a reduced binding affinity for human Fcγ receptors compared to the native human IgG1 Fc region.

[0180]

[136] The method according to any one of

[130] to

[135] , wherein the Fc region shows enhanced FcRn binding activity under acidic pH conditions (e.g., pH 5.8) compared to the native IgG Fc region.

[0181]

[137] The method according to any one of

[130] to

[136] , wherein the Fc region comprises: Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering.

[0182]

[138] The method according to any one of

[130] to

[137] , wherein the Fc region comprises: Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering.

[0183]

[139] The method according to any one of

[130] to

[138] , wherein the Fc region further comprises: Ile or Leu at position 428; and / or Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering.

[0184]

[140] The method according to any one of

[130] to

[139] , wherein the Fc region comprises a combination of amino acid substitutions selected from the group consisting of:

[0185] (a) N434A / Q438R / S440E;

[0186] (b) N434A / Q438R / S440D;

[0187] (c) N434A / Q438K / S440E;

[0188] (d) N434A / Q438K / S440D;

[0189] (e) N434A / Y436T / Q438R / S440E;

[0190] (f) N434A / Y436T / Q438R / S440D;

[0191] (g) N434A / Y436T / Q438K / S440E;

[0192] (h) N434A / Y436T / Q438K / S440D;

[0193] (i) N434A / Y436V / Q438R / S440E;

[0194] (j) N434A / Y436V / Q438R / S440D;

[0195] (k) N434A / Y436V / Q438K / S440E;

[0196] (l) N434A / Y436V / Q438K / S440D;

[0197] (m) N434A / R435H / F436T / Q438R / S440E;

[0198] (n) N434A / R435H / F436T / Q438R / S440D;

[0199] (o) N434A / R435H / F436T / Q438K / S440E;

[0200] (p) N434A / R435H / F436T / Q438K / S440D;

[0201] (q) N434A / R435H / F436V / Q438R / S440E;

[0202] (r) N434A / R435H / F436V / Q438R / S440D;

[0203] (s) N434A / R435H / F436V / Q438K / S440E;

[0204] (t) N434A / R435H / F436V / Q438K / S440D;

[0205] (u) M428L / N434A / Q438R / S440E;

[0206] (v) M428L / N434A / Q438R / S440D;

[0207] (w) M428L / N434A / Q438K / S440E;

[0208] (x) M428L / N434A / Q438K / S440D;

[0209] (y) M428L / N434A / Y436T / Q438R / S440E;

[0210] (z) M428L / N434A / Y436T / Q438R / S440D;

[0211] (aa) M428L / N434A / Y436T / Q438K / S440E;

[0212] (ab) M428L / N434A / Y436T / Q438K / S440D;

[0213] (ac) M428L / N434A / Y436V / Q438R / S440E;

[0214] (ad) M428L / N434A / Y436V / Q438R / S440D;

[0215] (ae) M428L / N434A / Y436V / Q438K / S440E;

[0216] (af) M428L / N434A / Y436V / Q438K / S440D;

[0217] (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[0218] (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E,

[0219] According to EU numbering.

[0220]

[141] The method according to any one of

[130] to

[136] , wherein the Fc region comprises a combination of amino acid substitutions M428L / N434A / Q438R / S440E.

[0221]

[142] The method according to any one of

[130] to

[141] , wherein the Fc region comprises a combination of one or more amino acid substitutions that promote Fc region multimerization.

[0222]

[143] The method according to

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

[0223]

[144] The method according to

[142] or

[143] , wherein the multimerization is hexamerization.

[0224]

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

[52] , [A1] to [A24], [B1] to [B15], and

[101] to

[144] , wherein the antigen-binding molecule comprises an amino acid residue resulting from the substitution of at least one cysteine residue in its hinge region.

[0225]

[146] The method according to

[145] , wherein the cysteine residue is present at EU numbering position 226 and / or position 229 in the hinge region.

[0226]

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

[52] , [A1] to [A24], [B1] to [B15], and

[101] to

[146] , wherein the antigen-binding molecule is a multispecific antigen-binding molecule.

[0227]

[148] The method according to

[147] , wherein the multispecific antigen-binding molecule is a bispecific antigen-binding molecule or a trispecific antigen-binding molecule.

[0228]

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

[52] , [A1] to [A24], [B1] to [B15], and

[101] to

[148] , wherein the antigen-binding molecule is an antibody.

[0229]

[150] The method according to

[149] , wherein the antibody is an IgG antibody, preferably an IgG1, IgG2, IgG3 or IgG4 antibody.

[0230] The present disclosure also relates to the following inventions:

[0231]

[201] The method according to any one of

[101] to

[150] , wherein at least one disulfide bond in the region other than the hinge region is formed between amino acid residues at the same position present in the first antigen-binding domain and the second antigen-binding domain.

[0232]

[202] The method according to any one of

[101] to

[150] , wherein at least one disulfide bond in the region other than the hinge region is formed between amino acid residues at different positions present in the first antigen-binding domain and the second antigen-binding domain.

[0233]

[203] The method according to any one of

[101] to

[150] ,

[201] and

[202] , wherein at least one disulfide bond in the region other than the hinge region is formed between the heavy chains of the first antigen-binding domain and the second antigen-binding domain, between the light chains of the first antigen-binding domain and the second antigen-binding domain, or between any combination of the CH1 region, CL region, VL region, VH region or VHH region of the first antigen-binding domain and the CH1 region, CL region, VL region, VH region or VHH region of the second antigen-binding domain.

[0234]

[204] The method according to

[203] , wherein at least one disulfide bond is formed between the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain.

[0235]

[205] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 regions of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU number 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.

[0236]

[206] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number 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.

[0237]

[207] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number positions 134, 135, 136, 137, 191, 192, 193, 194, 195 and 196.

[0238]

[208] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number positions 135, 136 and 191.

[0239]

[209] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number positions 119, 120, 121, 122 and 123.

[0240]

[210] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number positions 131, 132, 133, 134, 135, 136, 137, 138, 139 and 140.

[0241]

[211] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU number positions 148, 149 and 150.

[0242]

[212] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167.

[0243]

[213] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178.

[0244]

[214] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197.

[0245]

[215] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.

[0246]

[216] The method according to

[204] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 218 and 219.

[0247]

[217] The method according to any one of

[204] to

[216] , wherein the positions of the amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain differ by 3 amino acids or less.

[0248]

[218] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 135, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at any one of EU numbering positions 132 to 138.

[0249]

[219] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 136, and the amino acid residue in the CH1 region of the second antigen-binding domain is any one of the amino acid residues at EU numbering positions 133 to 139.

[0250]

[220] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 191, and the amino acid residue in the CH1 region of the second antigen-binding domain is any one of the amino acid residues at EU numbering positions 188 to 194.

[0251]

[221] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 135, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 135.

[0252]

[222] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 136, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 136.

[0253]

[223] The method according to

[204] , wherein the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 191, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 191.

[0254]

[224] The method according to any one of

[204] to

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

[0255]

[225] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein the antigen-binding molecule comprises one, two or more additional disulfide bonds between the first antigen-binding domain and the second antigen-binding domain, and the additional disulfide bonds are formed via the amino acid residues at the following positions according to EU numbering in the respective CH1 regions of the first antigen-binding domain and the second antigen-binding domain:

[0256] (a) between the amino acid residues at any one of positions 131 to 138, 194 and 195 in each of the two antigen-binding domains;

[0257] (b) between the amino acid residues at position 131 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0258] (c) between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0259] (d) between the amino acid residues at position 133 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0260] (e) between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0261] (f) between the amino acid residues at position 135 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0262] (g) between the amino acid residues at position 136 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0263] (h) between the amino acid residues at position 137 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0264] (i) between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0265] (j) between the amino acid residues at position 131 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0266] (k) between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0267] (l) between the amino acid residues at position 133 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0268] (m) between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0269] (n) between the amino acid residues at position 135 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0270] (o) between the amino acid residues at position 136 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0271] (p) between the amino acid residues at position 137 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains; and

[0272] (q) between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains.

[0273]

[226] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more charged amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two or more amino acid residues with opposite charges at EU numbering positions 193 to 195 in the CH1 region.

[0274]

[227] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more positively charged amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two or more negatively charged amino acid residues at EU numbering positions 193 to 195 in the CH1 region.

[0275]

[228] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more negatively charged amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more positively charged amino acid residues at EU numbering positions 193 to 195 in the CH1 region.

[0276]

[229] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more of the following amino acid residues in the CH1 region:

[0277] (a) the amino acid residue at EU numbering position 136 is glutamic acid (E) or aspartic acid (D);

[0278] (b) the amino acid residue at EU numbering position 137 is glutamic acid (E) or aspartic acid (D); and

[0279] (c) the amino acid residue at EU numbering position 138 is glutamic acid (E) or aspartic acid (D);

[0280] and wherein the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more of the following amino acid residues in the CH1 region:

[0281] (d) the amino acid residue at EU numbering position 193 is lysine (K), arginine (R) or histidine (H);

[0282] (e) the amino acid residue at EU numbering position 194 is lysine (K), arginine (R) or histidine (H); and

[0283] (f) the amino acid residue at EU numbering position 195 is lysine (K), arginine (R) or histidine (H).

[0284]

[230] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more of the following amino acid residues in the CH1 region:

[0285] (a) the amino acid residue at EU numbering position 136 is lysine (K), arginine (R) or histidine (H);

[0286] (b) The amino acid residue at EU number position 137 is lysine (K), arginine (R), or histidine (H); and

[0287] (c) The amino acid residue at EU number position 138 is lysine (K), arginine (R), or histidine (H);

[0288] and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more of the following amino acid residues in the CH1 region:

[0289] (d) The amino acid residue at EU number position 193 is glutamic acid (E) or aspartic acid (D);

[0290] (e) The amino acid residue at EU number position 194 is glutamic acid (E) or aspartic acid (D); and

[0291] (f) The amino acid residue at EU number position 195 is glutamic acid (E) or aspartic acid (D).

[0292]

[231] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein each of the first antigen-binding domain and the second antigen-binding domain contains any one of the combinations of specific charged amino acids (according to EU number) in the CH1 region shown in Table 1, 2, or 3.

[0293]

[232] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain and the second antigen-binding domain contains one, two, or more hydrophobic amino acid residues at EU number positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more hydrophobic amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0294]

[233] The method according to

[232] , wherein the hydrophobic amino acid residue is alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp).

[0295]

[234] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein each of the first antigen-binding domain and the second antigen-binding domain contains any one of the combinations of specific hydrophobic amino acids (according to EU number) in the CH1 region shown in Table 4.

[0296]

[235] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises a "pestle" amino acid residue at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more "mortar" amino acid residues at EU numbering positions 193 to 195 in the CH1 region.

[0297]

[236] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more "mortar" amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises a "pestle" amino acid residue at EU numbering positions 193 to 195 in the CH1 region.

[0298]

[237] The method according to

[236] , wherein the "pestle" amino acid residue is selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "mortar" amino acid residue is selected from the group consisting of alanine (Ala), valine (Val), threonine (Thr) and serine (Ser).

[0299]

[238] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more aromatic amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more positively charged amino acid residues at EU numbering positions 193 to 195 in the CH1 region.

[0300]

[239] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein either the first antigen-binding domain or the second antigen-binding domain comprises one, two or more positively charged amino acid residues at EU numbering positions 136 to 138 in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more aromatic amino acid residues at EU numbering positions 193 to 195 in the CH1 region.

[0301]

[240] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein the aromatic amino acid residue is selected from the group consisting of tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe); and the positively charged amino acid residue is selected from the group consisting of lysine (Lys), arginine (Arg), and histidine (His).

[0302]

[241] The method according to

[203] , wherein at least one disulfide bond in the region other than the hinge region is formed between the CL region of the first antigen-binding domain and the CL region of the second antigen-binding domain.

[0303]

[242] The method according to

[241] , wherein the positions of the amino acid residues in the CL regions of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbering positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213.

[0304]

[243] The method according to

[241] , wherein the positions of the amino acid residues in the CL regions of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbering positions 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213.

[0305]

[244] The method according to

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

[0306]

[245] The method according to

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

[0307]

[246] The method according to

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

[0308]

[247] The method according to

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

[0309]

[248] The method according to

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

[0310]

[249] The method according to

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

[0311]

[250] The method according to

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

[0312]

[251] The method according to any one of

[241] to

[250] , wherein the positions of the amino acid residues in the CL region of the first antigen-binding domain and the second antigen-binding domain differ by 3 amino acids or less.

[0313]

[252] The method according to

[241] , wherein the amino acid residue in the CL region of the first antigen-binding domain is the amino acid residue at Kabat numbering position 126, and the amino acid residue in the CL region of the second antigen-binding domain is the amino acid residue at Kabat numbering position 126.

[0314]

[253] The method according to

[203] , wherein at least one disulfide bond in the region other than the hinge region is formed between the amino acid residue in the CH1 region of the first antigen-binding domain and the amino acid residue in the CL region of the second antigen-binding domain.

[0315]

[254] The method according to

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

[0316]

[255] The method according to

[253] , wherein the amino acid residue in the CH1 region is the amino acid residue at EU numbering position 191, and the amino acid residue in the CL region is the amino acid residue at Kabat numbering position 126.

[0317]

[256] The method according to any one of

[101] to

[150] and

[201] to

[224] , wherein each of the first antigen-binding domain and the second antigen-binding domain independently comprises lysine (K), arginine (R), or histidine (H) at Kabat numbering positions 123 and / or 124 in the CL region, and independently comprises glutamic acid (E) or aspartic acid (D) at EU numbering positions 147 and / or 213 in the CH1 region.

[0318]

[257] The method according to

[255] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises arginine (R) and lysine (K) at Kabat numbering positions 123 and 124 in the CL region, respectively, and comprises glutamic acid (E) at EU numbering positions 147 and 213 in the CH1 region.

[0319]

[258] The method according to any one of

[241] to

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

[0320]

[259] The method according to

[203] , wherein at least one disulfide bond in the region other than the hinge region is formed between the amino acid residues in the VH region of the first antigen-binding domain and the amino acid residues in the VH region of the second antigen-binding domain.

[0321]

[260] The method according to

[259] , wherein the positions of the amino acid residues in the VH regions of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: EU numbering positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b.

[0322]

[261] The method according to

[203] , wherein at least one disulfide bond in a region other than the hinge region is formed between an amino acid residue in the VL region of the first antigen-binding domain and an amino acid residue in the VL region of the second antigen-binding domain.

[0323]

[262] The method according to

[261] , wherein the positions of the amino acid residues in the VL regions (κ subclass) of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbered positions 21, 27, 58, 77, 100, 105, and 107.

[0324]

[263] The method according to

[261] , wherein the positions of the amino acid residues in the VL regions (λ subclass) of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbered positions 6, 19, 33, and 34.

[0325]

[264] The method according to

[203] , wherein at least one disulfide bond in a region other than the hinge region is formed between an amino acid residue in the VHH region of the first antigen-binding domain and an amino acid residue in the VHH region of the second antigen-binding domain.

[0326]

[265] The method according to

[264] , wherein the positions of the amino acid residues in the VHH regions of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of Kabat numbered 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.

[0327] The present disclosure also relates to the following inventions:

[0328]

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

[52] , [A1] to [A24], [B1] to [B15],

[101] to

[150] , and

[201] to

[265] , wherein the antigen-binding molecule comprises:

[0329] a first antigen-binding domain and a second antigen-binding domain capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137, and

[0330] a third antigen-binding domain capable of binding to DLL3, preferably human DLL3.

[0331]

[302] The method according to

[301] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, which may be the same as or different from each other, and comprises an antibody variable region independently selected from the group consisting of the following (a1) to (a4):

[0332] (a1) An antibody variable region comprising:

[0333] A heavy chain variable region comprising:

[0334] Heavy chain complementarity-determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO: 27,

[0335] Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 28, and

[0336] Heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 29; and

[0337] A light chain variable region comprising:

[0338] Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO: 30,

[0339] Light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 31, and

[0340] Light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 32;

[0341] (a2) An antibody variable region comprising:

[0342] A heavy chain variable region comprising:

[0343] Heavy chain complementarity-determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO: 33,

[0344] Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 34, and

[0345] Heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 35; and

[0346] A light chain variable region comprising:

[0347] Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO: 30,

[0348] Light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 31, and

[0349] The light chain CDR3, which comprises the amino acid sequence of SEQ ID NO:32;

[0350] (a3) An antibody variable region that binds to an epitope that is the same as the epitope to which the antibody variable region of (a1) or (a2) binds; and

[0351] (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

[0352]

[303] The method according to

[301] or

[302] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region that can be the same as or different from each other, and comprise an antibody variable region independently selected from the group consisting of the following (a1) to (a4):

[0353] (a1) An antibody variable region comprising:

[0354] A heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:36, and

[0355] A light chain variable region that comprises the amino acid sequence of SEQ ID NO:37; and

[0356] (a2) An antibody variable region comprising:

[0357] A heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:38, and

[0358] A light chain variable region that comprises the amino acid sequence of SEQ ID NO:37;

[0359] (a3) An antibody variable region that binds to an epitope that is the same as the epitope to which the antibody variable region of (a1) or (a2) binds; and

[0360] (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

[0361]

[304] The method according to any one of

[301] to

[303] , wherein the third antigen-binding domain comprises an antibody variable region independently selected from any one of the following (a1) to (a4):

[0362] (a1) An antibody variable region comprising:

[0363] A heavy chain variable region that comprises:

[0364] A heavy chain complementarity determining region (CDR) 1 that comprises the amino acid sequence of SEQ ID NO:46,

[0365] Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:47, and

[0366] Heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:48; and

[0367] Light chain variable region, which comprises:

[0368] Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO:49,

[0369] Light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:50, and

[0370] Light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:51;

[0371] (a2) An antibody variable region comprising:

[0372] Heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:52, and

[0373] Light chain variable region, which comprises the amino acid sequence of SEQ ID NO:53;

[0374] (a3) An antibody variable region that binds to an epitope identical to the epitope to which the antibody variable region of (a1) or (a2) binds; and

[0375] (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

[0376]

[305] The method according to any one of

[301] to

[304] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0377] Heavy chain variable region, which comprises:

[0378] Heavy chain complementarity determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO:27,

[0379] Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:28, and

[0380] Heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:29; and

[0381] Light chain variable region, which comprises:

[0382] Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO:30,

[0383] The light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:31, and

[0384] The light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:32.

[0385]

[306] The method according to any one of

[301] to

[305] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0386] The heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:36, and

[0387] The light chain variable region, which comprises the amino acid sequence of SEQ ID NO:37.

[0388]

[307] The method according to any one of

[301] to

[306] , wherein the third antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0389] The heavy chain variable region, which comprises:

[0390] The heavy chain complementarity determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO:46;

[0391] The heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:47, and

[0392] The heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:48; and

[0393] The light chain variable region, which comprises:

[0394] The light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO:49,

[0395] The light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:50, and

[0396] The light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:51.

[0397]

[308] The method according to any one of

[301] to

[307] , wherein the third antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0398] The heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:52, and

[0399] The light chain variable region, which comprises the amino acid sequence of SEQ ID NO:53.

[0400]

[309] The method according to any one of

[301] to

[308] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0401] The heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:36, and

[0402] The light chain variable region, which comprises the amino acid sequence of SEQ ID NO:37, and wherein the third antigen-binding domain comprises an antibody variable region, and the antibody variable region comprises:

[0403] The heavy chain variable region, which comprises SEQ ID NO:52, and

[0404] The light chain variable region, which comprises SEQ ID NO:53.

[0405]

[310] The method according to any one of

[301] to

[309] , wherein each of the first antigen-binding domain and the second antigen-binding domain is a Fab having a cysteine residue at EU numbering position 191 in the heavy chain and having a disulfide bond formed by two cysteine residues.

[0406]

[311] The method according to any one of

[301] to

[310] , wherein each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a Fab, and the Fab comprises:

[0407] A heavy chain comprising a VH region and a CH1 region, and

[0408] A light chain comprising a VL region and a CL region,

[0409] wherein the C-terminus of the CH1 region of the heavy chain of the third antigen-binding domain is directly or via a peptide linker fused to the N-terminus of the VH region of the Fab heavy chain of the first antigen-binding domain or the second antigen-binding domain.

[0410]

[312] The method according to

[311] , wherein the peptide linker comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0411]

[313] The method according to any one of

[301] to

[312] , wherein the third antigen-binding domain is a crossed Fab in which the VH region is linked to the CL region and the VL region is linked to the CH1 region, and wherein each of the first antigen-binding domain and the second antigen-binding domain is a conventional Fab in which the VH region is linked to the CH1 region and the VL region is linked to the CL region.

[0412]

[314] The method according to any one of

[301] to

[313] , wherein:

[0413] in the CL region of each of the first antigen-binding domain and the second antigen-binding domain, the amino acid residues at Kabat numbering positions 123 and 124 are arginine and lysine, respectively, and

[0414] in the CH1 region of each of the first antigen-binding domain and the second antigen-binding domain, the amino acid residues at EU numbering positions 147 and 213 are both glutamate.

[0415]

[315] The method according to any one of

[301] to

[314] , wherein the antigen-binding molecule further comprises an Fc region.

[0416]

[316] The method according to

[315] , wherein the Fc region comprises a first Fc region subunit and a second Fc region subunit,

[0417] wherein the first Fc region subunit is selected from the group consisting of:

[0418] an Fc region polypeptide comprising alanine at each of positions 234 and 235;

[0419] an Fc region polypeptide comprising alanine at each of positions 234, 235 and 297; and

[0420] an Fc region polypeptide comprising alanine at each of positions 234, 235 and 297, cysteine at position 354 and tryptophan at position 366, and

[0421] the second Fc region subunit is selected from the group consisting of:

[0422] an Fc region polypeptide comprising alanine at each of positions 234 and 235;

[0423] an Fc region polypeptide comprising alanine at each of positions 234, 235 and 297; and

[0424] An Fc region polypeptide comprising alanine at each of positions 234, 235 and 297, cysteine at position 349, serine at position 366, alanine at position 368 and valine at position 407,

[0425] wherein all positions are according to EU numbering.

[0426]

[317] The method according to any one of

[316] or

[317] , wherein the Fc region comprises any one of the following:

[0427] (a) A first Fc region subunit comprising the amino acid sequence of SEQ ID NO:23, and a second Fc region subunit comprising the amino acid sequence of SEQ ID NO:24;

[0428] (b) A first Fc region subunit comprising the amino acid sequence of SEQ ID NO:25, and a second Fc region subunit comprising the amino acid sequence of SEQ ID NO:26; or

[0429] (c) A first Fc region subunit comprising the amino acid sequence of SEQ ID NO:58, and a second Fc region subunit comprising the amino acid sequence of SEQ ID NO:59.

[0430]

[318] The method according to any one of

[301] to

[317] , wherein the antigen-binding molecule comprises any combination of five polypeptide chains selected from the group consisting of:

[0431] (a1) A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:39, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:41, and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO:42 (chain 4 and chain 5);

[0432] (a2) A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:43, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:44, and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO:42 (chain 4 and chain 5); and

[0433] A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 44, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 42;

[0434] wherein, preferably, the five polypeptide chains (chains 1 to 5) are linked and / or associated with each other according to the orientation shown in Figure 3 and / or associated with each other according to the orientation shown therein.

[0435]

[319] The method according to

[301] , wherein the antigen-binding molecule comprises the following five polypeptide chains: a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 54, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 55, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 57;

[0436] wherein, preferably, the five polypeptide chains (chains 1 to 5) are linked and / or associated with each other according to the orientation shown in Figure 3 and / or associated with each other according to the orientation shown therein.

[0437]

[320] The method according to

[301] , wherein the antigen-binding molecule comprises the following five polypeptide chains:

[0438] A polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 54 (chain 1),

[0439] A polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 55 (chain 2),

[0440] A polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 60 (chain 3), and

[0441] Two polypeptide chains that each comprise the amino acid sequence of SEQ ID NO: 57 (chain 4 and chain 5);

[0442] wherein, preferably, the five polypeptide chains (chains 1 to 5) are linked and / or associated with each other according to the orientation shown in Figure 3 and / or associated with each other according to the orientation shown therein.

[0443] The present disclosure also relates to the following inventions:

[0444]

[401] According to the method described in any one of [1] to

[52] , [A1] to [A24], [B1] to [B15],

[101] to

[150] ,

[201] to

[265] , and

[301] to

[320] , the method further comprises quantifying the ratio (LINC ratio) of an antigen-binding molecule having at least one disulfide bond in a region other than the hinge region in the preparation (LINC form) to the sum of the following:

[0445] (i) an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC form), and

[0446] (ii) mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule (unLINC form).

[0447]

[402] According to the method described in

[401] , the step of quantification comprises electrophoresis and chromatography.

[0448]

[403] According to the method described in

[402] , the electrophoresis is selected from the group consisting of non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and non-reducing capillary SDS gel electrophoresis (CE-SDS).

[0449]

[404] According to the method described in

[402] , the chromatography is hydrophobic interaction chromatography (HIC).

[0450]

[405] According to the method described in any one of

[401] to

[404] , the method further comprises adding a protease to the preparation before the quantification step.

[0451]

[406] According to the method described in

[405] , the protease digests the antigen-binding molecule between T and H in the amino acid sequence KSCDKT / HTCPPCP of the antigen-binding molecule.

[0452]

[407] According to the method described in

[406] , the antigen-binding molecule is a human IgG1 antibody.

[0453]

[408] According to the method described in

[406] or

[407] , the protease is IdgE.

[0454]

[409] According to the method described in

[408] , IdgE is derived from Streptococcus agalactiae.

[0455]

[410] According to the method described in any one of

[405] to

[409] , the step of quantification comprises non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC).

[0456]

[411] The method according to

[410] , wherein the quantification step comprises non-reducing capillary SDS gel electrophoresis (CE-SDS), and wherein the method further comprises performing non-reducing capillary SDS gel electrophoresis (CE-SDS) on a formulation without added protease and a sample containing only protease.

[0457]

[412] The method according to

[411] , the method further comprising preparing electrophoretograms of a formulation with added protease, a formulation without added protease, and a sample containing only protease.

[0458]

[413] The method according to

[412] , wherein the LINC ratio is determined by the ratio of (the peak from the LINC form) to (the peak from the formulation with added protease, including the peak from the unLINC form and the peak from protease) - (the peak from the formulation without added protease, the peak not including the peak from the unLINK form) – (the peak from the sample containing only protease) + (the peak from the LINC form).

[0459]

[414] The method according to

[410] , wherein the quantification step comprises hydrophobic interaction chromatography, and wherein the LINC ratio is determined by the ratio of the peak from the LINC form to the sum of the peak from the LINC form and the peaks from all non-LINC forms.

[0460]

[415] The method according to any one of

[405] to

[414] , wherein the chromatography in [1] to

[52] , [B1] to [B15],

[101] to

[150] ,

[201] to

[265] , and

[301] to

[320] is membrane chromatography.

[0461]

[416] The method according to any one of

[405] to

[415] , wherein the method further comprises culturing the formulation after adding protease.

[0462] The present disclosure also relates to the following inventions:

[0463]

[501] A formulation comprising an antigen-binding molecule produced by the method according to any one of [1] to

[52] , [A1] to [A24], [B1] to [B15],

[101] to

[150] ,

[201] to

[265] ,

[301] to

[320] , and

[401] to

[416] , the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

[0464]

[502] A pharmaceutical composition, the pharmaceutical composition comprising a preparation containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a hinge region other than the hinge region, wherein the preparation is prepared by the method according to any one of [1] to

[52] , [A1] to [A24], [B1] to [B15],

[101] to

[150] ,

[201] to

[265] ,

[301] to

[320] , and

[401] to

[416] .

[0465] [Advantages of the Invention]

[0466] The present disclosure provides an effective and easy method for producing and purifying an antigen-binding molecule having an appropriate disulfide bond in a region other than the hinge region. The method in the present disclosure enables the easy, rapid, and high-yield acquisition of the LINC-Ig form. The method in the present disclosure has the advantages of a wide range of types, concentrations, pH values, and reaction times of reducing agents.

[0467] In addition, compared with such production and purification in a tank, the production and purification of the LINC-Ig form in a column (such as those in the method of the present disclosure) have advantages such as reducing the possibility of aggregation of the antigen-binding molecule, short reaction time, easy operation, the possibility of automation by using programmed liquid chromatography (LC), the possibility of maintaining the concentration of the reducing agent in the reaction at a constant level, and easy reproducibility across scales. Brief Description of the Drawings

[0468] Figure 1 The analysis results of the Cys-reduced samples are shown. Starting from the left, a control (1 sample) that skipped the reduction / oxidation step and performed affinity chromatography, 0.1 to 100 mmol / L Cys solutions at pH 7.0 (4 samples), and 0.1 to 100 mmol / L Cys solutions at pH 8.0 (4 samples) are shown. At pH 7.0 and 8.0, especially in the concentration range of 0.1 to 10 mmol / L, an effective increase in the LINC ratio was confirmed. For the results of 100 mmol / L, no bands could be confirmed on the gel, and since the accurate LINC ratio could not be calculated, the symbol "-" was used.

[0469] Figure 2The analysis results of the TCEP-reduced sample are shown. Starting from the left, a control (1 sample) that skipped the reduction / oxidation step and underwent affinity chromatography, 0.001 to 1 mmol / L TCEP solutions at pH 7.0 (4 samples), and 0.001 to 1 mmol / L TCEP solutions at pH 8.0 (4 samples) are shown. For the results of 0.1 to 1 mmol / L, bands could not be confirmed on the gel, and since the accurate LINC ratio could not be calculated, the symbol "-" was used.

[0470] Figure 3 is a figure showing the design and naming rules for trivalent antibodies in the dual / LINC (1+2) form.

[0471] Figure 4 An electrophoretogram from CE-SDS is shown.

[0472] Figure 5 A chromatogram from HIS analysis is shown, including peak names. The standard chromatogram (upper figure) and the magnified chromatogram (lower figure) are shown. Detailed Description of the Invention

[0473] Those skilled in the art generally easily understand and commonly use conventional methods to utilize the technologies and procedures described or referenced herein, such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Antibodies, A Laboratory Manual edited by Harlow and Lane (1988), and Animal Cell Culture (edited by R.I. Freshney (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R.I. Freshney), 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A.D. Doyle, J.B. Griffiths, and D.G. Newell, 1993 - 8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.widely used methods as described in Calos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J.E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., ed., J.B. Lippincott Company, 1993).

[0474] The following definitions and detailed descriptions are provided to assist in understanding the present disclosure as shown herein.

[0475] I. Definitions

[0476] As used herein, when describing the site of an amino acid change, the meaning of the term "and / or" includes each combination of the appropriate combinations of "and" and "or". Specifically, for example, "the amino acid at position 33, 55, and / or 96 is substituted" includes the following variants of amino acid changes: (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) the amino acid at positions 33, 55, and 96.

[0477] Unless otherwise specified, amino acid residues in the light chain constant regions herein are numbered according to Kabat et al., and amino acid residues in the heavy chain constant regions are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0478] Amino acid

[0479] In this text, amino acids are described by single-letter codes or three-letter codes or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I or Val / V.

[0480] Alteration of amino acid

[0481] For amino acid changes (also described herein as "amino acid substitutions" or "amino acid mutations") in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. In addition, several known methods can also be used as amino acid change methods to substitute unnatural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003) 100(11), 6353-6357). For example, a cell-free translation system containing tRNA (Clover Direct (Protein Express)) suitable for use with an unnatural amino acid that binds to a complementary amber suppressor tRNA for one of the stop codons (codon UAG (amber codon)) can be used.

[0482] In addition, in the present text, as an expression indicating an amino acid change, an expression that shows the one-letter or three-letter code of the amino acids before and after the change, respectively, before and after the number indicating a specific position may be appropriately used. For example, when substituting an amino acid contained in the variable region of an antibody, the change N100bL or Asn100bLeu indicates the substitution of Asn with Leu at position 100b (according to Kabat numbering). That is, the number shows the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number shows the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number shows the amino acid after substitution. Similarly, when substituting an amino acid in the Fc region contained in the constant region of an antibody, the change P238D or Pro238Asp indicates the substitution of Pro with Asp at position 238 (according to EU numbering). That is, the number shows the amino acid position according to EU numbering, the one-letter or three-letter amino acid code written before the number shows the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number shows the amino acid after substitution.

[0483] Polypeptide

[0484] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by an amide bond (also called a peptide bond). The term "polypeptide" refers to any chain having two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain having two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of any one of these terms, or may be used interchangeably with any one of these terms. The term "polypeptide" also is intended to refer to products of post-expression modification of polypeptides, which post-expression modification includes, but is not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques and not necessarily translated from a designated nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. The size of the polypeptides as described herein may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are called folded; and polypeptides that do not have a defined three-dimensional structure but can adopt a large number of different conformations are called unfolded.

[0485] Percent amino acid sequence identity (%)

[0486] "Percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes herein, the sequence comparison computer program ALIGN-2 is used to generate the percent amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not changed. In the case of amino acid sequence comparison using ALIGN-2, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to a given amino acid sequence B) is calculated as follows:

[0487] 100 times the fraction X / Y

[0488] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. Unless otherwise specifically indicated, all values of percent amino acid sequence identity used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0489] Antigen-binding molecule

[0490] As used herein, the term "antigen-binding molecule" in its broadest sense refers to any molecule that specifically binds to an antigenic determinant (epitope) and contains an antigen-binding domain or has binding activity to an antigen, and can also refer to molecules such as peptides or proteins that are about five amino acids or longer in length. Peptides and proteins are not limited to those derived from organisms; for example, they can be polypeptides produced from sequences designed artificially. They can also be any one of naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Scaffold molecules that contain known stable conformational structures such as α / β barrels as scaffolds and in which part of the molecule is regarded as an antigen-binding site are also an embodiment of the antigen-binding molecules described herein. In one embodiment, the antigen-binding molecule can comprise two or more (e.g., 2, 3, 4, 5 or more) polypeptide chains. In one embodiment, the polypeptide chains constituting the antigen-binding molecule can be antibody heavy chains or antibody light chains. Specifically, in one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In one embodiment, the antigen-binding molecule is a trispecific antibody comprising chains 1 to 5, as shown in Figure 3 of the present application. In one embodiment, the antigen-binding molecule is a non-antibody protein or a fragment or a derivative thereof.

[0491] Multispecific antigen-binding molecule

[0492] A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to more than one antigen. The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. The term "trispecific" means that the antigen-binding molecule is capable of specifically binding to at least three different antigenic determinants.

[0493] In certain embodiments, the multispecific antigen-binding molecule of the present application is a trispecific antigen-binding molecule that is capable of binding to either CD3 or CD137, but not both antigens simultaneously, and is capable of specifically binding to DLL3.

[0494] Antigen-binding domain

[0495] As used herein, an "antigen-binding domain" refers to a region that specifically binds to and is complementary to all or a portion of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, the antigen-binding domain can only bind to a specific portion of the antigen. This specific portion is referred to as an "epitope". In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain comprises both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", and "Fab'". In other embodiments, the antigen-binding domain comprises a non-antibody protein that binds to a specific antigen or a fragment thereof. In a specific embodiment, the antigen-binding domain comprises one or two Fabs that include a CH1 region. In a specific embodiment, the antigen-binding domain comprises a hinge region.

[0496] As used herein, the term "antigen-binding domain" can direct the entity to which it is attached to a target site, such as to a specific type of tumor cell expressing a cancer antigen (DLL3). The antigen-binding domain can activate signal transduction through its target antigens, such as a T cell receptor complex antigen (especially CD3) and / or a co-stimulatory receptor (CD137).

[0497] As used herein, the terms "first", "second", and "third" with respect to antigen-binding domains and the like are used for convenience in distinguishing when there is more than one of each type of moiety. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation on the antigen-binding molecule.

[0498] As used herein, "specifically binds" means binding in a state where one of the molecules involved in specific binding does not exhibit any significant binding to molecules other than the single or multiple binding partner molecules. In addition, it is also used when the antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. When the epitope bound by the antigen-binding domain is contained in multiple different antigens, the antigen-binding molecule comprising the antigen-binding domain can bind to various antigens having that epitope.

[0499] In the present disclosure, the expression "binds to the same epitope" means that the epitopes bound by two antigen-binding domains 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%.

[0500] Antibody

[0501] As used herein, the term "antibody" 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 or trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0502] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons, which is composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also referred to as the variable heavy chain domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also referred to as the heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also referred to as the variable light chain domain or light chain variable domain), followed by a constant light chain (CL) domain (also referred to as the light chain constant region). The heavy chains of immunoglobulins can be assigned to one of five types, which are designated as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types based on the amino acid sequence of their constant domains: called κ and λ. An immunoglobulin is substantially composed of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

[0503] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody preparation, such variants typically being present in minor amounts). 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 modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies 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 locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0504] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region as defined herein.

[0505] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0506] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, an antibody that binds a particular antigen can be isolated using the VH or VL domain from an antibody that binds that antigen to screen a library for the complementary VL or VH domain. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0507] The term "hypervariable region" or "HVR" as used herein refers to each of the following: the regions of an antibody variable domain that are hypervariable in sequence ("complementary determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contact residues ("antigen contact points"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0508] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0509] (b) CDRs present 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));

[0510] (c) Antigen - contact points present 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

[0511] (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).

[0512] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al., supra.

[0513] HVR - H1, HVR - H2, HVR - H3, HVR - L1, HVR - L2, and HVR - L3 are also referred to as "H - CDR1", "H - CDR2", "H - CDR3", "L - CDR1", "L - CDR2", and "L - CDR3", respectively.

[0514] "Framework" or "FR" refers to the variable - domain residues other than the hypervariable - region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally occur in VH (or VL) in the following sequence: FR1 - H1 (L1) - FR2 - H2 (L2) - FR3 - H3 (L3) - FR4.

[0515] The "human consensus framework" is a framework that represents the amino acid residues that are most often present in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as that in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0516] The term "hinge region" refers to the antibody heavy chain polypeptide portion that connects the CH1 domain and the CH2 domain in the wild-type antibody heavy chain, e.g., from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system. It is known that in native IgG antibodies, the cysteine residue at position 220 according to 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 two antibody heavy chains, disulfide bonds are formed between the cysteine residues at position 226 and at position 229 according to EU numbering in the hinge region. Generally, the "hinge region" is defined as extending from 216 to 238 (EU numbering) or from 226 to 251 (Kabat numbering) in human IgG1. This hinge can be further divided into three different regions: the upper hinge, the central hinge, and the lower hinge. In human IgG1 antibodies, these regions are typically defined as follows:

[0517] Upper hinge: 216 to 225 (EU numbering) or 226 to 238 (Kabat numbering), central hinge: 226 to 230 (EU numbering) or 239 to 243 (Kabat numbering), lower hinge: 231 to 238 (EU numbering) or 244 to 251 (Kabat numbering).

[0518] By placing the first and last cysteine residues that form the inter-heavy chain SS bond in the same position, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence (e.g., Brekke et al., 1995, Immunol (see Table 1 in Today 16:85-90). The hinge region herein includes the wild-type hinge region and variants in which the amino acid residues in the wild-type hinge region are altered by substitution, addition, or deletion.

[0519] The terms (amino acid residues) "able to connect" and (disulfide bonds) "able to form" include cases where disulfide bonds have already formed, as well as cases where they have not formed but can subsequently form under appropriate conditions.

[0520] The term "disulfide bond formed between amino acids not in the hinge region" (or "disulfide bond formed between amino acid residues in a region outside the hinge region") means a disulfide bond formed, connected, or linked by amino acids in any antibody region located outside the "hinge region" as defined above. For example, such disulfide bonds are formed, connected, or linked by amino acids at any position outside the hinge region in an antibody (e.g., from approximately position 216 to approximately position 230 according to the EU numbering system, or from approximately position 226 to approximately position 243 according to the Kabat numbering system). In some embodiments, such disulfide bonds are formed, connected, or linked by amino acids located in the CH1 region, CL region, VL region, VH region, and / or VHH region. In some embodiments, such disulfide bonds are formed, connected, or linked by amino acids at positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214 in the CH1 region according to EU numbering. In some embodiments, such disulfide bonds are formed, connected, or linked by amino acids at positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214 in the CH1 region according to EU numbering. In some embodiments, such disulfide bonds are formed, connected, or linked by amino acids at positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 in the CH1 region according to EU numbering. In a preferred embodiment, such disulfide bonds are formed, connected, or linked by amino acids at position 191 in the CH1 region according to EU numbering.

[0521] The term "misdisulfide-bonded form" means an antigen-binding molecule in which a disulfide bond is formed between amino acid residues different from the desired amino acid residues. When an antigen-binding molecule contains a disulfide bond formed between the desired amino acid residues and a disulfide bond formed between amino acid residues different from the desired amino acid residues, such an antigen-binding molecule is included in the misdisulfide-bonded forms herein.

[0522] The term "non - disulfide - bonded form" refers to an antigen - binding molecule in which cysteine residues remain free or in which cysteine residues form disulfide bonds with molecules different from the molecules forming the antigen - binding molecule (such as impurity molecules). The "non - disulfide - bonded forms" described herein only need to have such characteristics, and for example, even if they contain disulfide bonds (required disulfide bonds) formed between amino acid residues in regions other than the hinge region, or disulfide bonds different from the required disulfide bonds (mis - disulfide - bonded portions), as long as they have the above - mentioned characteristics, they are included in the "non - disulfide - bonded forms" herein.

[0523] The constant region is preferably an antibody constant region, more preferably a constant region of IgG1, IgG2, IgG3, and IgG4 type antibodies, and even more preferably a constant region of human IgG1, IgG2, IgG3, and IgG4 type antibodies. In addition, the constant region is preferably a heavy - chain constant region, more preferably a heavy - chain constant region of IgG1, IgG2, IgG3, and IgG4 type, and even more preferably a heavy - chain constant region of human IgG1, IgG2, IgG3, and IgG4 type. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, multiple allotypic sequences with genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91 - 3242, and any of them can be used in the present invention. The amino - acid - modified constant region can contain other amino - acid mutations or modifications as long as they include the amino - acid mutations of the present invention.

[0524] In the present text, the term "Fc region" or "Fc domain" refers to a region containing a fragment composed of a hinge or a part thereof and CH2 and CH3 domains in an antibody molecule. The Fc region of the IgG class means but is not limited to the region from, for example, cysteine 226 (EU number (also referred to as EU index) in this text) to the C - terminus or from proline 230 (EU number) to the C - terminus. The Fc region can preferably be obtained by partially digesting, for example, an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a proteolytic enzyme such as pepsin, and then re - eluting the fraction adsorbed on a protein A column or a protein G column. Such proteolytic enzymes are not particularly limited as long as the enzyme can digest the whole antibody under appropriately set reaction conditions (such as pH) to restrictively form Fab or F(ab')2. Examples thereof can include pepsin and papain.

[0525] An Fc region derived from, for example, a naturally occurring IgG can be used as the "Fc region". As used herein, a naturally occurring IgG refers to a polypeptide that contains the same amino acid sequence as a naturally occurring IgG and belongs to the antibody class that is encoded substantially by immunoglobulin gamma genes. A naturally occurring human IgG refers to, for example, a naturally occurring human IgG1, a naturally occurring human IgG2, a naturally occurring human IgG3, or a naturally occurring human IgG4. Naturally occurring IgG also includes variants and the like that are spontaneously derived therefrom. In the target immunological protein sequences of NIH Publication No. 91-3242, multiple allotypic sequences based on gene polymorphisms are described as the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, any of which can be used in the present invention. In particular, the sequence of human IgG1 can have DEL or EEM as the amino acid sequence at EU numbering positions 356 to 358.

[0526] The Fc domain of an antigen-binding molecule is composed of a pair of polypeptide chains that contain the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit of the dimer contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the antigen-binding molecule described herein contains no more than one Fc domain.

[0527] As used herein, the Fc domain of an antigen-binding molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further particular embodiment, the Fc domain is a human IgG1 Fc region.

[0528] Chimeric antibody

[0529] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy chain and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy chain and / or light chain variable region is derived from a different source or species.

[0530] Humanized antibody

[0531] A "humanized" antibody is a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will generally comprise all of at least one, and usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in a "humanized form", e.g., a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0532] Antibody fragment

[0533] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody which binds 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-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg 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 a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that comprise all or a portion of the heavy-chain variable domain or all or a portion of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0534] Fv (variable fragment)

[0535] In this text, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain composed of a pair of the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies could be prepared from the periplasmic fraction of Escherichia coli by inserting the antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in Escherichia coli (Science (1988) 240(4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH associates with VL in an antigen-binding manner.

[0536] scFv, single-chain antibody, and sc(Fv)2

[0537] In this text, the terms "scFv", "single-chain antibody", and "sc(Fv)2" all refer to antibody fragments containing a single polypeptide chain with variable regions derived from the heavy and light chains but not the constant regions. Generally, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of the required structure that is thought to permit antigen binding. Pluckthun discussed single-chain antibodies in detail in "The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, 269-315 (1994)". See also International Patent Publication WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and / or humanized.

[0538] An scFv is an antigen-binding domain in which the VH and VL that form the Fv are joined together by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85(16), 5879-5883). The VH and VL can be held in very close proximity by the peptide linker.

[0539] sc(Fv)2 is a single-chain antibody in which four variable regions of two VLs and two VHs are joined by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and two VLs can be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes, for example, bispecific sc(Fv)2, which recognizes two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by joining scFv with a linker such as a peptide linker.

[0540] In the present text, the form of the antigen-binding domain forming sc(Fv)2 includes an antibody in which two VH units and two VL units start from the N-terminus of a single-chain polypeptide and are arranged in the order of VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). 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. Examples of the order of the form are listed below.

[0541] [VL]-linker-[VH]-linker-[VH]-linker-[VL]

[0542] [VH]-linker-[VL]-linker-[VL]-linker-[VH]

[0543] [VH]-linker-[VH]-linker-[VL]-linker-[VL]

[0544] [VL]-linker-[VL]-linker-[VH]-linker-[VH]

[0545] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0546] The molecular form of sc(Fv)2 is also described in detail in WO 2006 / 132352. According to these descriptions, those skilled in the art can appropriately prepare the required sc(Fv)2 to produce the polypeptide complex disclosed herein.

[0547] In addition, the antigen-binding molecule or antibody in the present disclosure can be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, a sugar chain addition sequence is preferably inserted into the antigen-binding molecule or antibody so that the sugar chain produces a desired effect.

[0548] The linker for connecting the variable regions of an antibody includes any peptide linker, synthetic linker, and linker such as those disclosed in Protein Engineering, 9(3), 299-305, 1996, which can be introduced by genetic engineering. However, peptide linkers are preferred in the present disclosure. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. The length is preferably five amino acids or more (no particular limitation, and the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, their lengths can all be the same or different.

[0549] For example, such peptide linkers include:

[0550] Ser,

[0551] Gly-Ser,

[0552] Gly-Gly-Ser,

[0553] Ser-Gly-Gly,

[0554] Gly-Gly-Gly-Ser (SEQ ID NO:5),

[0555] Ser-Gly-Gly-Gly (SEQ ID NO:6),

[0556] Gly-Gly-Gly-Gly-Ser (SEQ ID NO:7),

[0557] Ser-Gly-Gly-Gly-Gly (SEQ ID NO:8),

[0558] Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:9),

[0559] Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:10),

[0560] Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:11),

[0561] Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:12),

[0562] (Gly-Gly-Gly-Gly-Ser (SEQ ID NO:7))n and

[0563] (Ser-Gly-Gly-Gly-Gly(SEQ ID NO:8))n,

[0564] where n is an integer of 1 or greater. Those skilled in the art can accordingly select the length or sequence of the peptide linker according to the purpose.

[0565] Synthetic linkers (chemical crosslinking agents) are commonly used to crosslink peptides, and examples include:

[0566] N-hydroxysuccinimide (NHS),

[0567] Disuccinimidyl suberate (DSS),

[0568] Bis(sulfosuccinimidyl)suberate (BS3),

[0569] Dithiobis(succinimidyl propionate) (DSP),

[0570] Dithiobis(sulfosuccinimidyl propionate) (DTSSP),

[0571] Ethylene glycol bis(succinimidyl succinate) (EGS),

[0572] Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),

[0573] Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

[0574] Bis[2-(succinimidyloxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available.

[0575] Generally, three linkers are required to connect four antibody variable regions together. The linkers used can be of the same type or different types.

[0576] Fab, F(ab')2, and Fab'

[0577] "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain"). The heavy chain of a wild-type Fab molecule cannot form a disulfide bond with another heavy chain molecule. In this article, in addition to wild-type Fab molecules, it also includes Fab variants in which the amino acid residues in the wild-type Fab molecule are altered by substitution, addition, or deletion. In a specific embodiment, the mutant amino acid residues contained in the Fab variant (e.g., cysteine residues or lysine residues after substitution, addition, or insertion) can form a disulfide bond with another heavy chain molecule or a part thereof (e.g., a Fab molecule).

[0578] scFab is an antigen-binding domain in which a single light chain forming a Fab and the CH1 region and variable region from a single heavy chain are linked together by a peptide linker. The light chain, the CH1 region from the heavy chain, and the variable region can be kept in close proximity by the peptide linker.

[0579] "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 digesting an immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions present in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions in each of the two H chains to generate two homologous antibody fragments, in which the L chain containing VL (L-chain variable region) and CL (L-chain constant region) is linked via a disulfide bond in its C-terminal region to an H-chain fragment containing VH (H-chain variable region) and CHγ1 (γ1 region in the H-chain constant region). Each of these two homologous antibody fragments is called Fab'.

[0580] "F(ab')2" consists of two light chains and two heavy chains, and the heavy chain contains the constant region of the CH1 domain and a part of the CH2 domain, thereby forming a disulfide bond between the two heavy chains. The F(ab')2 disclosed herein can preferably be produced as follows. A complete monoclonal antibody or a monoclonal antibody containing the desired antigen-binding domain is partially digested with a protease such as pepsin; and the Fc fragment is removed by adsorption to a protein A column. The protease is not particularly limited as long as it can selectively cleave the entire antibody under appropriate set enzyme reaction conditions such as pH to produce F(ab')2. Such proteases include, for example, pepsin and ficin.

[0581] Fusion

[0582] "Fusion" means that components (e.g., Fab molecules and Fc domain subunits) are directly or via one or more peptide linkers linked by peptide bonds.

[0583] "Crossed" Fab

[0584] A "crossed" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged, i.e., a crossed Fab molecule contains a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossed Fab molecule in which the variable regions of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain constant region is referred to herein as the "heavy chain" of the crossed Fab molecule. Conversely, in a crossed Fab molecule in which the constant regions of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain variable region is referred to herein as the "heavy chain" of the crossed Fab molecule.

[0585] "Conventional" Fab

[0586] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native form, i.e., it contains a heavy chain (VH-CH1) composed of a heavy chain variable region and a constant region, and a light chain (VL-CL) composed of a light chain variable region and a constant region.

[0587] Single-domain antibody

[0588] In this article, the term "single-domain antibody" refers to an antibody whose structure is not particularly limited as long as the domain itself can exhibit antigen-binding activity. A common antibody such as an IgG antibody exhibits antigen-binding activity in a state where the variable region is formed by pairing of VH and VL. In contrast, it is known that a single-domain antibody can exhibit antigen-binding activity only through its own domain structure without pairing with another domain. Single-domain antibodies generally have a relatively low molecular weight and exist in monomeric form.

[0589] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that are naturally lacking in light chains, such as VHH of camelids and V NAR , and antibody fragments that contain all or part of an antibody VH domain or all or part of an antibody VL domain. Examples of single-domain antibodies that are antibody fragments containing all or part of an antibody VH / VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from human antibody VH or human antibody VL, as described, for example, in U.S. Patent No. 6,248,516 B1. A single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0590] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which genes capable of producing single-domain antibodies have been introduced. Camelids include camels, llamas, alpacas, dromedaries, guanacos, etc. Examples of transgenic animals into which genes capable of producing single-domain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 or U.S. Patent Publication No. US2011 / 0123527A1. Humanized single-chain antibodies can also be obtained by replacing the framework sequence of a single-domain antibody obtained from an animal with a human germline sequence or a sequence similar thereto. Humanized single-domain antibodies (e.g., humanized VHH) are an embodiment of single-domain antibodies.

[0591] Alternatively, single-domain antibodies can be obtained from a polypeptide library containing single-domain antibodies by ELISA, panning, etc. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, primary antibody libraries 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)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2(528 - 536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1(35 - 43), Journal of Biological Chemistry 2016 291:24(12641 - 12657), and AIDS 2016 30:11(1691 - 1701)).

[0592] As used herein, an "agonist" antigen-binding molecule or an "agonist" antibody is an antigen-binding molecule or antibody that significantly enhances the biological activity of the antigen to which it binds.

[0593] As used herein, a "blocking" antigen-binding molecule or a "blocking" antibody, or an "antagonistic" antigen-binding molecule or an "antagonistic" antibody is an antigen-binding molecule or antibody that significantly inhibits (partially or completely) the biological activity of the antigen to which it binds.

[0594] Antigen

[0595] As used herein, the term "antigen" refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) to which an antigen-binding portion binds, thereby forming an antigen-binding portion-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in free substances in serum and / or in the extracellular matrix (ECM). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., CD3, CD137, DLL3) can be any naturally occurring form of the protein from any vertebrate source, which vertebrate source includes mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is human CD3, human CD137, or human DLL3. When referring to a specific protein herein, the term encompasses the "full-length", unprocessed protein, as well as any form of the protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0596] Fc region with reduced Fcγ receptor-binding activity

[0597] As used herein, "reduced Fcγ receptor binding activity" means, for example, based on the above-described assay methods, that the competitive activity of a test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the competitive activity of a control antigen-binding molecule or antibody.

[0598] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3 or IgG4 antibodies can be suitably used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in SEQ ID NO: 1 (A is added to the N-terminus of RefSeq accession number AAC82527.1), 2 (A is added to the N-terminus of RefSeq accession number AAB59393.1), 3 (A is added to the N-terminus of RefSeq accession number CAA27268.1) and 4 (A is added to the N-terminus of RefSeq accession number AAB59394.1). In addition, when using an antigen-binding molecule or antibody containing a mutant of the Fc domain of an antibody of a specific isotype as a test substance, an antigen-binding molecule or antibody containing the Fc domain of the same isotype is used as a control to evaluate the effect of the mutation of the mutant on the Fcγ receptor binding activity. As described above, an antigen-binding molecule or antibody containing a mutant of the Fc domain judged to have reduced Fcγ receptor binding activity is suitably prepared.

[0599] Such known mutants include, for example, mutants having a deletion of amino acids 231A to 238S (EU numbering) (WO2009 / 011941), and mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V and L235A (Blood (2007) 109, 1185-1192).

[0600] Specifically, preferred antigen-binding molecules or antibodies include antigen-binding molecules or antibodies containing an Fc domain, in the amino acids of the Fc domain of an antibody forming a specific isotype, the Fc domain having a mutation (such as substitution) of at least one amino acid selected from the following amino acid positions: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, 332, 439, 445, 449 or 451 (EU numbering). The isotype of the antibody from which the Fc domain is derived is not particularly limited, and a suitable Fc domain derived from monoclonal IgG1, IgG2, IgG3 or IgG4 antibodies can be used. An Fc domain derived from an IgG1 antibody is preferably used.

[0601] Preferred antigen-binding molecules or antibodies include, for example, antigen-binding molecules or antibodies comprising an Fc domain having any of the substitutions shown below, the positions of which are designated according to EU numbering in the amino acids forming the Fc domain of an IgG1 antibody (each number represents the position of an amino acid residue in the EU numbering; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, while the single-letter amino acid symbol after the number represents the amino acid residue after substitution):

[0602] (a) L234F, L235E, P331S;

[0603] (b) C226S, C229S, P238S;

[0604] (c) C226S, C229S; or

[0605] (d) C226S, C229S, E233P, L234V, L235A;

[0606] (e) L439M, A445N, R449Q, E451S

[0607] (f) M428L, N434A, Q438R, S440E,

[0608] and antigen-binding molecules or antibodies comprising an Fc domain having a deletion of the amino acid sequence at positions 231 to 238.

[0609] In addition, preferred antigen-binding molecules or antibodies also include antigen-binding molecules or antibodies comprising an Fc domain having any of the substitutions shown below, the positions of which are designated according to EU numbering in the amino acids forming the Fc domain of an IgG2 antibody:

[0610] (e) H268Q, V309L, A330S and P331S;

[0611] (f) V234A;

[0612] (g) G237A;

[0613] (h) V234A and G237A;

[0614] (i) A235E and G237A; or

[0615] (j) V234A, A235E and G237A. Each number represents the position of an amino acid residue in the EU numbering; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, while the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0616] In addition, preferred antigen-binding molecules or antibodies also include antigen-binding molecules or antibodies comprising an Fc domain having any one of the substitutions shown below, the positions of which are designated according to EU numbering in the amino acids forming the Fc domain of an IgG3 antibody:

[0617] (k) F241A;

[0618] (l) D265A; or

[0619] (m) V264A. Each number represents the position of the amino acid residue in the EU numbering; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, while the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0620] In addition, preferred antigen-binding molecules or antibodies also include antigen-binding molecules or antibodies comprising an Fc domain having any one of the substitutions shown below, the positions of which are designated according to EU numbering in the amino acids forming the Fc domain of an IgG4 antibody:

[0621] (n) L235A, G237A and E318A;

[0622] (o) L235E; or

[0623] (p) F234A and L235A. Each number represents the position of the amino acid residue in the EU numbering; and the single-letter amino acid symbol before the number represents the amino acid residue before substitution, while the single-letter amino acid symbol after the number represents the amino acid residue after substitution.

[0624] Other preferred antigen-binding molecules or antibodies include, for example, antigen-binding molecules or antibodies comprising an Fc domain in which any amino acid at positions 233, 234, 235, 236, 237, 327, 330 or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is replaced by the amino acid at the corresponding position in the EU numbering in IgG2 or IgG4.

[0625] Preferred antigen-binding molecules or antibodies also include, for example, antigen-binding molecules or antibodies comprising an Fc domain in which any one or more of the amino acids at positions 234, 235 and 297 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody are replaced by other amino acids. The type of the amino acid after substitution is not particularly limited; however, antigen-binding molecules or antibodies comprising an Fc domain in which any one or more of the amino acids at positions 234, 235 and 297 are replaced by alanine are particularly preferred.

[0626] Preferred antigen-binding molecules or antibodies also include, for example, antigen-binding molecules or antibodies comprising an Fc domain, wherein the amino acid at position 265 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is replaced by another amino acid. The type of the replaced amino acid is not particularly limited; however, an antigen-binding molecule or antibody comprising an Fc domain in which the amino acid at position 265 is replaced by alanine is particularly preferred.

[0627] Preferential enrichment (or increase)

[0628] The term "preferential enrichment (or increase)" means an increase in the relative abundance of the desired form, or an increase in the relative proportion of the desired form, or an increase in the population of the desired form (structural isoform). In some embodiments, the methods described herein increase the relative abundance of antibody structural isoforms, such as antibodies having at least one disulfide bond formed between amino acid residues outside the hinge region. In one embodiment, the at least one disulfide bond is formed between amino acid residues at position 191 (EU numbering) in the respective CH1 regions of the first and second antigen-binding domains. In certain embodiments, the method produces a homogeneous antibody preparation having at least 50%, 60%, 70%, 80%, 90%, preferably at least 95% molar ratio of the antibody having at least one disulfide bond formed outside the hinge region.

[0629] Homogeneous

[0630] A "homogeneous" population of antibodies means an antibody population that mainly comprises a single form of antibody, e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99% or 100% of the antibodies in a solution or composition are in the correctly folded form. Similarly, a "homogeneous" population of antibodies having at least one disulfide bond formed outside the hinge region means a population of the antibodies that mainly comprises a single, correctly folded form (e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99% or 100% molar ratio of the antibodies having at least one disulfide bond formed outside the hinge region). In a preferred embodiment, the "homogeneous" population of the antibodies comprises at least one disulfide bond formed between amino acid residues at position 191 (EU numbering) in the respective CH1 regions of the first and second antigen-binding domains (i.e., the "paired cysteines" at position 191 in the CH1 regions according to EU numbering).

[0631] Any one of a variety of analytical and / or qualitative techniques can be used to determine whether an antibody population is homogeneous and the relative abundance or proportion of protein / antibody conformations in a mixture. If two conformations resolve differently during a separation technique such as chromatography, electrophoresis, filtration, or other purification techniques, such purification techniques can be used to determine the relative proportion of conformations in the mixture. For example, at least two different conformations of recombinant IgG can be resolved by hydrophobic interaction chromatography. In addition, since far-UV circular dichroism has been used to estimate the secondary structure composition of proteins (Perczel et al., 1991, Protein Engrg. 4:669-679), such a technique can determine whether alternative conformations of the protein exist. Another technique for determining conformation is fluorescence spectroscopy, which can be used to determine complementary differences in the tertiary structure assignable to tryptophan and tyrosine fluorescence. Other techniques that can be used to determine differences in conformation and thus the relative proportion of conformations are: online SEC for measuring the aggregation state, differential scanning calorimetry for measuring the melting transition (Tm) and component enthalpy, and chaotropic agent unfolding. Another technique that can be used to determine differences in conformation and thus the relative proportion of conformations is LC / MS detection to determine protein heterogeneity.

[0632] Alternatively, if there are differences in activity between the conformations of the antibody / protein, the relative proportion of conformations in the mixture can be determined by an activity assay (e.g., binding to a ligand, enzyme activity, biological activity, etc.). The biological activity of the protein can also be used. Alternatively, a binding assay can be used, where the activity is expressed as activity units / mg protein.

[0633] To determine the heterogeneity of the antibody / protein, IEC chromatography is used. In this case, the antibody is purified or considered to be "homogeneous", which means that no polypeptide peaks or fractions corresponding to other polypeptides are detected upon analysis by IEC chromatography. In certain embodiments, the antibody is purified or considered to be "homogeneous" such that no polypeptide bands corresponding to other polypeptides are detected upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the art will recognize that multiple bands corresponding to polypeptides can be visualized by SDS-PAGE due to differential glycosylation, differential post-translational processing, etc. Most preferably, the polypeptide is purified to be substantially homogeneous, as indicated by a single polypeptide band after SDS-PAGE analysis. The polypeptide bands can be visualized by silver staining, Coomassie blue staining, and / or (if the polypeptide is radioactively labeled) by autoradiography.

[0634] Examples of the conditions for SDS-PAGE analysis herein are as follows. Use 4% to 20% Mini-PROTEAN (registered trademark) TGX Stain-Free TMThe precast gel (Bio-Rad) was subjected to non-reducing SDS-PAGE with 1x Tris / Glycine / SDS running buffer (Bio-Rad). The monoclonal antibody sample was heated at 70 °C for 10 minutes. 0.2 μg was loaded and electrophoresed at 200 V for 90 minutes. The proteins were visualized using a Chemidoc imaging system (Bio-Rad). The percentage of individual bands was analyzed by Image Lab software version 6.0 (Bio-Rad), where the intensity % of an individual band (e.g., the faster migrating (lower band) and the slower migrating (upper band)) was calculated by dividing the band intensity by the sum of the intensities of the two bands. Then, the gel could be stained with CBB, and the gel image could be captured, and the bands could be quantified using an imaging device. In the gel image, several bands, e.g., two bands, namely the "upper band" and the "lower band", could be observed for the antibody variant sample. In this case, the molecular weight of the upper band could correspond to the molecular weight of the parental antibody (before modification). Structural changes (such as disulfide cross-linking via the Fab) could be caused by cysteine substitutions, which could lead to changes in electrophoretic mobility. In this case, the lower band could be considered to correspond to the antibody with one or more engineered disulfide bonds formed between the CH1 regions. Compared to the control sample, the antibody variant sample with additional cysteine substitutions could show a higher lower band to upper band ratio. The additional cysteine substitutions could enhance / facilitate disulfide cross-linking of the Fab; and could increase the percentage or structural homogeneity of the antibody preparation with engineered disulfide bonds formed at the mutation site; and could decrease the percentage of the antibody preparation that does not form engineered disulfide bonds at the mutation site. As used herein, the term "lower band to upper band ratio" refers to the ratio between the quantity / intensity of the lower band and the upper band that can be quantified during the above SDS-PAGE experiment.

[0635] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation that is in a form in which the bioactivity of the active ingredient contained therein is effective and that contains no additional components that are toxic to the subject to whom the formulation is to be administered.

[0636] A "pharmaceutical carrier" refers to a non-toxic component of a pharmaceutical formulation other than the active ingredient. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0637] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In certain embodiments, the individual or subject is a human.

[0638] II. Methods for producing a preparation comprising an antigen-binding molecule

[0639] The present disclosure relates to a method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. The method in the present disclosure includes a step of subjecting a mixture to chromatography in the presence of a reducing agent, wherein the mixture comprises an antigen-binding molecule and a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule, and the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region (hereinafter, the mixture may be referred to as "a mixture comprising an antigen-binding molecule", "a mixture of antigen-binding molecules", etc.). In the method of the present disclosure, the mixture comprising an antigen-binding molecule may be subjected to one or more types of chromatography. Alternatively, each of the mixtures comprising an antigen-binding molecule may be subjected to one or more (e.g., two, three, or more) types (e.g., 2, 3, or more types) of chromatography. When multiple chromatography treatments are performed, these treatments may be carried out sequentially or non-sequentially.

[0640] In the method of the present disclosure, chromatography well-known to those skilled in the art can be used. Examples include, but are not limited to, column chromatography, membrane chromatography, and thin-layer chromatography.

[0641] In the production method of the present disclosure, the step of subjecting the mixture comprising an antigen-binding molecule to chromatography is carried out in the presence of a reducing agent. In the production method of the present disclosure, there is no limitation on the embodiment of the step of subjecting the mixture comprising an antigen-binding molecule to chromatography, as long as a reducing agent is present. For example, the antigen-binding molecule can be loaded onto a chromatography matrix in advance and then contacted with a reducing agent, or a substance obtained by mixing the mixture comprising an antigen-binding molecule with a reducing agent can be passed through the chromatography.

[0642] In one aspect, when column chromatography is used, the method of the present disclosure may include a step of contacting a chromatography matrix packed in a column for column chromatography with a mixture comprising an antigen-binding molecule and a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule, and the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region. In one aspect, when membrane chromatography is used, the method of the present disclosure may include a step of contacting a chromatography matrix coated on a membrane for membrane chromatography with a mixture comprising an antigen-binding molecule and a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule, and the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region. The mixture comprising an antigen-binding molecule may be contacted with a reducing agent in advance.

[0643] In the present disclosure, the step of chromatographing a mixture comprising an antigen-binding molecule and mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule, wherein the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region, may comprise the steps of:

[0644] (a) contacting the mixture comprising the antigen-binding molecule and mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule with a solution comprising a reducing agent, wherein the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region other than the hinge region; and

[0645] (b) removing the reducing agent.

[0646] In one aspect, the methods of the present disclosure produce or purify a population of homogeneous antigen-binding molecules or a preparation or concentrate of homogeneous antigen-binding molecules by the steps described herein.

[0647] In one aspect, relative to antigen-binding molecules having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region loaded onto a matrix (such antigen-binding molecules include antigen-binding molecules that appropriately form at least one disulfide bond between amino acid residues in a region other than the hinge region, and antigen-binding molecules that do not appropriately form at least one disulfide bond between amino acid residues in a region other than the hinge region (i.e., mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule)), the methods of the present disclosure can increase the proportion of antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

[0648] In one aspect, relative to antigen-binding molecules having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region loaded onto a matrix (such antigen-binding molecules include antigen-binding molecules that appropriately form at least one disulfide bond between amino acid residues in a region other than the hinge region, and antigen-binding molecules that do not appropriately form at least one disulfide bond between amino acid residues in a region other than the hinge region (or in other words, mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule)), the methods of the present disclosure produce antigen-binding molecules having at least one disulfide bond between amino acid residues in a region other than the hinge region at a molar ratio of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0649] Accordingly, in one embodiment, the present disclosure relates to methods for producing a formulation comprising an antigen-binding molecule having an increased concentration, the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. In one embodiment, the formulation produced by the methods of the present disclosure can be a concentrate of an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. Additionally, in one embodiment, the present disclosure relates to methods for increasing the concentration of an antigen-binding molecule in a mixture of antigen-binding molecules, the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. In further embodiments, the present disclosure relates to methods for purifying a formulation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region from a mixture comprising the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mis-disulfide bonded and / or non-disulfide bonded forms of the antigen-binding molecule. In the present disclosure, antigen-binding molecules that do not have a disulfide bond formed between amino acid residues in a region other than the hinge region include antigen-binding molecules in which cysteine residues in a region other than the hinge region are capped or that exist only as cysteine.

[0650] In the methods of the present disclosure, a mixture of antigen-binding molecules is contacted with a solution comprising a reducing agent. In a specific embodiment, a mixture of antigen-binding molecules loaded onto a matrix is contacted with a solution comprising a reducing agent in chromatography. In one aspect, examples of the chromatography matrix of the present disclosure include, but are not limited to, an affinity chromatography matrix, an ion exchange chromatography matrix, a hydrophobic interaction chromatography matrix, a matrix for multimodal chromatography comprising both ion exchange chromatography and hydrophobic interaction chromatography, and a hydroxyapatite matrix. In the present disclosure, "matrix", "resin", "ligand", and "substrate" can be used interchangeably.

[0651] In the present disclosure, the affinity chromatography matrix is not limited as long as it exhibits affinity chromatography. In one aspect, examples of the affinity chromatography matrix include, but are not limited to, a Protein A matrix, a Protein G matrix, a Protein L matrix, a sequence-selective peptide matrix, a matrix that selectively binds to an antigen-binding molecule, etc.

[0652] In the methods of the present disclosure, commercially available products can be used as affinity chromatography matrices. In specific aspects, but not limited to the following items, MabSelect SuRe (registered trademark) (Cytiva Corporation), MabSelect Xtra (registered trademark) (Cytiva Corporation), MabSelect SuRe (registered trademark) LX (Cytiva Corporation), MabSelect SuRe (registered trademark) pcc (Cytiva Corporation), MabSelect PrismA (registered trademark) (Cytiva Corporation), MabSpeed (registered trademark) rP202 (Cytiva Corporation), TOYOPEARL (registered trademark) r Protein A HC-650F (TOSOH Corporation), Praesto (registered trademark) Jetted A50 (Purolite Corporation), Amsphere (registered trademark) A3 (JSR Corporation), Amsphere (registered trademark) Protein A JWT203 (JSR Corporation), Eshmuno A (registered trademark) (Merck Millipore Corporation), Capto L (registered trademark) (Cytiva Corporation), and MabSelect (registered trademark) VL (Cytiva Corporation) can be used as examples of affinity chromatography matrices.

[0653] In one aspect, the ion exchange chromatography matrices in the present disclosure include an anion exchange resin (anion exchange ligand) or a cation exchange resin (cation exchange ligand). The anion exchange resin and cation exchange resin in the present disclosure are not limited as long as they exhibit anion exchange and cation exchange effects, respectively.

[0654] In the method of the present disclosure, commercially available products can be used as anion exchange chromatography matrices. In specific aspects, without being limited to the following items, YMC-BioPro (YMC Corporation), Q Sepharose (registered trademark) High Performance (Cytiva Corporation), Q Sepharose (registered trademark) Fast Flow (Cytiva Corporation), Q Sepharose (registered trademark) XL (Cytiva Corporation), Q Sepharose (registered trademark) Big Beads (Cytiva Corporation), Capto (registered trademark) Q ImpRes (Cytiva Corporation), Capto (registered trademark) Q (Cytiva Corporation), Capto (registered trademark) Q XP (Cytiva Corporation), Capto (registered trademark) DEAE (Cytiva Corporation), SOURCE (registered trademark) 30Q (Cytiva Corporation), SOURCE (registered trademark) 15Q (Cytiva Corporation), DEAE Sepharose (registered trademark) Fast Flow (Cytiva Corporation), ANX Sepharose (registered trademark) 4 Fast Flow (Cytiva Corporation), POROS (registered trademark) 50PI (Thermo Fisher Corporation), POROS (registered trademark) 50HQ (Thermo Fisher Corporation), POROS (registered trademark) HQ (Thermo Fisher Corporation), POROS (registered trademark) D (Thermo Fisher Corporation), POROS (registered trademark) PI (Thermo Fisher Corporation), Eshumuno (registered trademark) Q (Merck Millipore Corporation), Fractogel (registered trademark) TMAE (Merck Millipore Corporation), Fractogel (registered trademark) DEAE (Merck Millipore Corporation), Macro-Prep (registered trademark) Q (Bio-Rad Laboratories Corporation), Macro-Prep (registered trademark) DEAE (Bio-Rad LaboratoriesExamples of anion exchange chromatography matrices include Giga Cap (registered trademark) Q-650M (TOSOH Corporation), Giga Cap (registered trademark) DEAE-650M (TOSOH Corporation), and QHyperCel (registered trademark) (PALL Corporation).

[0655] Commercially available products can also be used for cation exchange chromatography matrices. In specific aspects, and not limited to the following items, examples of cation exchange chromatography matrices include Capto (registered trademark) S (Cytiva Corporation), Capto (registered trademark) SPImpRes (Cytiva Corporation), Capto (registered trademark) S ImpaAct (Cytiva Corporation), SP Sepharose (registered trademark) Fast Flow (Cytiva Corporation), CM Sepharose (registered trademark) Fast Flow (Cytiva Corporation), SP Sepharose (registered trademark) High Performance (Cytiva Corporation), CM Sepharose (registered trademark) High Performance (Cytiva Corporation), SP Sepharose (registered trademark) XL (Cytiva Corporation), SP Sepharose (registered trademark) Big Beads (Cytiva Corporation), Eshumuno (registered trademark) CPX (Merck Millipore Corporation), Eshumuno (registered trademark) CP-FT (Merck Millipore Corporation), POROS (registered trademark) 50HS (Thermo Fisher Corporation), and POROS (registered trademark) XS (Thermo Fisher Corporation).

[0656] In the present disclosure, the matrix for hydrophobic interaction chromatography is not limited as long as it exhibits hydrophobic interaction chromatography. In one aspect, the hydrophobic interaction chromatography matrix includes a hydrophobic ligand.

[0657] In the method of the present disclosure, commercially available products can be used as hydrophobic interaction chromatography matrices. In specific aspects, without limitation to the following items, Phenyl Sepharose (registered trademark) High Performance (Cytiva Corporation), Butyl Sepharose (registered trademark) High Performance (Cytiva Corporation), Phenyl Sepharose (registered trademark) 6 Fast Flow (Cytiva Corporation), Butyl-S Sepharose (registered trademark) 6 Fast Flow (Cytiva Corporation), Butyl Sepharose (registered trademark) 4 Fast Flow (Cytiva Corporation), Octyl Sepharose (registered trademark) 4 Fast Flow (Cytiva Corporation), Capto (registered trademark) Phenyl ImpRes (Cytiva Corporation), Capto (registered trademark) Phenyl (Cytiva Corporation), Capto (registered trademark) Phenyl (High Sub) (Cytiva Corporation), Capto (registered trademark) Butyl (Cytiva Corporation), Capto (registered trademark) Butyl ImpRes (Cytiva Corporation), Capto (registered trademark) Octyl (Cytiva Corporation), Phenyl Sepharose (registered trademark) 6 Fast Flow (Low Sub) (Cytiva Corporation), Phenyl Sepharose (registered trademark) 6 Fast Flow (High Sub) (Cytiva Corporation), POROS (registered trademark) Ethyl (Thermo Fisher Corporation), Fractogel (registered trademark) Phenyl (Merck Millipore Corporation), Fractogel (registered trademark) Propyl (Merck Millipore Corporation), TOYOPEARL (registered trademark) Butyl (TOSOH Corporation), TOYOPEARL (registered trademark) Ether (TOSOH Corporation), TOYOPEARL (registered trademark) Hexyl (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl (TOSOH Corporation), TOYOPEARL (registered trademark) PPG (TOSOH Corporation), TOYOPEARL (registered trademark) Super Butyl (TOSOHExamples of hydrophobic interaction chromatography matrices include, but are not limited to, TOYOPEARL (registered trademark) Butyl-600 (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650C (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650M (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650S (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-600M (TOSOH Corporation), and Macro-Prep (registered trademark) HIC (Bio-Rad Laboratories Corporation).

[0658] In one aspect, the multimodal chromatography matrix of the present disclosure may include a matrix combining a cation exchange ligand and a hydrophobic ligand, or a matrix combining an anion exchange ligand and a hydrophobic ligand. In the methods of the present disclosure, commercially available products can be used for the multimodal chromatography matrix. Examples of matrices for multimodal chromatography include, but are not limited to, Capto (registered trademark) adhere (Cytiva Corporation), Capto (registered trademark) adhere ImpRes (Cytiva Corporation), Capto (registered trademark) MMC (Cytiva Corporation), Capto (registered trademark) MMC ImpRes (Cytiva Corporation), and Eshmuno (registered trademark) CMX (Merck Millipore Corporation).

[0659] In one aspect, the hydroxyapatite chromatography matrix may comprise hydroxyapatite or a derivative thereof (such as fluorapatite). In the methods of the present disclosure, commercially available products may also be used for the hydroxyapatite chromatography matrix. In specific embodiments, without limitation to the following items, Ceramic Hydroxyapatite (registered trademark) Type I (Bio-Rad Laboratories Corporation), Ceramic Hydroxyapatite (registered trademark) Type II (Bio-Rad Laboratories Corporation), Ceramic Fluoroapatite (registered trademark) (Bio-Rad Laboratories Corporation), MPC (registered trademark) Ceramic Hydroxyfluorapatite (Bio-Rad Laboratories Corporation), HA Ultrogel (registered trademark) (PALL Corporation), and Ca++ Pure-HA (registered trademark) (TOSOH Corporation) may be exemplified as matrices for hydroxyapatite chromatography.

[0660] In the methods of the present disclosure, commercially available products may also be used for membrane chromatography. In specific embodiments, without limitation to the following items, Mustang (registered trademark) Q (PALL Corporation), Mustang (registered trademark) S (PALL Corporation), Sartobind (registered trademark) Q (Sartorius Corporation), Sartobind (registered trademark) S (Sartorius Corporation), Sartobind (registered trademark) STIC (Sartorius Corporation), Sartobind (registered trademark) Protein A (Sartorius Corporation), Fibro (Cytiva Corporation), Protein Capture (GORE Corporation), Natrix (registered trademark) Q (Merck Millipore Corporation), Purexa (registered trademark)-MQ (Purilogics Corporation), Purexa (registered trademark)-A (Purilogics Corporation), and Purexa (registered trademark)-DMAE (Purilogics Corporation) may be exemplified as products for membrane chromatography.

[0661] In the present disclosure, the terms "reducing reagent", "reductant", and "solution containing a reductant" are used interchangeably. In some embodiments, the above-mentioned reductant is a free thiol. In addition, in one aspect, the reductant in the present disclosure can be a monothiol, dithiol, phosphine, or inorganic reagent. Examples of monothiols in the present disclosure include glutathione, cysteine, lipoic acid, 2-mercaptoethanol, and 2-MEA, but are not limited thereto. Examples of dithiols in the present disclosure include dithiothreitol (DTT), DTE, and DTBA, but are not limited thereto. Examples of phosphines in the present disclosure include tris(2-carboxyethyl)phosphine (TCEP) and THPP, but are not limited thereto. Examples of inorganic reagents in the present disclosure include sodium sulfite and sodium metabisulfite, but are not limited thereto.

[0662] The reducing reagent preferably consists of a compound selected from the group consisting of glutathione (GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-aminoethanethiol (2-MEA), tris(2-carboxyethyl)phosphine (TCEP), dithionitrobenzoate, cysteine, sodium sulfite, sodium metabisulfite, and Na2SO3. In some embodiments, TCEP, 2-MEA, DTT, cysteine, GSH, or Na2SO3 can be used. In some preferred embodiments, cysteine can be used. In some preferred embodiments, TCEP can be used. In the method of the present disclosure, one or more types (e.g., 2 types, 3 types, or more types) of reducing agents can be used.

[0663] In one embodiment, the reducing agent can be in the form of a solution, or preferably a buffer. Examples of buffers include buffers for chromatography techniques, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer, but are not limited thereto. In addition, examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris.

[0664] In the present disclosure, "contacting" means subjecting or exposing a mixture of antigen-binding molecules to a solution containing a reducing agent, or mixing a mixture of antigen-binding molecules with a solution containing a reducing agent. In the method of the present disclosure, a mixture of antigen-binding molecules can be contacted with a solution containing a reducing agent while the molecules are in a state of being bound to a solid matrix (e.g., a column chromatography column or a membrane chromatography membrane).

[0665] While not bound by the following theory, the presence of unLINC forms (i.e., divalent or trivalent antigen-binding molecules that do not have engineered disulfide bonds or "paired cysteines") may be due to unpaired Cys residues often forming disulfide bonds with molecules containing free thiol groups, such as cysteinylation and glutathioneylation that prevent LINC formation (formation of engineered disulfide bonds in regions outside the hinge region) by "capping" unpaired Cys residues. To remove molecules with unpaired capped cysteines, a reducing agent can help de-cap the surface cysteines, and further re-oxidation of the de-capped antigen-binding molecules (e.g., by removing the reducing agent) can promote disulfide bond formation between the de-capped cysteines for LINC formation. Thus, removal of cysteinylation from unpaired thiols in the unLINC format by reduction and re-oxidation can remove the unLINC form and improve the homogeneity of the antibody.

[0666] In one aspect, in the methods of the present disclosure, contacting a mixture of antigen-binding molecules with a solution comprising a reducing agent causes disulfide bonds formed between amino acid residues capable of forming disulfide bonds in the antigen-binding molecules to break. In one aspect, contacting a mixture of antigen-binding molecules with a solution comprising a reducing agent causes de-capping of capped cysteine residues in the antigen-binding molecules.

[0667] In one aspect, in the methods of the present disclosure, removal of the reducing agent causes disulfide bonds to form between amino acid residues capable of forming disulfide bonds in the antigen-binding molecules.

[0668] Contacting a mixture of antigen-binding molecules with a solution comprising a reducing agent is carried out for a time sufficient to increase the relative proportion of the desired conformation. Any relative increase in the proportion is desirable, including, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the antigen-binding molecules having an undesired conformation being converted to antigen-binding molecules having the desired conformation.

[0669] In one aspect, in the methods of the present disclosure, a solution comprising a reducing agent is flowed through a chromatography system containing a matrix carrying the antigen-binding molecules. In a specific embodiment, the antigen-binding molecules constitute a stationary phase immobilized to the chromatography matrix, and the solution comprising the reducing agent is part of the mobile phase. In such a case, the contacting can be carried out as part of a chromatographic purification procedure.

[0670] The reducing agent is present at a concentration sufficient to increase the relative proportion of the desired conformation (e.g., an antigen-binding molecule in the "paired cysteine" form, which has one or more engineered disulfide bonds between two Fabs of an antibody (e.g., between regions other than the hinge region)). The optimal absolute concentration and molar ratio of the reducing agent depend on the total concentration of the antigen-binding molecule and, in some cases, also on the concentration of a particular antigen-binding molecule. Additionally, the reducing agent concentration depends on the number and accessibility of unpaired cysteines in the antigen-binding molecule.

[0671] In one aspect, examples of the concentration of the reducing agent include ranges that can be specified by any combination of a lower limit selected from the group consisting of about 0.0001 mM, about 0.0005 mM, about 0.001 mM, about 0.005 mM, about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.5 mM, and about 1.0 mM, and an upper limit selected from the group consisting of about 100.0 mM, about 75.0 mM, about 50.0 mM, about 25.0 mM, about 10.0 mM, about 5.0 mM, about 1.0 mM, about 0.5 mM, about 0.1 mM, and about 0.01 mM. In some embodiments, as the concentration of the reducing agent, ranges of about 0.00001 mM to about 10.0 mM, about 0.00005 mM to about 5.0 mM, about 0.0001 mM to about 1.0 mM, about 0.0005 mM to about 0.5 mM, about 0.001 mM to about 0.1 mM, or about 0.001 mM to about 0.01 mM can be exemplified. In further embodiments, as the concentration of the reducing agent, ranges of about 0.001 mM to about 100.0 mM, about 0.005 mM to about 75.0 mM, about 0.01 mM to about 50.0 mM, about 0.05 mM to about 25.0 mM, or about 0.1 mM to about 10 mM can be exemplified. In additional embodiments, as the concentration of the reducing agent, ranges of about 0.0001 mM to about 100.0 mM, about 0.0005 mM to about 50.0 mM, about 0.001 mM to about 10.0 mM, about 0.005 mM to about 5.0 mM, or about 0.01 mM to about 1.0 mM can be exemplified. In a specific embodiment, the concentration of the reducing agent can be about 0.001 mM, about 0.01 mM, about 0.1 mM, about 0.15 mM, about 1.0 mM, or about 10.0 mM.

[0672] In some preferred embodiments, when the reducing agent is cysteine, the concentration of the reducing agent can be from about 0.01 mM to about 100.0 mM, from about 0.05 mM to about 50.0 mM, from about 0.1 mM to about 10.0 mM, or from about 0.5 mM to about 5.0 mM. In further preferred embodiments, when the reducing agent is cysteine, the concentration of the reducing agent can be from about 0.001 mM to about 100.0 mM, from about 0.005 mM to about 75.0 mM, from about 0.01 mM to about 50.0 mM, from about 0.05 mM to about 25.0 mM, or from about 0.1 mM to about 10 mM. In further preferred embodiments, when the reducing agent is cysteine, the concentration of the reducing agent can be from about 0.0001 mM to about 100.0 mM, from about 0.001 mM to about 75.0 mM, from about 0.01 mM to about 50.0 mM, or from about 0.1 mM to about 10.0 mM. In some preferred embodiments, when the reducing agent is dithiol, the concentration of the reducing agent can be from about 0.001 mM to about 10 mM. In some preferred embodiments, when the reducing agent is phosphine, the concentration of the reducing agent can be from about 0.0001 mM to about 1 mM or from about 0.001 mM to about 0.01 mM. In some preferred embodiments, when the reducing agent is TCEP, the concentration of the reducing agent can be from about 0.00001 mM to about 10.0 mM, from about 0.00005 mM to about 5.0 mM, from about 0.0001 mM to about 1 mM, from about 0.0005 mM to about 0.5 mM, from about 0.001 mM to about 0.1 mM, or from about 0.001 mM to about 0.01 mM. In some preferred embodiments, when the reducing agent is an inorganic reagent, the concentration of the reducing agent can be from about 0.0001 mM to about 10 mM or from about 0.001 mM to about 0.1 mM.

[0673] In a specific embodiment, when the reducing agent is cysteine, the concentration of the reducing agent can be about 0.1 mM, about 0.15 mM, about 1.0 mM, about 10.0 mM or about 100 mM. In a specific embodiment, when the reducing agent is TCEP, the concentration of the reducing agent can be about 0.001 mM, about 0.01 mM, about 0.1 mM or about 1.0 mM.

[0674] To maximize the yield of antigen-binding molecules having the desired conformation, the pH of the solution containing the reducing agent is selected to protect the stability of the antigen-binding molecules and is most suitable for disulfide exchange. In one aspect, the pH of the solution containing the reducing agent of the present disclosure can be from about 4.5 to about 10.0, from about 5.0 to about 9.0, from about 6.5 to about 8.5, or from about 7.0 to about 8.0. In non-limiting embodiments of the present invention, the optimal pH is found to be about 7.0, about 7.5 or about 8.0. However, the optimal pH in a particular embodiment of the present invention can be readily determined by those skilled in the art through experimentation.

[0675] In the method of the present disclosure, the contact of the chromatography matrix, the antigen-binding molecule, and the reducing agent occurs during chromatography. In one embodiment, in the method of the present disclosure, the contact between the antigen-binding molecule loaded on the matrix and the reducing agent occurs during chromatography. In other words, in the method of the present disclosure, the contact between the reducing agent and the antigen-binding molecule can be carried out by flowing a solution containing the reducing agent through a column chromatography column or a membrane chromatography device containing the antigen-binding molecule loaded on the matrix.

[0676] In some embodiments, a solution containing the reducing agent is flowed through a column chromatography column or a membrane chromatography device for a residence time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes. In a specific embodiment, a solution containing the reducing agent is flowed through a column chromatography column or a membrane chromatography device for a residence time of about 12 minutes or about 30 seconds. In some embodiments, the flow can be temporarily stopped while the column or device is filled with the solution containing the reducing agent.

[0677] In one aspect, when using column chromatography, a solution containing the reducing agent is flowed through the column for a residence time of about 2 minutes to about 80 minutes or about 4 minutes to about 24 minutes. In a specific embodiment, when using column chromatography, a solution containing the reducing agent is flowed through the column for a residence time of about 12 minutes. In one aspect, the flow can be temporarily stopped while the column is filled with the solution containing the reducing agent. In one aspect, when using column chromatography, the solution containing the reducing agent of the present disclosure can be flowed through the column at a rate of about 25 cm / hour to about 500 cm / hour, about 50 cm / hour to about 400 cm / hour, about 75 cm / hour to about 350 cm / hour, or about 100 cm / hour to about 300 cm / hour. In some embodiments, the solution containing the reducing agent of the present disclosure can be flowed through the column at a rate of about 100 cm / hour. In one aspect, the flow can be temporarily stopped while the column is filled with the solution containing the reducing agent.

[0678] In one aspect, when using membrane chromatography, a solution containing the reducing agent is flowed through the membrane device for a residence time of about 2 seconds to about 60 minutes or about 3 seconds to about 6 minutes, such as about 30 seconds. In some embodiments, the flow can be temporarily stopped while the membrane device is filled with the solution containing the reducing agent. In one aspect, when using membrane chromatography, the solution containing the reducing agent in the present disclosure can be flowed through the membrane device at a rate of about 0.017 MV / minute to about 30 MV / minute, about 0.17 MV / minute to about 20 MV / minute, about 1 MV / minute to about 10 MV / minute, or about 2 MV / minute to about 5 MV / minute. In a specific embodiment, the solution containing the reducing agent in the present disclosure can be flowed through the membrane device at a rate of about 2 MV / minute. The flow can be temporarily stopped while the membrane device is filled with the solution.

[0679] The amount of the solution containing a reducing agent that is passed through the column is not particularly limited. For example, when using column chromatography, the flow volume of the solution containing a reducing agent can be about 0.1-fold (about 0.1 column volume (CV)) to about 100-fold (about 100 CV), about 1-fold (about 1 CV) to about 20-fold (about 20 CV), or about 3-fold (about 3 CV) to about 10-fold (about 10 CV) of the column volume. In some embodiments, the amount of the solution containing a reducing agent that is passed through the column can be about 10-fold column volume (about 10 CV). Additionally, when using membrane chromatography, the flow volume of the solution containing a reducing agent can be about 1-fold (about 1 membrane volume (MV)) to about 500-fold (about 500 MV), or about 2-fold (about 2 MV) to about 100-fold (about 100 MV) of the membrane device volume. In some embodiments, the amount of the solution containing a reducing agent that is passed through the column can be about 15-fold column volume (about 15 MV).

[0680] The reaction time (contact time) between the mixture of antigen-binding molecules and the solution containing a reducing agent is not particularly limited, but examples include about 6 seconds to about 1440 minutes, about 18 seconds to about 300 minutes, about 5 minutes to about 180 minutes, about 10 minutes to about 150 minutes, or about 12 minutes to about 120 minutes. In certain embodiments, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 120 minutes or about 7.5 minutes.

[0681] In one aspect, when using column chromatography, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 4 minutes to about 1440 minutes, about 8 minutes to about 300 minutes, about 30 minutes to about 240 minutes, about 60 minutes to about 180 minutes, or about 120 minutes. In specific embodiments, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 120 minutes.

[0682] In one aspect, when using membrane chromatography, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 6 seconds to about 600 minutes or about 18 seconds to about 120 minutes, such as about 7.5 minutes.

[0683] In the methods of the present disclosure, the antigen-binding molecules can be contacted with the reducing agent in a variety of suitable volumes, such as at the analytical scale (1 mL to 50 mL), preparative scale (50 mL to 10 L), and manufacturing scale (10 L or more). In one aspect, the amount of antigen-binding molecules loaded onto the substrate can be about 5 to 100 g, about 7 to 70 g, about 10 to 40 g, such as about 10 g or about 25 g per 1 L of the chromatographic matrix containing the substrate.

[0684] In one aspect, when using column chromatography, the amount of antigen-binding molecule loaded onto the substrate can be about 5 g to about 80 g, about 7 g to about 70 g, or about 10 g to about 40 g, such as about 10 g, per 1 L of the chromatography matrix containing the substrate.

[0685] In one aspect, when using membrane chromatography, the amount of antigen-binding molecule loaded onto the membrane can be about 5 g to about 100 g, about 7 g to about 50 g, or about 10 g to about 40 g, such as about 25 g, per 1 L of the membrane device volume.

[0686] The methods of the present disclosure include a step of removing a reducing agent. In the present disclosure, removing the reducing agent includes completely removing the reducing agent, reducing the concentration of the reducing agent, and chemically inactivating the reducing agent. Removal of the reducing agent promotes re-oxidation of disulfide bonds in regions other than the hinge region between the two Fabs that make up the antigen-binding molecule.

[0687] In one aspect, the reducing agent can be removed by contacting the antigen-binding molecule with a solution that does not contain a reducing agent. In one aspect, the reducing agent can be removed by flowing a solution that does not contain a reducing agent through a column chromatography column or through a membrane chromatography device.

[0688] In some embodiments, a solution that does not contain a reducing agent is flowed through a column chromatography column or through a membrane chromatography device for a residence time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes. In a specific embodiment, a solution that does not contain a reducing agent is flowed through a column chromatography column or through a membrane chromatography device for a residence time of about 4 minutes. In some embodiments, the flow can be temporarily stopped while the column or device is filled with the solution.

[0689] In some embodiments, when using column chromatography, a solution that does not contain a reducing agent is flowed through the column for a residence time of about 2 minutes to about 80 minutes or about 4 minutes to about 24 minutes. In a particular embodiment, when using column chromatography, a solution that does not contain a reducing agent is flowed through the column for a residence time of about 4 minutes. In some embodiments, the flow can be temporarily stopped while the column is filled with the solution.

[0690] In some embodiments, when using column chromatography, a solution that does not contain a reducing agent can be flowed through the column at a rate of about 25 cm / hour to about 500 cm / hour, about 50 cm / hour to about 450 cm / hour, about 200 cm / hour to about 400 cm / hour, or about 250 cm / hour to about 350 cm / hour. Additionally, in a specific embodiment, a solution that does not contain the reducing agent of the present disclosure can be flowed through the column at a rate of about 300 cm / hour. The flow can be temporarily stopped while the column is filled with the solution.

[0691] In some embodiments, when using membrane chromatography, a solution that does not contain a reducing agent is flowed through the membrane device for a residence time of about 2 seconds to about 60 minutes or about 3 seconds to about 6 minutes, such as about 30 seconds. In some embodiments, the flow can be temporarily stopped while the device is filled with the solution.

[0692] In some embodiments, when using membrane chromatography, a solution that does not contain a reducing agent can be flowed through the membrane device at a rate of about 0.017 MV / minute to about 30 MV / minute, about 0.17 MV / minute to about 20 MV / minute, about 1 MV / minute to about 10 MV / minute, or about 2 MV / minute to about 5 MV / minute. Additionally, in a specific embodiment, a solution that does not contain the reducing agent of the present disclosure can be flowed through the membrane device at a rate of about 2 MV / minute. The flow can be temporarily stopped while the membrane device is filled with the solution.

[0693] The solution that does not contain a reducing agent is not particularly limited, but in one example, a buffer solution can be used, and more specifically, buffer solutions commonly used in chromatography techniques, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer, can be used. Additionally, examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris. In one aspect, in the method of the present disclosure, for the solution that does not contain a reducing agent, the same type of solution as the solution containing a reducing agent used in the contacting step can be used, except that the reducing agent is absent.

[0694] In one aspect, when using column chromatography, the flow volume of the solution that does not contain a reducing agent can be about 0.1 times (about 0.1 CV) to about 100 times (about 100 CV), about 1 time (about 1 CV) to about 20 times (about 20 CV), or about 3 times (about 3 CV) to about 10 times (about 10 CV) the column volume. In some embodiments, the flow volume of the solution that does not contain a reducing agent can be about 5.0 times the column volume (about 5.0 CV).

[0695] In one aspect, when using membrane chromatography, the flow volume of the solution that does not contain a reducing agent can be about 1 time (1 MV) to about 500 times (500 MV), or about 2 times (2 MV) to about 100 times (100 MV) the membrane device volume, such as about 15 times the membrane device volume (15 MV).

[0696] In one aspect, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution that does not contain a reducing agent can be from about 6 seconds to about 1440 minutes, from about 18 seconds to about 300 minutes, from about 5 minutes to about 60 minutes, from about 10 minutes to about 50 minutes, or from about 12 minutes to about 40 minutes. In certain embodiments, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution that does not contain a reducing agent can be about 20 minutes.

[0697] In one aspect, when using column chromatography, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution that does not contain a reducing agent can be from about 4 minutes to about 1440 minutes or from about 8 minutes to about 300 minutes. In certain embodiments, when using column chromatography, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution that does not contain a reducing agent can be about 20 minutes.

[0698] In one aspect, when using membrane chromatography, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution that does not contain a reducing agent can be from about 6 seconds to about 600 minutes or from about 18 seconds to about 120 minutes, such as about 7.5 minutes.

[0699] The methods of the present invention can be carried out over a wide range of temperatures. For example, the methods of the present invention can be carried out at about 4°C to about 37°C or at about 15°C to about 37°C. A typical temperature for contacting a formulation of partially or fully purified antigen-binding molecules is about 4°C to about 25°C (ambient temperature), or preferably at 23°C, but this contacting can also be carried out at lower and higher temperatures. In some embodiments, the methods of the present disclosure can be carried out at a temperature of about 20°C to 37°C, preferably at 23°C, 25°C, or 37°C, and more preferably at 23°C.

[0700] In addition, for a solution that does not contain a reducing agent, the optimal pH can be readily determined by one of ordinary skill in the art through experimentation. Examples can be from about 4.5 to about 10.0, from about 5.0 to about 9.0, from about 6.5 to about 8.5, or from about 7.0 to about 8.0. In addition, in a specific embodiment, the optimal pH can be about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, or about 7.9.

[0701] In one aspect, in the method of the present disclosure, before the step of contacting the mixture of antigen-binding molecules with a solution containing a reducing agent, it may include a step of contacting the mixture with a chromatography matrix to fill the interior of the column with the antigen-binding molecules or immobilize the antigen-binding molecules on a membrane. In a specific embodiment, when the mixture is a cell culture fluid containing antigen-binding molecules (harvested cell culture fluid: HCCF), before the step of contacting the cell culture fluid with a solution containing a reducing agent, it may include a step of contacting the cell culture fluid with a chromatography matrix to fill the interior of the column with the antigen-binding molecules or immobilize the antigen-binding molecules on a membrane. In one aspect, the mixture of antigen-binding molecules includes antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (i.e., LINC form) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (i.e., unLINC form).

[0702] In certain aspects, the antigen-binding molecules may be at least partially purified before the step of contacting with a solution containing a reducing agent. In some embodiments, the production method of the present disclosure may include a step of performing affinity chromatography (preferably protein A chromatography) on the cell culture fluid containing antigen-binding molecules before the contacting step.

[0703] In one aspect, the method of the present disclosure may include a step of removing impurities in the chromatography before the contacting step. As described in an example as an instance, the impurities can be removed by methods well known to those skilled in the art (such as flowing a buffer through a column carrying a substrate adsorbed with antigen-binding molecules).

[0704] In one aspect, the method of the present disclosure may further include a step of collecting or eluting antigen-binding molecules having at least one disulfide bond in a region other than the hinge region and / or a step of purifying the antigen-binding molecules before or after the step of removing the reducing agent. In one embodiment, the collection and elution of the antigen-binding molecules can be performed by eluting the antigen-binding molecules from the chromatography column by methods well known to those skilled in the art (such as using an elution buffer). Those skilled in the art can appropriately adjust the pH and composition of the elution buffer according to the characteristics of the antigen-binding molecules to be purified.

[0705] In one embodiment, formulations of the antigen-binding molecules produced as described herein can be further purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions for purifying a particular antigen-binding molecule will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, which will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antigen-binding molecule binds can be used. For example, for affinity chromatography purification of an antigen-binding molecule, a matrix having protein A or G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antigen-binding molecule. The purity of the antigen-binding molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis and high performance liquid chromatography.

[0706] In certain embodiments, antigen-binding molecules having at least one disulfide bond in a region other than the hinge region, produced or purified by the methods described herein, are further processed in a separate processing step using a chaotropic denaturant such as sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride (GuHCl). A substantial amount of the chaotrope is required to observe appreciable unfolding. In some embodiments, the processing step uses a chaotrope between 0.1 M and 2 M, which produces an effect equivalent to using 0.1 M to 2 M guanidine hydrochloride. In a specific embodiment, oxidative refolding is achieved in the presence of about 1.0 M guanidine hydrochloride or an amount of another chaotrope that produces the same or a similar amount of refolding as 1 M guanidine hydrochloride. In some embodiments, the method uses a chaotrope between about 1.5 M and 0.5 M. The amount of chaotrope used is based on the structural stability of the antigen-binding molecule in the presence of the chaotrope. Sufficient chaotrope needs to be present to disrupt the local tertiary structure and / or quaternary structure of the domain interactions of the antigen-binding molecule, but less than the amount required to completely unfold the molecule and / or the secondary structure of the individual domains. To determine the point at which the antigen-binding molecule will begin to unfold by equilibrium denaturation, a person of ordinary skill in the art titrates the chaotrope into a solution containing the antigen-binding molecule and monitors the structure by techniques such as circular dichroism or fluorescence. There are other parameters that can be used to unfold or slightly disrupt the structure of the antigen-binding molecule that can be used in place of the chaotrope. Temperature and pressure are two fundamental parameters previously used to alter the structure of antigen-binding molecules and can be used in place of the chaotrope when in contact with an oxidizing agent and / or a reducing agent. The inventors anticipate that a person of ordinary skill in the art can use any parameter that has been demonstrated to denature or disrupt the structure of the antigen-binding molecule in place of the chaotrope.

[0707] The method of the present disclosure increases the "LINC ratio", which is the ratio of antigen-binding molecules in LINC form relative to the antigen-binding molecules loaded on the matrix (i.e., the sum of antigen-binding molecules in LINC form having at least one disulfide bond in a region other than the hinge region, and antigen-binding molecules in non-LINC form). For example, as described in WO2021 / 157679, the LINC ratio can be calculated by separating crosslinked LINC form from non-crosslinked unLINC form (open form) by non-reducing SDS-PAGE and comparing the respective abundance ratios.

[0708] In one embodiment, a formulation of antigen-binding molecules produced as described herein can be further subjected to techniques such as electrophoresis and chromatography to determine the LINC ratio. More specifically, after the step of purification by chromatography, the method of the present disclosure can include a step of quantifying the LINC ratio in the formulation obtained from the purification step (i.e., the ratio of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC form) relative to the sum of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC form) and (ii) mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule (unLINC form)). In a certain embodiment, the method of the present disclosure can include a step of quantifying the ratio of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC form) relative to the sum of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC form) and (ii) non-disulfide-bonded forms of the antigen-binding molecule.

[0709] In one embodiment, the step of quantifying the LINC ratio includes electrophoresis, chromatography, etc. In one embodiment, non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), non-reducing capillary SDS gel electrophoresis (CE-SDS), etc. can be examples of electrophoresis. In addition, in one embodiment, hydrophobic interaction chromatography (HIC), etc. can be examples of chromatography.

[0710] In one embodiment, before the step of quantifying the LINC ratio, the method of the present disclosure may include the step of adding a protease to a preparation of an antigen-binding molecule produced as described herein. In one embodiment, the protease of the present disclosure may be, for example, IgdE, or preferably IgdE derived from Streptococcus agalactiae. IgdE is a protease that recognizes the upper part of the hinge region of the human IgG1 antibody (KSCDKT / HTCPPCP), and is an enzyme that digests the antibody between T and H. This enzyme can degrade the antibody to generate Fab and Fc regions. Streptococcus agalactiae-derived IgdE is an enzyme disclosed in documents such as Spoerry C et al., (2016) PLoS ONE 11(10):e0164809. Doi:10.1371 / journal.pone.0164809, and can be easily obtained by those skilled in the art using well-known techniques. In addition, Streptococcus agalactiae-derived IgdE can be commercially obtained, for example, from Genovis Inc. as "FabALACTICA TM (IgdE)". The inventors added Streptococcus agalactiae-derived IgdE to a preparation of an antigen-binding molecule produced as described herein, and found that the LINC ratio of the preparation can be determined by electrophoresis, chromatography, etc. of the preparation containing IgdE.

[0711] The concentration of the protease added to the preparation is not particularly limited, but in one embodiment, examples include 0.6 to 1.8 units-enzyme / μg-protein.

[0712] In certain embodiments, when the method of the present invention includes the step of adding a protease to a preparation of an antigen-binding molecule produced as described herein, non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC) can be used in the step of quantifying the LINC ratio. In addition, in certain embodiments, the chromatography for obtaining the preparation of the antigen-binding molecule can be membrane chromatography.

[0713] In one embodiment, in the methods of the present disclosure, the LINC ratio can be calculated as follows. For example, when non-reducing capillary SDS gel electrophoresis (CE-SDS) is used in the quantification step, electrophoretograms are prepared for the formulation with added protease, the formulation without added protease, and the sample containing only protease. In this case, the LINC ratio is determined as the ratio of the peak area derived from the LINC form to the value obtained by subtracting the peak area derived from the formulation without added protease (excluding the peak area derived from the unLINC form) and the peak area derived from the sample containing only protease from the "peak area derived from the formulation with added protease (including the peak area derived from the unLINC form and the protease-derived peak)", and then adding the "peak area derived from the LINC form". The sample containing only protease is preferably a sample having the same components as the formulation with added protease, except that the sample does not contain the formulation of the antigen-binding molecule obtained by the methods of the present disclosure.

[0714] In different embodiments, in the methods of the present disclosure, the LINC ratio can be calculated as follows. For example, when hydrophobic interaction chromatography (HIC) is used in the quantification step, the LINC ratio is determined by the ratio of the peak derived from the LINC form to the sum of the peak derived from the LINC form and the peaks derived from all non-LINC forms. Mobile phase A and mobile phase B in hydrophobic interaction chromatography (HIC) can be appropriately prepared by those skilled in the art based on common technical knowledge. In addition, the chromatography matrix can be appropriately selected by those skilled in the art and, for example, the matrices described herein can be used.

[0715] In one embodiment, the methods of the present disclosure can further include the step of culturing the formulation after adding protease. The culture conditions are not particularly limited, but can be, for example, incubation at 37 °C for 16 to 20 hours, such as 18 hours, in a phosphate buffer at pH 6 to 8.

[0716] In addition, in the present disclosure, the antigen-binding molecule that has undergone chromatography for purification can contain a signal sequence. Specifically, when an antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region is recombinantly expressed in a cell, sometimes in order to promote expression and / or secretion, the antigen-binding molecule can have a signal sequence. In one embodiment, when the translation product from the nucleic acid sequence for recombinantly expressing the antigen-binding molecule contains a signal sequence, the methods of the present disclosure can include the step of cleaving the signal sequence from the antigen-binding molecule. The signal sequence can be cleaved before or after the step of subjecting the antigen-binding molecule to chromatography for purification, or before or after the step of quantifying the LINC ratio of the substance purified by chromatography. Cleavage of the signal sequence can be carried out by methods well known to those skilled in the art.

[0717] In one aspect, the amino acid residue capable of forming a disulfide bond is a mutated, substituted, introduced, or engineered cysteine residue.

[0718] In one aspect, at least one disulfide bond in a region outside the hinge region is formed between polypeptides constituting the antigen-binding molecule. More specifically, in a specific embodiment, at least one disulfide bond in a region outside the hinge region in the present disclosure is an interchain disulfide bond. In some embodiments, at least one disulfide bond in a region outside the hinge region is 1, 2, 3, 4, 5, or more interchain disulfide bonds.

[0719] In one aspect, at least one disulfide bond in a region outside the hinge region is an engineered disulfide bond that does not exist in wild-type IgG.

[0720] III. Antigen-binding molecule

[0721] In one aspect, the antigen-binding molecule of the present disclosure comprises a first antigen-binding domain and a second antigen-binding domain that can be connected to each other via at least one disulfide bond. In one aspect, in the antigen-binding molecule produced or purified by the method of the present disclosure, the first antigen-binding domain and the second antigen-binding domain are connected via at least one disulfide bond. In an embodiment of the above aspect, the first antigen-binding domain and the second antigen-binding domain are connected via two, three, four, or more disulfide bonds.

[0722] In an embodiment, at least one of the first antigen-binding domain and the second antigen-binding domain itself has the activity of binding to an antigen (i.e., a single antigen-binding domain independently has antigen-binding activity). In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain itself has the activity of binding to an antigen.

[0723] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain in the present disclosure may have a Fab, Fab', scFab, Fv, scFv, or VHH structure.

[0724] In a certain aspect, each of the first antigen-binding domain and the second antigen-binding domain is a Fab molecule, and the antigen-binding molecule comprises at least one disulfide bond formed between the first antigen-binding domain and the second antigen-binding domain. Preferably, at least one disulfide bond is formed between amino acid residues (cysteines) that do not exist in the hinge region, or preferably formed between amino acid residues (cysteines) in the CH1 region of each antigen-binding domain.

[0725] In an embodiment of the above aspect, both the first antigen-binding domain and the second antigen-binding domain comprise a Fab and a hinge region.

[0726] In certain embodiments, at least one of the amino acid residues that form a disulfide bond between the antigen-binding domains is a mutant amino acid residue that is not present in the wild-type Fab or hinge region, and for example, the amino acid residue is a cysteine residue that is not present in the wild-type Fab or hinge region. Such a mutant amino acid residue can be introduced into the wild-type Fab or hinge region by methods such as amino acid substitution.

[0727] Alternatively, in another embodiment, in the antigen-binding molecules of the present disclosure, amino acid residues (e.g., cysteine residues) that are present in the wild-type Fab or hinge region and may be involved in the disulfide bond between the antigen-binding domains can be replaced or deleted with other amino acid residues. Examples of such cysteine residues include cysteine residues at positions 220, 226, and 229 according to EU numbering in the hinge region, and cysteine residue at position 214 in the CL region.

[0728] In one embodiment of the above aspect, at least one of the first antigen-binding domain and the second antigen-binding domain comprises an antibody fragment that binds to a specific antigen. In certain embodiments, the antibody fragment is Fab, Fab', scFab, Fv, scFv, or a single-domain antibody. In certain embodiments, the first and / or second antigen-binding domain comprises a hinge region. Amino acid residues that form a disulfide bond are present in the first antigen-binding domain and the second antigen-binding domain, respectively, and the bond between the antigen-binding domains is formed by the linkage between these amino acid residues. In certain embodiments, at least one amino acid residue that forms a disulfide bond between the antigen-binding domains is present within the antibody fragment.

[0729] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain in the present disclosure may or may not comprise a hinge region. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain in the present disclosure may each comprise a Fab and a hinge region that form an F(ab')2 structure.

[0730] In the embodiments of the above aspect, both the first antigen-binding domain and the second antigen-binding domain bind to the same antigen. In certain embodiments, both the first antigen-binding domain and the second antigen-binding domain bind to the same epitope on the same antigen. In certain other embodiments, each of the first antigen-binding domain and the second antigen-binding domain binds to a different epitope on the same antigen. In certain embodiments, the antigen-binding molecule in the present disclosure is a bispecific antigen-binding molecule (e.g., a bispecific antibody) that targets a specific antigen.

[0731] In another embodiment of the above aspect, each of the first antigen-binding domain and the second antigen-binding domain binds to a different antigen.

[0732] In another embodiment of the above aspect, the antigen-binding molecule in the present disclosure is a clamp antigen-binding molecule (e.g., a clamp antibody). The clamp antigen-binding molecule herein 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 (which binds to antigen A), and thereby enhances the binding activity of the antigen-binding molecule that binds to antigen A to antigen A (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 clamp antibody specifically binds to an antigen-antibody complex formed between CD3 and an antibody with reduced binding ability to CD3 (a CD3 antibody with reduced binding), and thereby can enhance the binding activity of the CD3 antibody with reduced binding to CD3 (alternatively, stabilizes the antigen-antibody complex formed by CD3 and the CD3 antibody with reduced binding). In certain embodiments, the first and / or second antigen-binding domain in the antigen-binding molecule in the present disclosure can be an antigen-binding domain (a clamp antigen-binding domain) derived from a clamp antigen-binding molecule.

[0733] In an embodiment of the above aspect, both the first antigen-binding domain and the second antigen-binding domain have the same amino acid sequence. In another embodiment, each of the first antigen-binding domain and the second antigen-binding domain has a different amino acid sequence.

[0734] In another embodiment of the above aspect, the antigen-binding molecule in the present disclosure has the activity of regulating the interaction between two antigen molecules. Without being bound by a particular theory, the activity of regulating the interaction is considered to be generated by the antigen-binding molecule in the present disclosure holding the two antigen molecules in a spatially closer position and reducing their mobility. In certain embodiments, compared with a control antigen-binding molecule, the antigen-binding molecule in the present disclosure can enhance or weaken the interaction between two antigen molecules. The control antigen-binding molecule differs from the antigen-binding molecule in the present disclosure only in that the control antigen-binding molecule has one less disulfide bond between the two antigen-binding domains. In a further embodiment, the one less disulfide bond can be selected from bonds derived from mutant amino acid residues that do not exist in the wild-type Fab or hinge region (e.g., cysteine residues that do not exist in the wild-type Fab or hinge region). The mutant amino acid residues are, for example, artificially mutated, substituted, introduced, or engineered cysteine residues.

[0735] In one embodiment, the antigen molecules are selected from the group consisting of: receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[0736] In certain embodiments, the two antigen molecules bound by the antigen-binding molecules in the present disclosure can be a ligand and its receptor, respectively. The antigen-binding molecules in the present disclosure have the activity of promoting the activation of the receptor through the ligand. In certain other embodiments, the two antigen molecules bound by the antigen-binding molecules in the present disclosure can be an enzyme and its substrate, respectively. The antigen-binding molecules in the present disclosure have the activity of promoting the catalytic reaction between the enzyme and the substrate.

[0737] In addition, in certain other embodiments, both of the two antigen molecules bound by the antigen-binding molecules in the present disclosure can be antigens (such as proteins) present on the cell surface. The antigen-binding molecules in the present disclosure have the activity of promoting the interaction between cells expressing the first antigen and cells expressing the second antigen. For example, the cells expressing the first antigen and the cells expressing the second antigen can be cytotoxic cells and their target cells, respectively. The antigen-binding molecules in the present disclosure promote the damage of target cells by cytotoxic cells. The cytotoxic cells are, for example, T cells, NK cells, monocytes, or macrophages.

[0738] In the embodiments of the above aspects, the antigen-binding molecules in the present disclosure are resistant to protease cleavage. In certain embodiments, the antigen-binding molecules in the present disclosure have increased resistance to protease cleavage compared to control antigen-binding molecules. In certain embodiments, in the antigen-binding molecules in the present disclosure, the proportion of the full-length molecules (such as full-length IgG molecules) remaining after protease treatment is increased compared to control antigen-binding molecules. In certain embodiments, in the antigen-binding molecules in the present disclosure, the proportion of specific fragments (such as Fab monomers) generated after protease treatment is decreased compared to control antigen-binding molecules.

[0739] In the embodiments of the above aspects, when the antigen-binding molecules in the present disclosure are treated with protease, dimers of the antigen-binding domain or its fragments (such as cross-linked Fab dimers) are excised. In one embodiment, the protease can cleave the hinge region of the antigen-binding molecule.

[0740] In one aspect, at least one of the first antigen-binding domain and the second antigen-binding domain in the present disclosure can bind to a soluble protein. In another embodiment, at least one of the first antigen-binding domain and the second antigen-binding domain in the present disclosure can bind to a membrane protein.

[0741] The first antigen-binding domain and the second antigen-binding domain in the present disclosure can bind to the first antigen and the second antigen, respectively. In some embodiments, the first antigen and the second antigen are derived from human, mouse, rat, monkey, rabbit, or dog. In some embodiments, examples of the first antigen and the second antigen include, but are not limited to, for example, immune cell surface molecules (e.g., T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules, and macrophage surface molecules), or antigens that are expressed not only on tumor cells, tumor blood vessels, stromal cells, etc., but also on normal tissues (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.), receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0742] In some embodiments, either the first antigen or the second antigen can be, for example, a molecule specifically expressed on T cells, and the other antigen can be a molecule expressed on the surface of T cells or any other immune cells. In another embodiment of the combination of the first antigen and the second antigen, preferably, either the first antigen or the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on immune cells and different from the initially selected antigen.

[0743] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. In particular, CD3 is preferably used. For example, in the case of human CD3, the site in CD3 to which the antigen-binding molecule in the present disclosure binds can be any epitope present in the sequences of the γ chain, δ chain, or ε chain that constitutes human CD3. In particular, an epitope present in the extracellular region of the ε chain in the human CD3 complex is preferably used. The polynucleotide sequences constituting the γ chain, δ chain, and ε chain structures of CD3 are NM_000073.2, NM_000732.4, and NM_000733.3, and their polypeptide sequences are NP_000064.1, NP_000723.1, and NP_000724.1 (RefSeq registration numbers). Examples of other antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[0744] In one embodiment, the first antigen can be a molecule specifically expressed on T cells, preferably a T cell receptor complex molecule such as CD3, more preferably human CD3. In another embodiment, the second antigen can be a molecule expressed on T cells or any other immune cells, preferably a cell surface regulator on immune cells, more preferably a co-stimulatory molecule expressed on T cells, and even more preferably a protein of the "TNF superfamily" or "TNF receptor superfamily", including but not limited to human CD137 (4-1BB), CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL. In a preferred embodiment, the first antigen can be CD3 and the second antigen can be CD137. Herein, the first antigen and the second antigen can be defined interchangeably.

[0745] In certain embodiments, the antigen-binding molecules in the present disclosure specifically bind to all or a portion of a partial peptide of CD3. In a particular embodiment, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In a particular embodiment, the antigen-binding molecule is cross-reactive with (i.e., specifically binds to) human and cynomolgus monkey CD3. In some embodiments, the antigen-binding molecule is capable of specifically binding to the ε subunit of CD3, particularly the human CD3ε subunit of CD3 shown in SEQ ID NO:13 (NP_000724.1) (the RefSeq registration number is shown in parentheses). In some embodiments, the antigen-binding molecule is capable of specifically binding to the CD3ε chain expressed on the surface of eukaryotic cells. In some embodiments, the antigen-binding molecule binds to the CD3ε chain expressed on the surface of T cells.

[0746] In certain embodiments, the antigen-binding molecules in the present disclosure specifically bind to all or a portion of a partial peptide of CD137. The term "CD137" herein is also known as 4-1BB and is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0747] In certain embodiments, the CD137 is human CD137. In some embodiments, advantageous examples of the antigen-binding molecules in the present disclosure include antigen-binding molecules that bind to the same epitope on human CD137 as the antibody bound to an epitope selected from the group consisting of:

[0748] In the human CD137 protein, an antibody that recognizes a region containing the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO:14),

[0749] an antibody that recognizes a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO:15),

[0750] an antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO:16), and

[0751] an antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (SEQ ID NO:17).

[0752] In an embodiment of the above aspect, at least one of the first antigen-binding domain and the second antigen-binding domain comprises a non-antibody protein that binds to a specific antigen or a fragment thereof. In certain embodiments, the non-antibody protein is either a ligand or a receptor of a pair of ligands and receptors that specifically bind to each other. Such receptors include, for example, receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0753] In one aspect, the antigen-binding molecule of the present disclosure may further comprise a third antigen-binding domain. In one aspect, the third antigen-binding domain may be fused to either the first antigen-binding domain or the second antigen-binding domain.

[0754] In one aspect, the third antigen-binding domain may be a Fab or scFv; in this case, the third antigen-binding domain may optionally be fused at its C-terminus via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of either the first antigen-binding domain or the second antigen-binding domain.

[0755] In certain embodiments, each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain of the present disclosure may be a Fab molecule; in this case, the third antigen-binding domain may optionally be fused at the C-terminus of its Fab heavy chain (CH1 region) via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of either the first antigen-binding domain or the second antigen-binding domain.

[0756] In one aspect, an example of the peptide linker of the present disclosure may be a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, but is not limited thereto.

[0757] In one aspect, the third antigen-binding domain of the present disclosure may be a cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first antigen-binding domain and the second antigen-binding domain may be a conventional Fab molecule.

[0758] In one aspect, the third antigen-binding domain of the present disclosure may bind to a third antigen different from the above-described first and second antigens. The third antigen-binding domain that binds to the third antigen may be an antigen-binding domain that recognizes any antigen. The third antigen-binding domain that binds to the third antigen in the present disclosure may be an antigen-binding domain that recognizes a molecule specifically expressed in cancer cells or cancer tissues.

[0759] In some embodiments, the third antigen is derived from a human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule specifically expressed on a cell or organ derived from a human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule not systematically expressed on the cell or organ. The third antigen is preferably, for example, a tumor cell-specific antigen and also includes antigens whose expression is associated with the malignant alteration of the cell, as well as abnormal sugar chains that appear on the cell surface or protein molecules during the malignant transformation of the cell. Specific examples thereof include the ALK receptor (pleiotrophin receptor), pleiotrophin, KS1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostate acid phosphatase, prostate-specific antigen (PSA), melanoma-associated antigen p97, melanoma antigen gp75, high-molecular-weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), Burkitt lymphoma antigen 38.13, CD19, human B-lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specific transplantation antigen (TSTA), T antigen, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, oncofetal antigen alpha-fetoprotein (e.g., oncofetal trophoblast glycoprotein 5T4 and oncofetal bladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neonatal glycoprotein, sphingolipid, breast cancer antigens (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16, and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens (such as I antigen found in fetal red blood cells; primary endoderm I antigen found in adult red blood cells; I(Ma) found in pre-transplant embryos or gastric cancer; M18 found in mammary epithelium; M39, SSEA-1 found in bone marrow cells; VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer; TRA-1-85 (blood group H), SCP-1 found in testicular and ovarian cancer; C14 found in colon cancer; F3 found in lung cancer; AH6 found in gastric cancer; Y hapten, Ley found in embryonal carcinoma cells; TL5 (blood group A), EGF receptor found in A431 cells; E1 series (blood group B) found in pancreatic cancer; FC10. found in embryonal carcinoma cells.2; gastric cancer antigen; CO-514 (blood group Lea) found in adenocarcinoma; NS-10 found in adenocarcinoma; CO-43 (blood group Leb), G49 found in the EGF receptor of A431 cells; MH2 (blood group ALeb / Ley) found in colon cancer; 19.9 found in colon cancer; gastric mucin; T5A7 found in bone marrow cells; R24 found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2 and M1:22:25:8 found in embryonal carcinoma cells; SSEA-3 and SSEA-4), skin T cell lymphoma-associated antigen, MART-1 antigen, sialylated Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic associated brain-testicular-carcinoma antigen (tumor neuron antigen MA2 and paraneoplastic neuron antigen), neuro-oncological ventral antigen 2 (NOVA2), hematopoietic cell carcinoma antigen gene 520, tumor associated antigen CO-029, tumor associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b MAGE-X2, cancer / testis antigen (NY-EOS-1), YKL-40 and any fragment of these polypeptides and their modified structures (the above-mentioned modified phosphate groups, sugar chains, etc.), EpCAM, EREG, CA19-9, CA15-3, sialylated SSEA-1 (SLX), HER2, PSMA, CEA and CLEC12A.

[0760] In a preferred embodiment, the third antigen is glypican 3 (GPC3). In yet another embodiment, the third antigen is DLL3 (delta-like 3). In certain embodiments, the third antigen binding domain can be a "DLL3 antigen binding domain" that binds to DLL3.

[0761] Unless otherwise indicated, as used herein, the term “DLL3” refers to any native DLL3 (delta-like 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses “full-length” unprocessed DLL3, as well as any form of DLL3 produced by processing in cells. The term also encompasses naturally occurring variants of DLL3, such as splice variants or allelic variants. The amino acid sequence of exemplary human DLL3 is designated as NCBI Reference Sequence (RefSeq) NM_016941.3, and the amino acid sequence of exemplary cynomolgus monkey DLL3 is designated as NCBI Reference Sequence XP_005589253.1, and the amino acid sequence of exemplary mouse DLL3 is designated as NCBI Reference Sequence NM_007866.2.

[0762] The human DLL3 protein contains a transmembrane (TM) region and an intracellular domain on the C-terminal side, and a DSL (Notch) domain on the N-terminal side. Additionally, DLL3 has an EGF domain containing six regions (EGF1 to EGF6 from the N-terminal side to the C-terminal side).

[0763] In some embodiments, the DLL3 antigen-binding domain specifically binds to the extracellular domain of DLL3. In some embodiments, the DLL3 antigen-binding domain specifically binds to an epitope within the extracellular domain of DLL3. In some embodiments, the DLL3 antigen-binding domain binds to the DLL3 protein expressed on the surface of eukaryotic cells. In some embodiments, the DLL3 antigen-binding domain binds to the DLL3 protein expressed on the surface of cancer cells.

[0764] In some embodiments, the DLL3 antigen-binding domains in the present disclosure bind to epitopes within the extracellular domain (ECD) (i.e., the domain from the N-terminus to the domain immediately preceding the TM region), but do not bind to the TM region or the C-terminal intracellular domain. The DLL3 antigen-binding domains in the present disclosure can bind to epitopes within any of the above domains / regions within the ECD. In a preferred embodiment, the DLL3 antigen-binding domains in the present disclosure bind to epitopes within the region from EGF6 to the region immediately preceding the TM region. More specifically, the DLL3 antigen-binding domains in the present disclosure can bind to epitopes within the region defined in SEQ ID NO:21 in human DLL3. In some embodiments, the DLL3 antigen-binding domains in the present disclosure bind to the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3 or the region from EGF6 to the region immediately preceding the TM region, or bind to epitopes within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3 or the region from EGF6 to the region immediately preceding the TM region. In some embodiments, the antigen-binding molecule or the DLL3 antigen-binding domain can be derived from previously reported anti-DLL3 antibodies (such as WO2019131988 and WO2011093097) in which the bound DLL3 epitopes have been characterized.

[0765] In human DLL3, the above domains / regions have the following amino acid residues (see, for example, http: / / www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746):

[0766] Extracellular domain (ECD): Amino acid residues at positions 1 to 492;

[0767] DSL domain: Amino acid residues at positions 176 to 215;

[0768] EGF domain: Amino acid residues at positions 216 to 465;

[0769] EGF1 region: Amino acid residues at positions 216 to 249;

[0770] EGF2 region: Amino acid residues at positions 274 to 310;

[0771] EGF3 region: Amino acid residues at positions 312 to 351;

[0772] EGF4 region: Amino acid residues at positions 353 to 389;

[0773] EGF5 region: Amino acid residues at positions 391 to 427;

[0774] EGF6 region: Amino acid residues at positions 429 to 465;

[0775] Region from EGF6 to the region immediately before the TM region: Amino acid residues at positions 429 to 492;

[0776] TM region: Amino acid residues at positions 493 to 513; and

[0777] C-terminal intracellular domain: Amino acid residues at positions 516 to 618 (or 516 to 587 in some isoforms). The above amino acid positions also refer to the amino acid positions in the amino acid sequence shown in SEQ ID NO:22.

[0778] Thus, the antigen-binding molecules in the present disclosure can bind to the above regions / domains in human DLL3 having amino acid residues at the above positions. That is, the antigen-binding molecules in the present disclosure can bind to epitopes within the above regions / domains in human DLL3 having amino acid residues at the above positions.

[0779] The DLL3 protein used in the present disclosure is not limited by its source, and is preferably a human or cynomolgus monkey DLL3 protein.

[0780] In some embodiments, for the DLL3 protein, a DLL3 ECD fragment protein (or ECD variant) can be used. Depending on the truncation site, the fragment / variant can include, from the N-terminal side to the C-terminal side, the DSL domain to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant can further include the region spanning from immediately after the EGF6 region to immediately before the TM region. A Flag tag can be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[0781] The CD3, CD137, or DLL3 protein in the present disclosure can be a protein having the above sequence, or can be a modified protein having a sequence derived from the above sequence by modification of one or more amino acids. Examples of modified proteins having a sequence derived from the above sequence by modification of one or more amino acids can include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more identity with the above amino acid sequence. Alternatively, partial peptides of these CD3, CD137, or DLL3 proteins can be used.

[0782] In certain embodiments, the antigen-binding molecules described herein bind to epitopes of CD3, CD137, or DLL3 that are conserved in CD3, CD137, or DLL3 from different species. In certain embodiments, the antigen-binding molecules in the present disclosure are trispecific antigen-binding molecules, i.e., the trispecific antigen-binding molecules are capable of specifically binding to three different antigens, capable of binding to one of CD3 or CD137, but not binding to both antigens simultaneously, and capable of specifically binding to DLL3.

[0783] In one aspect, the third antigen-binding domain is a conventional Fab molecule, and

[0784] (a) The first polypeptide comprises (from the N-terminus to the C-terminus) the VH of the third antigen-binding domain, the heavy-chain constant region (CH1); and the VH of the first antigen-binding domain, the heavy-chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3);

[0785] (b) The second polypeptide comprises (from the N-terminus to the C-terminus) the VL and the light-chain constant region (CL) of the third antigen-binding domain;

[0786] (c) The third polypeptide comprises (from the N-terminus to the C-terminus) the VH of the second antigen-binding domain, the heavy-chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3);

[0787] (d) The fourth polypeptide comprises (from the N-terminus to the C-terminus) the VL and the light-chain constant region (CL) of the second antigen-binding domain; and

[0788] (e) The fifth polypeptide comprises (from the N-terminus to the C-terminus) the VL and the light-chain constant region (CL) of the first antigen-binding domain.

[0789] In one aspect, the third antigen-binding domain is a VH / VL cross-Fab (where the variable regions of the Fab light chain and the Fab heavy chain are exchanged), and

[0790] (a) The first polypeptide comprises (from the N-terminus to the C-terminus) the VL of the third antigen-binding domain, the heavy-chain constant region (CH1); and the VH of the first antigen-binding domain, the heavy-chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3);

[0791] (b) The second polypeptide comprises (from the N-terminus to the C-terminus) the VH and the light-chain constant region (CL) of the third antigen-binding domain;

[0792] (c) The third polypeptide comprises (from the N-terminus to the C-terminus) the VH of the second antigen-binding domain, the heavy-chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3);

[0793] (d) The fourth polypeptide comprises (from the N-terminus to the C-terminus) the VL and the light chain constant region (CL) of the second antigen-binding domain; and

[0794] (e) The fifth polypeptide comprises (from the N-terminus to the C-terminus) the VL and the light chain constant region (CL) of the first antigen-binding domain.

[0795] In one aspect, the antigen-binding molecule in the present disclosure may further comprise an Fc region. In one aspect, the Fc region is composed of a first Fc region subunit and a second Fc region subunit that can stably associate.

[0796] In one aspect, in the antigen-binding molecule in the present disclosure, each of the first antigen-binding domain and the second antigen-binding domain is a Fab, wherein the first antigen-binding domain is fused to the N-terminus of the first or second subunit of the Fc region at the C-terminus of the Fab heavy chain, and the second antigen-binding domain is fused to the N-terminus of the remaining subunit of the Fc region at the C-terminus of the Fab heavy chain.

[0797] In one aspect, the Fc region in the present disclosure may be derived from human. In some embodiments, the Fc region in the present disclosure may be an IgG Fc region, preferably a human IgG Fc region, more preferably a human IgG1 Fc region.

[0798] In certain embodiments, the Fc region of the antigen-binding molecule is composed of a first Fc region subunit and a second Fc region subunit that can stably associate, and wherein the Fc region exhibits a reduced binding affinity for human Fcγ receptors as compared to the native human IgG1 Fc region.

[0799] In a particular embodiment, the Fc region of the antigen-binding molecule described herein comprises a modification that promotes the association of the first and second subunits of the Fc region. The modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc region and a "hole" modification in the other of the two subunits of the Fc region, as described in more detail below.

[0800] The technology of the pestle and mortar structure is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("pestle") at the interface of a first polypeptide and introducing a corresponding cavity ("mortar") in the interface of a second polypeptide such that the protrusion can be positioned in the cavity to facilitate the formation of heterodimers and impede the formation of homodimers. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine).

[0801] Thus, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain of an antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion within the CH3 domain of the first subunit, which protrusion can be positioned in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit, into which the protrusion within the CH3 domain of the first subunit can be positioned.

[0802] The protrusion and the cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0803] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), while in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0804] In yet another embodiment, additionally in the first subunit of the Fc domain, the serine residue at position 354 is replaced with a cysteine residue (S354C), and additionally in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0805] In a specific embodiment, in an Fc domain composed of a first Fc region subunit and a second Fc region subunit that can stably associate and exhibit a reduced binding affinity for human Fc-γ receptors compared to the native human IgG1 Fc domain, the first Fc region subunit is selected from the group consisting of:

[0806] (a1) an Fc region polypeptide comprising the mutations L234A, L235A;

[0807] (a2) an Fc region polypeptide comprising the mutations L234A, L235A, N297A; and

[0808] (a3) an Fc region polypeptide comprising the mutations L234A, L235A, N297A, S354C, T366W; and

[0809] The second Fc region polypeptide is selected from the group consisting of:

[0810] (a4) an Fc region polypeptide comprising the mutations L234A, L235A;

[0811] (a5) an Fc region polypeptide comprising the mutations L234A, L235A, N297A; and

[0812] (a6) an Fc region polypeptide comprising the mutations L234A, L235A, N297A, Y349C, T366S, L368A, Y407V (amino acid positions are numbered using the EU index numbering).

[0813] In certain embodiments, compared to the Fc region of native IgG, the Fc region of the antigen-binding molecule described herein exhibits enhanced FcRn binding activity at acidic pH conditions (e.g., pH 5.8). Such an Fc domain comprises: Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering.

[0814] In some embodiments, the Fc domain comprises: Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering.

[0815] In some embodiments, the Fc domain further comprises: Ile or Leu at position 428; and / or Ile, Leu, Val, Thr or Phe at position 436, according to EU numbering.

[0816] In an embodiment, the Fc domain comprises a combination of amino acid substitutions selected from the group consisting of:

[0817] (a) N434A / Q438R / S440E;

[0818] (b) N434A / Q438R / S440D;

[0819] (c) N434A / Q438K / S440E;

[0820] (d) N434A / Q438K / S440D;

[0821] (e) N434A / Y436T / Q438R / S440E;

[0822] (f) N434A / Y436T / Q438R / S440D;

[0823] (g) N434A / Y436T / Q438K / S440E;

[0824] (h) N434A / Y436T / Q438K / S440D;

[0825] (i) N434A / Y436V / Q438R / S440E;

[0826] (j) N434A / Y436V / Q438R / S440D;

[0827] (k) N434A / Y436V / Q438K / S440E;

[0828] (l) N434A / Y436V / Q438K / S440D;

[0829] (m) N434A / R435H / F436T / Q438R / S440E;

[0830] (n) N434A / R435H / F436T / Q438R / S440D;

[0831] (o) N434A / R435H / F436T / Q438K / S440E;

[0832] (p) N434A / R435H / F436T / Q438K / S440D;

[0833] (q) N434A / R435H / F436V / Q438R / S440E;

[0834] (r) N434A / R435H / F436V / Q438R / S440D;

[0835] (s) N434A / R435H / F436V / Q438K / S440E;

[0836] (t) N434A / R435H / F436V / Q438K / S440D;

[0837] (u) M428L / N434A / Q438R / S440E;

[0838] (v) M428L / N434A / Q438R / S440D;

[0839] (w) M428L / N434A / Q438K / S440E;

[0840] (x) M428L / N434A / Q438K / S440D;

[0841] (y) M428L / N434A / Y436T / Q438R / S440E;

[0842] (z) M428L / N434A / Y436T / Q438R / S440D;

[0843] (aa) M428L / N434A / Y436T / Q438K / S440E;

[0844] (ab) M428L / N434A / Y436T / Q438K / S440D;

[0845] (ac) M428L / N434A / Y436V / Q438R / S440E;

[0846] (ad) M428L / N434A / Y436V / Q438R / S440D;

[0847] (ae) M428L / N434A / Y436V / Q438K / S440E;

[0848] (af)M428L / N434A / Y436V / Q438K / S440D;

[0849] (ag)L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[0850] (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU numbering.

[0851] In some embodiments, the Fc region of the antigen-binding molecule comprises a combination of amino acid substitutions of M428L / N434A / Q438R / S440E.

[0852] In one aspect, the Fc region of the present disclosure may comprise a combination of one or more amino acid substitutions that promote multimerization of the Fc region. Examples of amino acid substitutions that promote multimerization include amino acid substitutions at at least one site selected from the group consisting of positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, 385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447 according to EU numbering (see, e.g., WO2016 / 164480). In specific embodiments, examples of multimers include, but are not limited to, dimers, trimers, and tetramers.

[0853] In one aspect, the antigen-binding molecule of the present disclosure may have an amino acid residue resulting from the substitution of at least one of the cysteine residues in its hinge region. In some embodiments, the cysteine residue may be present at position 226 and / or 229 according to EU numbering of the hinge region.

[0854] In embodiments, the Fc region comprises any of the following:

[0855] (a) a first Fc subunit comprising the amino acid sequence shown in SEQ ID NO:23 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO:24;

[0856] (b) a first Fc subunit comprising the amino acid sequence shown in SEQ ID NO:25 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO:26; or

[0857] (c) a first Fc region subunit comprising the amino acid sequence shown in SEQ ID NO:58 and a second Fc region subunit comprising the amino acid sequence shown in SEQ ID NO:59;

[0858] In one aspect, the antigen-binding molecules of the present disclosure are multispecific antigen-binding molecules. In some embodiments, the multispecific antigen-binding molecules are bispecific antigen-binding molecules or trispecific antigen-binding molecules.

[0859] In one aspect, the antigen-binding molecules of the present disclosure are antibodies. In specific embodiments, the antibodies of the present disclosure are IgG antibodies, preferably IgG1, IgG2, IgG3, or IgG4 antibodies.

[0860] IV. Disulfide bond

[0861] In the present disclosure, at least one disulfide bond in a region other than the hinge region can be described by the abbreviated term "LINC". Using this abbreviation, in some embodiments, the antigen-binding molecules in the present disclosure can be expressed as, for example, "bis / LINC", "DLL3-bis / LINC", "paired cysteine form", etc. Antigen-binding molecules in which the first antigen-binding domain and the second antigen-binding domain are not connected / have not been connected via at least one disulfide bond can be described by the abbreviated terms "unLINC" or "bis-LINC-Ig with unpaired cysteines", etc.

[0862] In one aspect of the present invention, each of the first antigen-binding domain and the second antigen-binding domain contains at least one cysteine residue (by mutation, substitution, or insertion) in a region other than the hinge region. In the "LINC" antigen-binding molecules in the present disclosure, at least one cysteine residue forms at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain.

[0863] In embodiments of the above aspect, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain can hold the two antigen-binding domains (i.e., the first antigen-binding domain and the second antigen-binding domain as described above) in spatially close positions. By virtue of the connection between the first antigen-binding domain and the second antigen-binding domain via a disulfide bond, the antigen-binding molecules in the present disclosure are capable of holding the two antigen-binding domains in a closer position than a control antigen-binding molecule, which differs from the antigen-binding molecules in the present disclosure only in that the control antigen-binding molecule does not introduce an additional bond between the two antigen-binding domains. In some embodiments, the term "spatially close position" or "closer position" includes the meaning that the first antigen-binding domain and the second antigen-binding domain as described above maintain a shortened distance and / or reduced flexibility.

[0864] Thus, the two antigen-binding domains of the antigen-binding molecule in the present disclosure (i.e., the first antigen-binding domain and the second antigen-binding domain as described above) can bind to antigens expressed on the same single cell. In other words, the corresponding two antigen-binding domains of the antigen-binding molecule in the present disclosure (i.e., the first antigen-binding domain and the second antigen-binding domain as described above) do not bind to antigens expressed on different cells to cause cross-linking of different cells. Such an antigen-binding mode of the antigen-binding molecule in the present disclosure can be referred to as "cis-binding", while the antigen-binding mode of the antigen-binding molecule in which the corresponding two antigen-binding domains of the antigen-binding molecule bind to antigens expressed on different cells, thereby causing cross-linking of different cells, can be referred to as "trans-binding". In some embodiments, the antigen-binding molecule in the present disclosure mainly binds to antigens expressed on the same single cell in a "cis-binding" manner.

[0865] In embodiments of the above aspect, by virtue of the disulfide linkage via a disulfide bond between the first antigen-binding domain and the second antigen-binding domain as described above, the antigen-binding molecule in the present disclosure can reduce and / or prevent unwanted cross-linking and activation of immune cells (e.g., T cells, NK cells, DC cells, etc.). That is, in some embodiments, the first antigen-binding domain of the antigen-binding molecule in the present disclosure binds to any signaling molecule (e.g., the first antigen) expressed on an immune cell (such as a T cell), and the second antigen-binding domain of the antigen-binding molecule in the present disclosure also binds to any signaling molecule (e.g., the first antigen or a second antigen different from the first antigen) expressed on an immune cell (such as a T cell). Thus, the first antigen-binding domain and the second antigen-binding domain of the antigen-binding molecule in the present disclosure can bind to either the first or second signaling molecule expressed on the same single immune cell (such as a T cell) (i.e., in a cis-binding manner), or to either the first or second signaling molecule expressed on different immune cells (such as T cells) (i.e., in a trans-binding manner). When the first antigen-binding domain and the second antigen-binding domain bind to signaling molecules expressed on different immune cells (such as T cells) in a trans-binding manner, those different immune cells (such as T cells) cross-link, and in some cases, such cross-linking of immune cells (such as T cells) may lead to unwanted activation of immune cells (such as T cells).

[0866] On the other hand, in the case of another embodiment of the antigen-binding molecule in the present disclosure, both the first antigen-binding domain and the second antigen-binding domain can bind to a signaling molecule expressed on the same single immune cell (such as a T cell) in a "cis-binding" manner, thereby reducing the cross-linking of different immune cells (such as T cells) via the antigen-binding molecule to avoid unwanted activation of immune cells.

[0867] In an embodiment of the above aspect, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain can be formed by connecting amino acid residues present at the same position in the first antigen-binding domain and the second antigen-binding domain to each other, or can be formed by connecting amino acid residues present at different respective positions to each other.

[0868] The positions of the amino acid residues in the antigen-binding domain can be shown according to the Kabat numbering or the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. For example, if the amino acid residues involved in the bond between the first antigen-binding domain and the second antigen-binding domain are present at corresponding same positions in the antigen-binding domain, the positions of these amino acid residues can be represented by the same number according to the Kabat numbering or the EU numbering system. Alternatively, if the amino acid residues involved in the bond between the first antigen-binding domain and the second antigen-binding domain are present at different non-corresponding positions in the antigen-binding domain, the positions of these amino acid residues can be represented by different numbers according to the Kabat numbering or the EU numbering system.

[0869] The amino acid residues from which the disulfide bond between the antigen-binding domains is derived are present in the first antigen-binding domain and the second antigen-binding domain, respectively, and the bond between the antigen-binding domains is formed by linking these amino acid residues. In an embodiment of the above aspect, at least one of the amino acid residues from which the disulfide bond between the antigen-binding domains is derived is an artificially mutated, substituted, introduced, or engineered mutant amino acid residue. For example, the amino acid residue is an artificially introduced cysteine residue. Such a mutant amino acid residue can be introduced into the wild-type antigen-binding domain by methods such as amino acid substitution. In some embodiments, at least one disulfide bond is formed between an amino acid residue in the heavy chain of the first antigen-binding domain and an amino acid residue in the heavy chain of the second antigen-binding domain, or is formed between an amino acid residue in the light chain of the first antigen-binding domain and an amino acid residue in the light chain of the second antigen-binding domain. In additional embodiments, the disulfide bond is formed between amino acid residues in any combination of the CH1 region, CL region, VH region, VL region, and VHH region of the first antigen-binding domain and the CH1 region, CL region, VH region, VL region, and VHH region of the second antigen-binding domain.

[0870] In one aspect, at least one disulfide bond is formed between 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 the second antigen-binding domain are each present at any one of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219 in the CH1 region according to EU numbering. In certain embodiments, the amino acid residue 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 in the CH1 region according to EU numbering. In certain embodiments, the amino acid residue is present at positions 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 in the CH1 region according to EU numbering. In certain embodiments, the amino acid residue is present at positions 135, 136, or 191 in the CH1 region according to EU numbering.

[0871] In embodiments of the above aspects, at least one disulfide bond is formed between 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 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 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. 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 148, 149, and 150 according to EU 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 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 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 amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to EU 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 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 the second antigen-binding domain are each independently selected from the group consisting of positions 218 and 219 according to EU numbering.

[0872] In embodiments of the above aspects, the positional difference (i.e., the distance therebetween) of the corresponding amino acid residues forming a disulfide bond in the first antigen-binding domain and the second antigen-binding domain is three amino acids or less (i.e., three amino acids, two amino acids, or one amino acid). This means that when comparing the positions of the amino acid residues forming a disulfide bond in the CH1 region of the first antigen-binding domain and the positions of the amino acid residues forming a disulfide bond in the CH1 region of the second antigen-binding domain according to EU numbering, the difference (i.e., the distance) is three amino acids or less. In certain embodiments, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residue at position 135 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at any one of positions 132 to 138 according to EU numbering in the CH1 region of the second antigen-binding domain. In certain embodiments, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residue at position 136 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at any one of positions 133 to 139 according to EU numbering in the CH1 region of the second antigen-binding domain.

[0873] In certain embodiments, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between 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 any one of positions 188 to 194 according to EU numbering in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residues at position 135 according to EU numbering in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residues at position 136 according to EU numbering in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residues at position 191 according to EU numbering in the CH1 regions of the two antigen-binding domains.

[0874] In some embodiments, the antigen-binding molecule in the present disclosure comprises one, two, or more additional disulfide bonds between the first antigen-binding domain and the second antigen-binding domain. The additional one, two, or more disulfide bonds are formed between the first antigen-binding domain and the second antigen-binding domain via the amino acid residues at the following positions according to EU numbering in each of the corresponding CH1 regions of the first antigen-binding domain and the second antigen-binding domain:

[0875] between the amino acid residues at any position of 131 to 138, 194, and 195 in each of the two antigen-binding domains;

[0876] (b) between the amino acid residues at position 131 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0877] (c) between the amino acid residues at position 132 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0878] (d) between the amino acid residues at position 133 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0879] (e) between the amino acid residues at position 134 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0880] (f) between the amino acid residues at position 135 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0881] (g) between the amino acid residues at position 136 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0882] (h) between the amino acid residues at position 137 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0883] (i) between the amino acid residues at position 138 in each of the two antigen-binding domains, and between the amino acid residues at position 194 in each of the two antigen-binding domains;

[0884] (j) between the amino acid residues at position 131 in each of the two antigen-binding domains, and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0885] (k) between the amino acid residues at position 132 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0886] (l) between the amino acid residues at position 133 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0887] (m) between the amino acid residues at position 134 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0888] (n) between the amino acid residues at position 135 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0889] (o) between the amino acid residues at position 136 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains;

[0890] (p) between the amino acid residues at position 137 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains; and

[0891] (q) between the amino acid residues at position 138 in each of the two antigen-binding domains and between the amino acid residues at position 195 in each of the two antigen-binding domains.

[0892] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more charged amino acid residues at positions 136 to 138 (according to EU numbering) in the corresponding CH1 region; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more amino acid residues with opposite charges at positions 193 to 195 (according to EU numbering) in the corresponding CH1 region.

[0893] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain comprises one, two, or more positively charged amino acid residues at positions 136 to 138 (according to EU numbering) in the corresponding CH1 region; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain comprises one, two, or more negatively charged amino acid residues at positions 193 to 195 (according to EU numbering) in the corresponding CH1 region.

[0894] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain comprises one, two, or more negatively charged amino acid residues at positions 136 to 138 (according to EU numbering) in the corresponding CH1 region; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain comprises one, two, or more positively charged amino acid residues at positions 193 to 195 (according to EU numbering) in the corresponding CH1 region.

[0895] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain comprises one, two, or more of the following amino acid residues in the corresponding CH1 region (according to EU numbering):

[0896] (a) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 136;

[0897] (b) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 137;

[0898] (c) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 138; and

[0899] the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain comprises one, two, or more of the following amino acid residues in the corresponding CH1 region (according to EU numbering):

[0900] (d) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 193;

[0901] (e) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 194; and

[0902] (f) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 195.

[0903] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain comprises one or more of the following amino acid residues in the corresponding CH1 region (according to EU numbering):

[0904] (a) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 136;

[0905] (b) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 137;

[0906] (c) an amino acid residue having lysine (K), arginine (R), or histidine (H) at position 138; and

[0907] the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain comprises one or more of the following amino acid residues in the corresponding CH1 region (according to EU numbering):

[0908] (d) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 193;

[0909] (e) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 194; and

[0910] (f) an amino acid residue having glutamic acid (E) or aspartic acid (D) at position 195.

[0911] In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain comprises any one of the combinations of specific charge mutations in the corresponding CH1 region (according to EU numbering) listed in Table 1, Table 2, or Table 3.

[0912] [Table 1]

[0913]

[0914]

[0915]

[0916]

[0917] [Table 2]

[0918]

[0919]

[0920] [Table 3]

[0921]

[0922] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more hydrophobic amino acid residues at positions 136 to 138 (according to EU numbering) in their respective CH1 regions; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more hydrophobic amino acid residues at positions 193 to 195 (according to EU numbering) in their respective CH1 regions.

[0923] In some embodiments of the above aspects, the hydrophobic amino acid residues are alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and / or tryptophan (Trp).

[0924] In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain contains any one of the combinations of hydrophobic amino acid mutations in the corresponding CH1 regions (according to EU numbering) listed in Table 4.

[0925] [Table 4]

[0926]

[0927]

[0928] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain contains a "pestle" amino acid residue at positions 136 to 138 (according to EU numbering) in their respective CH1 regions; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more "mortar" amino acid residues at positions 193 to 195 (according to EU numbering) in their respective CH1 regions.

[0929] In some embodiments of the above aspects, either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more "mortar" amino acid residues at positions 136 to 138 (according to EU numbering) in their respective CH1 regions; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains a "pestle" amino acid residue at positions 193 to 195 (according to EU numbering) in their respective CH1 regions.

[0930] In some embodiments, the "pestle" amino acid residues are selected from the group consisting of tryptophan (Trp) and phenylalanine (Phe); and the "mortar" amino acid residues are selected from the group consisting of alanine (Ala), valine (Val), threonine (Thr), and serine (Ser).

[0931] In some embodiments of the above aspect, either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more aromatic amino acid residues at positions 136 to 138 (according to EU numbering) in the corresponding CH1 region; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more positively charged amino acid residues at positions 193 to 195 (according to EU numbering) in the corresponding CH1 region.

[0932] In some embodiments of the above aspect, either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more positively charged amino acid residues at positions 136 to 138 (according to EU numbering) in the corresponding CH1 region; and the other antigen-binding domain of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more aromatic amino acid residues at positions 193 to 195 (according to EU numbering) in the corresponding CH1 region.

[0933] In some embodiments, the aromatic amino acid residues are selected from the group consisting of tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe); and the positively charged amino acid residues are selected from the group consisting of lysine (Lys), arginine (Arg), and histidine (His).

[0934] In one aspect, at least one disulfide bond is formed between 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. The amino acid residue is present, for example, at any one of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213 in the CL region according to Kabat numbering. 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 in the CL region according to Kabat numbering. In certain embodiments, the amino acid residue is present at position 126 in the CL region according to Kabat numbering.

[0935] In one embodiment of the above aspect, at least one disulfide bond is formed between 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 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 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 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 the second antigen-binding domain are each independently selected from the group consisting of positions 200, 201, 202, and 203 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 208, 209, 210, 211, 212, and 213 according to Kabat numbering.

[0936] In one embodiment of the above aspect, the difference in the positions (i.e., the distance therebetween) of the amino acid residues forming each of the disulfide bonds in the first antigen-binding domain and the second antigen-binding domain is within three amino acids (i.e., three amino acids, two amino acids, or one amino acid). This means that when comparing the positions of the amino acid residues forming the disulfide bond in the CL region of the first antigen-binding domain and the positions of the amino acid residues forming the disulfide bond in the CL region of the second antigen-binding domain according to EU numbering, the difference (i.e., the distance) is within three amino acids. In an exemplary embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed by connecting the amino acid residues at position 126 according to Kabat numbering in the CL regions of the two antigen-binding domains to each other.

[0937] In embodiments of the above aspects, at least one disulfide bond is formed between 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 residue in the CH1 region of the first antigen-binding domain is selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to EU numbering, and the amino acid residue in the CL region of the second antigen-binding domain is selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 according to Kabat numbering. In an exemplary embodiment, at least one disulfide bond is formed between 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.

[0938] In one aspect, the constant region is of human origin. 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.

[0939] In one aspect, in the constant domain CL of the light chain of each of the first antigen-binding portion and the second antigen-binding portion of the antigen-binding molecule of the present disclosure, the amino acid at position 123 and / or the amino acid at position 124 can be independently substituted with lysine (K), arginine (R), or histidine (H) (numbered according to Kabat). In other aspects, in the constant domain CH1 of the heavy chain of each of the first antigen-binding portion and the second antigen-binding portion of the antigen-binding molecule of the present disclosure, the amino acid at position 147 and / or the amino acid at position 213 can be independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to EU numbering).

[0940] In one aspect, in the constant domain CL of the light chain of each of the first antigen-binding portion and the second antigen-binding portion of the antigen-binding molecule of the present disclosure, the amino acids at positions 123 and 124 can each be arginine (R) and lysine (K) (numbered according to Kabat). In other embodiments, in the constant domain CH1 of the heavy chain of each of the first antigen-binding portion and the second antigen-binding portion of the antigen-binding molecule of the present disclosure, the amino acids at positions 147 and 213 can be glutamic acid (E) (numbered according to EU numbering).

[0941] In one embodiment of the above aspect, the constant region is of human origin. In certain embodiments, the subclass of the CL region is κ or λ.

[0942] In one embodiment of the above aspect, at least one disulfide bond is formed between an amino acid residue in the variable region of the first antigen-binding domain and an amino acid residue in the variable region of the second antigen-binding domain. In certain embodiments, at least one disulfide bond is formed between an amino acid residue in the VH region of the first antigen-binding domain and an amino acid residue in the VH region of the second antigen-binding domain. In certain embodiments, the amino acid residue is present at any position selected, for example, from the group consisting of positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b of the VH region according to EU numbering. In certain embodiments, at least one disulfide bond is formed between an amino acid residue in the VL region of the first antigen-binding domain and an amino acid residue in the VL region of the second antigen-binding domain. In certain embodiments, the aforementioned amino acid residue is present at any position selected, for example, from the group consisting of positions 21, 27, 58, 77, 100, 105, and 107 of the VL region (κ subclass) according to Kabat numbering and positions 6, 19, 33, and 34 of the VL region (λ subclass) according to Kabat numbering.

[0943] In one embodiment of the above aspect, at least one disulfide bond is formed between an amino acid residue in the VHH region of the first antigen-binding domain and an amino acid residue in the VHH region of the second antigen-binding domain. In certain embodiments, the amino acid residue is present at any position selected, for example, from the group consisting of positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 of the VHH region according to Kabat numbering.

[0944] In other embodiments, other techniques for promoting the association of H chains with the desired combination and between L chains and H chains can be applied to the antigen-binding molecules in the present disclosure.

[0945] For example, a technique for suppressing unwanted H chain association by introducing electrostatic repulsion at the interface of the second constant region or the third constant region (CH2 or CH3) of the antibody H chain can be applied to antigen-binding molecule association (WO2006 / 106905).

[0946] In techniques for suppressing undesired H-chain association by introducing electrostatic repulsion at the interface of CH2 or CH3, examples of amino acid residues that come into contact at the interface of another constant region of the H-chain include regions corresponding to the residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the ...

Claims

1. A method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises subjecting a mixture to chromatography in the presence of a reducing agent, the mixture comprising: an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule.

2. A method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, wherein the method comprises: (a) contacting a mixture with a reducing agent in chromatography, the mixture comprising: an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, and a mis-disulfide-bonded form and / or a non-disulfide-bonded form of the antigen-binding molecule; and (b) removing the reducing agent.

3. The method according to claim 1 or 2, wherein the antigen-binding molecule comprises two or more polypeptide chains, and the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the polypeptide chains.

4. The method according to claim 3, wherein either or both of the amino acid residues in the region other than the hinge region are cysteine residues that have been mutated, substituted or introduced.

5. The method according to any one of claims 1 to 4, wherein the chromatography comprises an affinity chromatography matrix, an ion exchange chromatography matrix, a hydrophobic interaction chromatography matrix, a multimodal chromatography matrix comprising both ion exchange chromatography and hydrophobic interaction chromatography, or a hydroxyapatite matrix.

6. The method according to claim 5, wherein the affinity chromatography matrix is selected from the group consisting of a protein A matrix, a protein G matrix, a protein L matrix, a sequence-selective peptide matrix, and a matrix that selectively binds to the antigen-binding molecule.

7. The method according to claim 5, wherein the ion exchange chromatography matrix is a cation exchange ligand or an anion exchange ligand.

8. The method according to claim 5, wherein the hydrophobic interaction chromatography matrix is a hydrophobic ligand.

9. The method according to claim 5, wherein the multimodal chromatography matrix is a matrix having a combination of a cation exchange ligand and a hydrophobic ligand, or a matrix having a combination of an anion exchange ligand and a hydrophobic ligand.

10. The method according to claim 5, wherein the hydroxyapatite chromatography matrix is hydroxyapatite or a derivative thereof.

11. The method according to any one of claims 5 to 10, wherein the chromatography matrix is packed into a column for column chromatography or coated on a membrane for membrane chromatography.

12. The method according to any one of claims 1 to 11, wherein the reducing agent is selected from the group consisting of monothiols, dithiols, phosphines, and inorganic reagents, and combinations of two or more thereof.

13. The method according to any one of claims 1 to 12, wherein the reducing agent is cysteine and / or TCEP.

14. The method according to any one of claims 1 to 13, wherein the reducing agent is TCEP and the concentration of the reducing agent is from 0.00001 mM to 10.0 mM.

15. The method according to any one of claims 1 to 13, wherein the reducing agent is cysteine and the concentration of the reducing agent is from 0.01 mM to 100 mM.

16. The method according to any one of claims 2 to 15, wherein the removal of the reducing agent comprises contacting the antigen-binding molecule with a solution free of the reducing agent.

17. The method according to any one of claims 1 to 16, wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain capable of being linked to each other via at least one disulfide bond.

18. The method according to claim 17, wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the first antigen-binding domain and the second antigen-binding domain.

19. The method according to claim 17 or 18, wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the heavy chain of the first antigen-binding domain and the heavy chain of the second antigen-binding domain.

20. The method according to any one of claims 17 to 19, wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain.

21. The method according to any one of claims 17 to 20, wherein the at least one disulfide bond formed between amino acid residues in a region other than the hinge region is formed between the amino acid residue at EU numbering position 191 in the heavy chain of the first antigen-binding domain and the amino acid residue at EU numbering position 191 in the heavy chain of the second antigen-binding domain.

22. The method according to any one of claims 17 to 21, wherein the first antigen-binding domain and the second antigen-binding domain bind to a first antigen and a second antigen, respectively, the first antigen and the second antigen being proteins present on the surface of a cell, and wherein the antigen-binding molecule has the activity of promoting the interaction between the cell expressing the first antigen and the cell expressing the second antigen.

23. The method according to claim 22, wherein the cell expressing the first antigen is a cell having cytotoxic activity, and the cell expressing the second antigen is a target cell of the cell having cytotoxic activity, and wherein the antigen-binding molecule promotes the damage of the target cell by the cell having cytotoxic activity.

24. The method according to claim 23, wherein the cell having cytotoxic activity is a T cell, an NK cell, a monocyte or a macrophage.

25. The method according to any one of claims 22 to 24, wherein the first antigen and the second antigen are independently selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

26. The method according to any one of claims 17 to 25, wherein the first antigen-binding domain and the second antigen-binding domain are each capable of binding to CD3 and / or CD137.

27. The method according to any one of claims 1 to 26, wherein the antigen-binding molecule further comprises a third antigen-binding domain.

28. The method according to claim 27, wherein the third antigen-binding domain is fused to the first antigen-binding domain or the second antigen-binding domain.

29. The method according to claim 27 or 28, wherein the third antigen-binding domain is a Fab or scFv.

30. The method according to any one of claims 27 to 29, wherein the third antigen-binding domain is optionally fused at its C-terminus via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of the first antigen-binding domain or the second antigen-binding domain.

31. The method according to any one of claims 27 to 30, wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each Fab molecules, and wherein the third antigen-binding domain is optionally fused at the C-terminus of its Fab heavy chain (CH1 region) via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of the first antigen-binding domain or the second antigen-binding domain.

32. The method according to claim 30 or 31, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:

20.

33. The method according to any one of claims 27 to 32, wherein the third antigen-binding domain is a cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the first antigen-binding domain and the second antigen-binding domain are conventional Fab molecules.

34. The method according to any one of claims 27 to 33, wherein the third antigen-binding domain is capable of binding to an antigen expressed on cancer cells or cancer tissues.

35. The method according to any one of claims 27 to 34, wherein the third antigen-binding domain is capable of binding to DLL3, preferably human DLL3.

36. The method according to any one of claims 1 to 35, wherein the antigen-binding molecule further comprises an Fc region.

37. The method according to any one of claims 17 to 36, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region that can be the same as or different from each other, and comprises an antibody variable region independently selected from the group consisting of the following (a1) to (a4): (a1) An antibody variable region comprising: A heavy chain variable region comprising: Heavy chain complementarity determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO: 27, Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 28, and heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 29; and A light chain variable region comprising: Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO: 30, Light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 31, and light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 32; (a2) An antibody variable region comprising: A heavy chain variable region comprising: Heavy chain complementarity determining region (CDR) 1, which comprises the amino acid sequence of SEQ ID NO: 33, Heavy chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 34, and heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 35; and A light chain variable region comprising: Light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO: 30, Light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO: 31, and light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO: 32; (a3) An antibody variable region that binds to an epitope that is the same as the epitope to which the antibody variable region of (a1) or (a2) binds; and (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

38. The method according to any one of claims 17 to 37, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region that can be the same as or different from each other, and comprises an antibody variable region independently selected from the group consisting of the following (a1) to (a4): (a1) An antibody variable region comprising: A heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 36, and A light chain variable region that comprises the amino acid sequence of SEQ ID NO: 37; (a2) An antibody variable region comprising: A heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 38, and A light chain variable region that comprises the amino acid sequence of SEQ ID NO: 37; (a3) An antibody variable region that binds to an epitope that is the same as the epitope to which the antibody variable region of (a1) or (a2) binds; and (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

39. The method according to any one of claims 27 to 38, wherein the third antigen-binding domain comprises an antibody variable region independently selected from the group consisting of the following (a1) to (a4): (a1) An antibody variable region comprising: A heavy chain variable region comprising: A heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 46, A heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 47, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 48; and A light chain variable region comprising: A light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 49, A light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 50, and a light chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 51; (a2) An antibody variable region comprising: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53; (a3) An antibody variable region that binds to the same epitope as the epitope to which the antibody variable region of (a1) or (a2) binds; and (a4) An antibody variable region that competes with the antibody variable region of (a1) or (a2) for binding to the antigen.

40. The method according to any one of claims 17 to 39, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: A heavy chain variable region comprising: A heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 27, A heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 28, and A heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising: A light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 30, A light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 31, and A light chain CDR 3 comprising the amino acid sequence of SEQ ID NO:

32.

41. The method according to any one of claims 17 to 40, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36, and A light chain variable region comprising the amino acid sequence of SEQ ID NO:

37.

42. The method according to any one of claims 27 to 41, wherein the third antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: A heavy chain variable region comprising: A heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 46, A heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 47, and A heavy chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:48; and a light chain variable region, which comprises: A light chain CDR 1, which comprises the amino acid sequence of SEQ ID NO:49, A light chain CDR 2, which comprises the amino acid sequence of SEQ ID NO:50, and A light chain CDR 3, which comprises the amino acid sequence of SEQ ID NO:

51.

43. The method according to any one of claims 27 to 42, wherein the third antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: A heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:52, and A light chain variable region, which comprises the amino acid sequence of SEQ ID NO:

53.

44. The method according to any one of claims 27 to 43, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising: A heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:36, and A light chain variable region, which comprises the amino acid sequence of SEQ ID NO:37, and wherein the third antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: A heavy chain variable region, which comprises SEQ ID NO:52, and A light chain variable region, which comprises SEQ ID NO:

53.

45. The method according to any one of claims 17 to 44, wherein the first antigen-binding domain and the second antigen-binding domain are each a Fab having a cysteine residue at EU numbering position 191 in the heavy chain and having a disulfide bond formed by two cysteine residues.

46. The method according to any one of claims 1 to 45, wherein the antigen-binding molecule comprises any combination of five polypeptide chains selected from the group consisting of: (a1) A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:39 (chain 1), A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:40 (chain 2), A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:41 (chain 3), and two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO:42 (chains 4 and 5); (a2) A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:43 (chain 1), A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:40 (chain 2), A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:44 (chain 3), and two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO:42 (chains 4 and 5); and (a3) A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:45 (chain 1), A polypeptide chain, which comprises the amino acid sequence of SEQ ID NO:40 (chain 2), A polypeptide chain comprising the amino acid sequence of SEQ ID NO:44 (chain 3), and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO:42 (chains 4 and 5); Among them, Preferably, the five polypeptide chains (chains 1 to 5) are linked and / or associated with each other according to the orientation shown in Figure 3.

47. A preparation produced by the method according to any one of claims 1 to 46, the preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

48. A pharmaceutical composition comprising a preparation produced by the method according to any one of claims 1 to 46, wherein the preparation comprises an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

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