Methods for analyzing antibodies

Abstract

In one embodiment, this document provides a method for measuring, determining, or quantifying the ratio of antigen-binding molecules in a composition having at least one disulfide bond formed in a region outside the hinge region to the sum of antigen-binding molecules having at least one disulfide bond formed in a region outside the hinge region and antigen-binding molecules not having disulfide bonds formed in a region outside the hinge region, using the protease FabALACTICA (registered trademark) and electrophoresis and / or chromatography.

Description

Technical Field

[0001] This disclosure relates to a method for producing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. This disclosure also relates to methods for increasing or enriching such antigen-binding molecules, methods for eliminating the heterogeneity of disulfide bonds in antigen-binding molecules, etc. Background Technology

[0002] Antigen-binding molecules with artificial disulfide bonds between amino acid residues in regions outside 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 antibody heavy chains constituting the antigen-binding molecule and the CH1 region of the other antibody heavy chain. However, when such an antigen-binding molecule is obtained by expressing it in cell culture medium, two forms (molecules with appropriate disulfide bonds between heavy chains (LINC form) and molecules without appropriate disulfide bonds, such as mis-disulfide-bonded and non-disulfide-bonded forms of the molecule (unLINC form)) will coexist in the cell culture medium. When preparing LINC-Ig, molecules in the cell culture medium that do not form appropriate disulfide bonds between heavy chains, such as open-type molecules with capped sulfur atoms, must be converted into molecules with appropriate disulfide bonds.

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

[0004] PTL 2 and PTL 4 disclose methods for efficiently obtaining antibodies with LINC, which involve treating a formulation containing molecules with disulfide bonds between heavy chains and molecules that do not form suitable disulfide bonds with a reducing agent, and then re-oxidizing the formulation by means of buffer exchange or the like to form disulfide bonds.

[0005] PTL 5 discloses the use of redox buffers (e.g., cysteine / cystine) to enable reduction-oxidation reactions in downstream processes to prevent antibody fragmentation.

[0006] PTL 6 discloses a method for refolding recombinant antibodies by contacting them with a reducing / oxidative coupling agent.

[0007] NPL 1 discloses the use of redox buffer to suppress batch-to-batch inhomogeneity caused by cysteine ​​esterification of a specific antibody (MAB007).

[0008] Furthermore, since unLINC form molecules can be classified as relatively toxic impurities in pharmaceuticals, methods for analyzing and measuring the amount of unLINC forms are needed as part of quality control. However, to date, no useful methods have been found for measuring, determining, or quantifying the amount or proportion of LINC and / or unLINC form molecules in cell culture media or their purified products (including LINC and unLINC forms).

[0009] [List of Citations]

[0010] [Patent Literature]

[0011] [PTL 1] WO2020 / 027330

[0012] [PTL 2] WO2021 / 157679

[0013] [PTL 3] WO2021 / 200898

[0014] [PTL 4] WO2021 / 201087

[0015] [PTL 5] WO2020 / 037016

[0016] [PTL 6] WO2006 / 047340

[0017] [PTL 7] WO2021 / 201202

[0018] [Non-patent literature]

[0019] [NPL 1] Pharm Sci. 2008 Feb;97(2):775-90, Removal ofCysteinylation from an Unpaired Sulfhydryl in the Variable Region of aRecombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, andBiological Activity

[0020] [NPL 2] Spoerry C et al., (2016) PLoS ONE 11(10): e0164809. doi:10.1371 / journal.pone.0164809 Summary of the Invention

[0021] [Technical Issues]

[0022] As described above, a method is known for efficiently obtaining antigen-binding molecules with LINC by treating a mixture containing antigen-binding molecules with suitable disulfide bonds between heavy chains and antigen-binding molecules without suitable disulfide bonds (e.g., capped molecules) with a reducing agent and then re-oxidizing the mixture. However, this method has problems from the perspective of productivity and ease of manufacture, including low yield and long reaction time.

[0023] To address the aforementioned problems, this disclosure aims to provide a method for the efficient and convenient production and purification of antigen-binding molecules having suitable disulfide bonds between antibody heavy chains. This disclosure relates to a method for increasing the structural homogeneity and relative abundance of antigen-binding molecules having one or more disulfide bonds formed between amino acid residues in regions outside the hinge region at each of the two antibody heavy chains. In other words, this disclosure relates to a method for reducing the relative abundance of antigen-binding molecules that do not have suitable disulfide bonds between amino acid residues in regions outside the hinge region.

[0024] This disclosure also aims to provide a method for measuring, determining, or quantifying the ratio of the LINC form to the sum of the LINC and unLINC forms in a composition comprising the LINC and unLINC forms.

[0025] [Solution to the problem]

[0026] As a result of investigations conducted to address the aforementioned problems, the inventors discovered that by contacting antigen-binding molecules with a reducing agent and then removing the reducing agent, at least one disulfide bond can be formed more effectively and reproducibly between amino acid residues in regions outside the hinge region. The antigen-binding molecules bind to an affinity column and have amino acid residues capable of forming at least one disulfide bond between amino acid residues in regions outside the hinge region (these antigen-binding molecules include those that form suitable disulfide bonds between amino acid residues in regions outside the hinge region, and those that do not form at least one suitable disulfide bond between amino acid residues in regions outside the hinge region). Specifically, the inventors developed a technique to stably obtain high yields of antigen-binding molecules (in the form of LINC-Ig (registered trademark)) having at least one disulfide bond between amino acid residues in regions outside the hinge region (harvested cell culture medium: HCCF) by inserting two steps (flowing a solution containing a reducing agent and removing the reducing agent) into the protein A affinity chromatography purification step (a commonly used technique for primary purification from cell culture medium).

[0027] The inventors have further discovered for the first time that a commercially available enzyme called FabALACTICA (registered trademark) (also known as IgdE) digests human IgG1 at a specific site above the hinge and produces intact and homogeneous Fab and Fc fragments, cleaving the unLINC form of the antigen-binding molecule in the hinge region but not the LINC form, wherein the antigen-binding molecule has cysteine ​​residues introduced into the CH1 region. Based on this discovery, the inventors have developed methods for separating the LINC and unLINC forms using devices such as electrophoresis and chromatography equipment, as well as methods for measuring, determining, or quantifying their content ratio.

[0028] This disclosure is based on these findings and specifically relates to the following:

[0029] [1] A method for producing a formulation comprising an antigen-binding molecule, said antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

[0030] The method includes subjecting a mixture to chromatography in the presence of a reducing agent, wherein the antigen-binding molecule has amino acid residues that can form at least one disulfide bond between amino acid residues in regions other than the hinge region.

[0031] [2] A method for producing 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, wherein the method comprises:

[0032] (a) Contacting an antigen-binding molecule with a solution containing a reducing agent in a chromatogram, said 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

[0033] (b) Remove the reducing agent.

[0034] [3] A method for producing a formulation comprising an antigen-binding molecule, said antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

[0035] The method includes subjecting a mixture to chromatography in the presence of a reducing agent, the mixture comprising:

[0036] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0037] The antigen-binding molecule is in an incorrect disulfide bond form and / or a non-disulfide bond form.

[0038] [4] A method for producing 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, wherein the method comprises:

[0039] (a) Contacting the mixture in chromatography with a solution containing a reducing agent, the mixture comprising:

[0040] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0041] The incorrect disulfide bond and / or non-disulfide bond form of the antigen-binding molecule;

[0042] as well as

[0043] (b) Remove the reducing agent.

[0044] [5] The method according to any one of [1] to [4], wherein the antigen-binding molecule comprises two or more polypeptide chains, and at least one disulfide bond formed between amino acid residues in the region outside the hinge region is formed between the polypeptide chains.

[0045] [6] According to the method of [5], either or both of the polypeptide chains contain mutated, substituted or introduced cysteine ​​residues in a region outside the hinge region.

[0046] [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 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.

[0047] [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 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.

[0048] [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.

[0049]

[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 the region outside the hinge region is formed between an amino acid residue at EU number position 191 in the heavy chain of the first antigen-binding domain and an amino acid residue at EU number position 191 in the heavy chain of the second antigen-binding domain.

[0050]

[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 multimode chromatography matrix comprising both ion exchange chromatography and hydrophobic interaction chromatography, or a hydroxyapatite matrix.

[0051]

[12] According to the method of

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

[0052]

[13] According to the method of

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

[0053]

[14] According to the method of

[11] , the hydrophobic interaction chromatographic matrix is ​​a hydrophobic ligand.

[0054]

[15] According to the method of

[11] , the multimode chromatographic matrix is ​​a matrix having a combination of cation exchange ligands and hydrophobic ligands, or a matrix having a combination of anion exchange ligands and hydrophobic ligands.

[0055]

[16] According to the method of

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

[0056]

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

[16] , wherein the method comprises:

[0057] Contact the mixture with a chromatographic matrix, the mixture comprising:

[0058] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0059] The incorrect disulfide bond and / or non-disulfide bond form of the antigen-binding molecule;

[0060] The chromatographic matrix is ​​loaded into a column for column chromatography or applied to a membrane for membrane chromatography.

[0061]

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

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

[0062]

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

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

[0063]

[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.

[0064]

[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 about 0.01 mM to about 100 mM, about 0.0001 mM to about 100.0 mM, 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.

[0065]

[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.

[0066]

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

[18] , wherein the reducing agent is 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.

[0067]

[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.

[0068]

[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.

[0069]

[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.

[0070]

[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.

[0071]

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

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

[0072]

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

[28] has a pH of about 7.0, about 7.5 or about 8.0 for the solution containing the reducing agent.

[0073]

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

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

[0074]

[31] According to the method of

[30] , the solution containing the reducing agent is passed 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, or the flow is temporarily stopped when the column or device is filled with the reducing agent.

[0075]

[32] According to the method of

[30] or

[31] , the solution containing the reducing agent is passed through a column for column chromatography or a device for membrane chromatography for a residence time of 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.

[0076]

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

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

[0077]

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

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

[0078]

[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.

[0079]

[36] According to the method of

[35] , the chromatography is column chromatography or membrane chromatography, and the contact of the mixture with the solution without reducing agent includes passing the solution without reducing agent through a column for column chromatography or a device for membrane chromatography.

[0080]

[37] The method according to

[35] or

[36] , wherein a solution without reducing agent is passed 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.

[0081]

[38] The method according to any one of

[35] to

[37] , wherein a solution without reducing agent is passed through a column for column chromatography or an apparatus 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 apparatus is filled with the solution.

[0082]

[39] The method according to any one of

[35] to

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

[0083]

[40] The method according to any one of

[35] to

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

[0084]

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

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

[0085]

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

[41] , wherein the method further comprises: prior to step (a),

[0086] Contact the mixture with a chromatographic matrix, the mixture comprising:

[0087] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0088] The incorrect disulfide bond and / or non-disulfide bond form of the antigen-binding molecule;

[0089] This allows for the loading of antigen-binding molecules onto a column used in column chromatography or the immobilization of antigen-binding molecules onto a membrane used in membrane chromatography.

[0090]

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

[42] , wherein the method further comprises: removing impurities in the chromatogram prior to step (a).

[0091]

[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 outside the hinge region before or after step (b).

[0092]

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

[44] , wherein contact between the antigen-binding molecule and the solution containing the reducing agent results in the breaking of disulfide bonds formed between amino acid residues that can form disulfide bonds, and / or the decapping of sulfur atoms in the amino acid residues that can form disulfide bonds.

[0093]

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

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

[0094]

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

[46] , wherein the amino acid residues that can form disulfide bonds are introduced or engineered into cysteine ​​residues.

[0095]

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

[47] , wherein at least one disulfide bond in the region outside the hinge region is an interchain disulfide bond.

[0096]

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

[48] , wherein at least one disulfide bond formed between amino acid residues in the region outside the hinge region is one, two, three, four or more interchain disulfide bonds.

[0097]

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

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

[0098]

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

[50] is used to increase the ratio (LINC ratio) of antigen-binding molecules (LINC form) having at least one disulfide bond in the region outside the hinge region to the sum of the following:

[0099] (i) An antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0100] (ii) Incorrect disulfide-bonded and / or non-disulfide-bonded (unLINC) forms of antigen-binding molecules.

[0101]

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

[51] is used to produce an antigen-binding molecule (LINC form) having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the sum of the following in a molar ratio:

[0102] (i) An antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region and

[0103] (ii) Incorrect disulfide-bonded and / or non-disulfide-bonded (unLINC) forms of antigen-binding molecules.

[0104] This disclosure also relates to the following inventions:

[0105] [A1] The method according to any one of [1] to

[52] , wherein the chromatography is column chromatography.

[0106] [A2] The method according to [A1], wherein the method includes:

[0107] Contact the mixture with a chromatographic matrix, the mixture comprising:

[0108] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0109] The incorrect disulfide bond and / or non-disulfide bond form of the antigen-binding molecule;

[0110] The chromatographic matrix is ​​loaded into the column.

[0111] [A3] The method according to [A1] or [A2], wherein the solution containing the reducing agent is flowed 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.

[0112] [A4] The method according to any one of [A1] to [A3], wherein a solution containing a reducing agent is flowed through a 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 a solution containing the reducing agent.

[0113] [A5] The method according to any one of [A1] to [A4], wherein a solution containing a reducing agent is flowed through a column for a residence time of about 12 minutes, and optionally the flow is temporarily stopped when the column is filled with a solution containing the reducing agent.

[0114] [A6] The method according to any one of [A1] to [A5], wherein a solution containing a reducing agent is flowed 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.

[0115] [A7] The method according to any one of [A1] to [A6], wherein a solution containing a reducing agent is flowed 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.

[0116] [A8] According to any one of [A1] to [A7], the flow volume of the solution containing the reducing agent is from about 0.1 times (about 0.1 CV (column volume)) to about 100 times (about 100 CV) of the column volume.

[0117] [A9] According to any one of [A1] to [A8], the flow volume of the solution containing the reducing agent is about 1 times (1 CV) to about 20 times (about 20 CV) of the column volume.

[0118] [A10] According to any one of [A1] to [A9], the flow volume of the solution containing the reducing agent is about 10.0 times the column volume (about 10.0 CV).

[0119] [A11] The method according to any one of [A1] to [A10], wherein the mixture is contacted with a solution containing the reducing agent for about 4 minutes to about 1440 minutes.

[0120] [A12] The method according to any one of [A1] to [A11], wherein the mixture is contacted with a solution containing the reducing agent for about 8 minutes to about 300 minutes.

[0121] [A13] The method according to any one of [A1] to [A12], wherein the mixture is contacted with a solution containing the reducing agent for about 120 minutes.

[0122] [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 matrix.

[0123] [A15] The method according to any one of [A1] to [A14], wherein a solution without reducing agent is passed through the column for a residence time of about 2 minutes to about 80 minutes, or the flow is temporarily stopped when the column is filled with the solution.

[0124] [A16] The method according to any one of [A1] to [A15], wherein a solution without reducing agent is passed through the column for 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.

[0125] [A17] The method according to any one of [A1] to [A16], wherein a solution without reducing agent is flowed through the column for a residence time of about 4 minutes, or the flow is temporarily stopped when the column is filled with the solution.

[0126] [A18] The method according to any one of [A1] to [A17], wherein a solution without reducing agent is flowed 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, or the flow is temporarily stopped when the column is filled with the solution.

[0127] [A19] The method according to any one of [A1] to [A18], wherein a solution without reducing agent is flowed through a 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.

[0128] [A20] The method according to any one of [A1] to [A19], wherein the flow volume of the solution without 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 times (about 1 CV) to about 20 times (about 20 CV) of the column volume.

[0129] [A21] The method according to any one of [A1] to [A20], wherein the flow volume of the solution without reducing agent is about 5.0 times the column volume (about 5.0 CV).

[0130] [A22] The method according to any one of [A1] to [A21], wherein the mixture is contacted with a solution free of the reducing agent for about 4 minutes to about 1440 minutes.

[0131] [A23] The method according to any one of [A1] to [A22], wherein the mixture is contacted with a solution free of the reducing agent for about 8 minutes to about 300 minutes.

[0132] [A24] The method according to any one of [A1] to [A23], wherein the mixture is contacted with a solution free of the reducing agent for about 20 minutes.

[0133] This disclosure also relates to the following inventions:

[0134] [B1] The method according to any one of [1] to

[52] , wherein the chromatography is membrane chromatography.

[0135] [B2] The method according to [B1], wherein the method includes:

[0136] Contact the mixture with a chromatographic matrix, the mixture comprising:

[0137] An antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0138] The incorrect disulfide bond and / or non-disulfide bond form of the antigen-binding molecule;

[0139] The chromatographic matrix is ​​applied or immobilized onto a membrane used for membrane chromatography.

[0140] [B3] According to the method of [B1] or [B2], wherein the solution containing the reducing agent is allowed to flow 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.

[0141] [B4] The method according to any one of [B1] to [B3], wherein the solution containing the reducing agent is allowed to flow through the membrane device for a residence time of about 3 seconds to about 6 minutes, for example about 30 seconds, or the flow is temporarily stopped when the membrane device is filled with the solution containing the reducing agent.

[0142] [B5] According to any one of [B1] to [B4], the flow volume of the solution containing the reducing agent is from about 1 times the volume of the membrane device (about 1 MV (membrane volume)) to about 500 times the volume of the membrane device (about 500 MV).

[0143] [B6] According to any one of [B1] to [B5], the flow volume of the solution containing the reducing agent is from about 2 times (about 2 MV) to about 100 times (about 100 MV) of the membrane device volume, for example about 15 times (about 15 MV) of the membrane device volume.

[0144] [B7] The method according to any one of [B1] to [B6], wherein the mixture is contacted with a solution containing the reducing agent for about 6 seconds to about 600 minutes.

[0145] [B8] The method according to any one of [B1] to [B7], wherein the mixture is contacted with a solution containing the reducing agent for about 18 seconds to about 120 minutes, for example about 7.5 minutes.

[0146] [B9] The method according to any one of [B1] to [B8], wherein the amount of antigen-binding molecules carried by the matrix is ​​about 5 g to about 100 g or about 7 g to about 70 g per 1 L membrane device volume, for example about 25 g.

[0147] [B10] The method according to any one of [B1] to [B9], wherein a solution without reducing agent is flowed 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.

[0148] [B11] The method according to any one of [B1] to [B10], wherein a solution without reducing agent is flowed through the membrane device for a residence time of about 3 seconds to about 6 minutes, for example about 30 seconds, or the flow is temporarily stopped when the membrane device is filled with the solution.

[0149] [B12] The method according to any one of [B1] to [B11], wherein the flow volume of the solution without reducing agent is from about 1 times (about 1 MV) to about 500 times (about 500 MV) the volume of the membrane device.

[0150] [B13] The method according to any one of [B1] to [B12] wherein the flow volume of the solution without reducing agent is about 2 times (about 2 MV) to about 100 times (about 100 MV) of the membrane device volume, for example about 15 times (about 15 MV) of the membrane device volume.

[0151] [B14] The method according to any one of [B1] to [B13], wherein the mixture is contacted with a solution free of the reducing agent for about 6 seconds to about 600 minutes.

[0152] [B15] The method according to any one of [B1] to [B14], wherein the mixture is contacted with a solution free of the reducing agent for about 18 seconds to about 120 minutes, for example about 7.5 minutes.

[0153] This disclosure also relates to the following inventions:

[101]

[0155] A method for measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules in the form having at least one disulfide bond formed in a region outside the hinge region (LINC form) to the sum of LINC forms and antigen-binding molecules in the form not having a disulfide bond formed in a region outside the hinge region (unLINC form), comprising the following steps:

[0156] (a) Composition (2) is prepared by adding a protease to composition (1) comprising both LINC and unLINC forms; and

[0157] (b) Perform electrophoresis or chromatography on composition (2).

[102]

[0159] According to the method described in

[101] , the protease is an enzyme that does not digest the LINC form but can digest the unLINC form.

[103]

[0161] The method according to

[101] or

[102] , wherein the protease is a cysteine ​​protease capable of digesting human IgG1 antibodies.

[104]

[0163] The method according to any one of

[101] to

[103] , wherein the protease is an enzyme capable of cleaving the hinge region of a human IgG1 antibody.

[105]

[0165] The method according to any one of

[101] to

[104] , wherein the protease is an enzyme that digests human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[106]

[0167] The method according to any one of

[101] to

[105] , wherein the protease is IgdE.

[107]

[0169] According to the method described in

[106] , IgdE is derived from Streptococcus agalactiae.

[108]

[0171] The method according to

[106] or

[107] , wherein IgdE is a protein selected from the group consisting of the following (a) to (d):

[0172] (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 61;

[0173] (b) A protein encoded by the base sequence shown in SEQ ID NO: 62;

[0174] (c) A protein comprising one or more amino acids substituted, deleted, added, and / or inserted relative to the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP; and

[0175] (d) A protein comprising an amino acid sequence having at least 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[109]

[0177] The method according to any one of

[101] to

[108] , wherein the protease is FabALACTICA (registered trademark).

[110]

[0179] According to any one of

[101] to

[109] ,

[0180] Step (a) further includes obtaining a composition (3) containing a protease but not in either the LINC or unLINC form.

[0181] In step (b), each of compositions (1) to (3) is subjected to non-reducing capillary SDS gel electrophoresis (CE-SDS) and electrophoretic images (1) to (3) of compositions (1) to (3) are obtained respectively.

[0182] The electrophoresis diagram (1) includes: peaks originating from unLINC form without protease digestion and peaks originating from LINC form, as well as peaks that are neither originating from unLINC form without protease digestion nor from LINC form (peak Z).

[0183] The electrophoresis diagram (2) includes: peaks originating from the LINC form (peak L), peaks originating from the unLINC form after protease digestion (peak unL), peaks originating from the protease (peak E), and peaks that are neither originating from the unLINC form after protease digestion, nor from the LINC form, nor from the protease (peak Z).

[0184] Electrophoresis diagram (3) includes a peak (peak E) derived from the protease.

[111]

[0186] The method according to

[110] further includes:

[0187] (c) Calculate the ratio (%) using the following formula (I);

[0188]

[0189] in,

[0190] A represents the area of ​​peak L in electrophoresis diagram (2).

[0191] B is the sum of the area values ​​of peaks unL, E, and Z in electrophoresis diagram (2).

[0192] C is the area value of peak Z in electrophoresis diagram (1), and

[0193] D is the area value of peak E in electrophoresis diagram (3).

[112]

[0195] According to any one of

[101] to

[109] , wherein step (b) involves performing hydrophobic interaction chromatography (HIC) on the composition (2) and obtaining a chromatogram.

[113]

[0197] The method according to

[112] further includes:

[0198] (c) Calculate the ratio (%) using the following formula (II);

[0199]

[0200] in,

[0201] A represents the area of ​​the peak originating from the LINC form in the chromatogram; and

[0202] B is the sum of the area values ​​of all peaks in the chromatogram originating from the unLINC form after protease digestion.

[0203] This disclosure also relates to the following inventions:

[0204]

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

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

[101] to

[113] , wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain that can be linked to each other via at least one disulfide bond.

[0205]

[202] According to the method of

[201] , the first antigen-binding domain and the second antigen-binding domain each comprise a Fab, Fab', scFab, Fv, scFv or VHH structure.

[0206]

[203] According to the method of

[201] or

[202] , wherein the first antigen-binding domain and the second antigen-binding domain each include or do not include a hinge region.

[0207]

[204] The method according to any one of

[201] to

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

[0208]

[205] The method according to any one of

[201] to

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

[0209]

[206] The method according to any one of

[201] to

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

[0210]

[207] The method according to any one of

[201] to

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

[0211]

[208] The method according to any one of

[201] to

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

[0212]

[209] The method according to any one of

[201] to

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

[0213]

[210] The method according to any one of

[201] to

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

[0214]

[211] The method according to any one of

[201] to

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

[0215]

[212] The method according to any one of

[201] to

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

[0216]

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

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

[101] to

[113] and

[201] to

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

[0217]

[214] The method according to any one of

[201] to

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

[0218]

[215] The method according to any one of

[201] to

[204] , wherein the first antigen-binding domain and the second antigen-binding domain are respectively bound to the enzyme and its substrate, and wherein the antigen-binding molecule has activity that promotes the catalytic reaction between the enzyme and the substrate.

[0219]

[216] The method according to any one of

[201] to

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

[0220]

[217] According to the method of

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

[0221]

[218] According to the method of

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

[0222]

[219] The method according to any one of

[205] to

[218] , 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, co-stimulatory molecules and cell adhesion molecules.

[0223]

[220] The method according to any one of

[201] to

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

[0224]

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

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

[101] to

[113] and

[201] to

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

[0225]

[222] The method according to any one of

[201] to

[221] , wherein the third antigen-binding domain is fused with either the first antigen-binding domain or the second antigen-binding domain.

[0226]

[223] According to the method of

[221] or

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

[0227]

[224] The method according to any one of

[202] to

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

[0228]

[225] The method according to any one of

[221] to

[224] , wherein the first antigen-binding domain, the second antigen-binding domain and the third antigen-binding domain are each Fab molecules, wherein the third antigen-binding domain is optionally fused 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 at the C-terminus of its Fab heavy chain (CH1 region).

[0229]

[226] The method according to

[224] or

[225] , 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.

[0230]

[227] The method according to any one of

[221] to

[226] , 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.

[0231]

[228] The method according to any one of

[221] to

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

[0232]

[229] The method according to any one of

[221] to

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

[0233]

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

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

[101] to

[113] and

[201] to

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

[0234]

[231] According to the method of

[230] , the Fc region is composed of a first Fc region subunit and a second Fc region subunit that can stably associate with each other.

[0235]

[232] According to the method of

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

[0236]

[233] The method according to any one of

[230] to

[232] , wherein the Fc region is derived from a human.

[0237]

[234] The method according to any one of

[230] to

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

[0238]

[235] The method according to any one of

[230] to

[234] , wherein the Fc region shows reduced binding affinity for human Fcγ receptors compared to the natural human IgG1 Fc region.

[0239]

[236] The method according to any one of

[230] to

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

[0240]

[237] The method according to any one of

[230] to

[236] , 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 number.

[0241]

[238] The method according to any one of

[230] to

[237] , 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 number.

[0242]

[239] The method according to any one of

[230] to

[238] , 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 number.

[0243]

[240] The method according to any one of

[230] to

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

[0244] (a) N434A / Q438R / S440E;

[0245] (b) N434A / Q438R / S440D;

[0246] (c) N434A / Q438K / S440E;

[0247] (d) N434A / Q438K / S440D;

[0248] (e) N434A / Y436T / Q438R / S440E;

[0249] (f) N434A / Y436T / Q438R / S440D;

[0250] (g) N434A / Y436T / Q438K / S440E;

[0251] (h) N434A / Y436T / Q438K / S440D;

[0252] (i) N434A / Y436V / Q438R / S440E;

[0253] (j) N434A / Y436V / Q438R / S440D;

[0254] (k) N434A / Y436V / Q438K / S440E;

[0255] (l) N434A / Y436V / Q438K / S440D;

[0256] (m) N434A / R435H / F436T / Q438R / S440E;

[0257] (n) N434A / R435H / F436T / Q438R / S440D;

[0258] (o) N434A / R435H / F436T / Q438K / S440E;

[0259] (p) N434A / R435H / F436T / Q438K / S440D;

[0260] (q) N434A / R435H / F436V / Q438R / S440E;

[0261] (r) N434A / R435H / F436V / Q438R / S440D;

[0262] (s) N434A / R435H / F436V / Q438K / S440E;

[0263] (t) N434A / R435H / F436V / Q438K / S440D;

[0264] (u) M428L / N434A / Q438R / S440E;

[0265] (v) M428L / N434A / Q438R / S440D;

[0266] (w) M428L / N434A / Q438K / S440E;

[0267] (x) M428L / N434A / Q438K / S440D;

[0268] (y) M428L / N434A / Y436T / Q438R / S440E;

[0269] (z) M428L / N434A / Y436T / Q438R / S440D;

[0270] (aa) M428L / N434A / Y436T / Q438K / S440E;

[0271] (ab) M428L / N434A / Y436T / Q438K / S440D;

[0272] (ac) M428L / N434A / Y436V / Q438R / S440E;

[0273] (ad) M428L / N434A / Y436V / Q438R / S440D;

[0274] (ae) M428L / N434A / Y436V / Q438K / S440E;

[0275] (af) M428L / N434A / Y436V / Q438K / S440D;

[0276] (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[0277] (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E,

[0278] According to EU number.

[0279]

[241] The method according to any one of

[230] to

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

[0280]

[242] The method according to any one of

[230] to

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

[0281]

[243] According to the method of

[242] , the amino acid substitution that promotes polymerization comprises an amino acid substitution at at least one position selected from the group consisting of: EU number positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440 and 447.

[0282]

[244] The method according to

[242] or

[243] , wherein the polymerization is hexamerization.

[0283]

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

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

[101] to

[113] and

[201] to

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

[0284]

[246] According to the method of

[245] , the cysteine ​​residue is present at EU number position 226 and / or position 229 in the hinge region.

[0285]

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

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

[101] to

[113] and

[201] to

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

[0286]

[248] According to the method of

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

[0287]

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

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

[101] to

[113] and

[201] to

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

[0288]

[250] The method according to

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

[0289]

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

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

[101] to

[113] and

[201] to

[250] , wherein the antigen-binding molecule has the amino acid sequence KSCDKTHTCPPCP in its hinge region.

[0290]

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

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

[101] to

[113] and

[201] to

[251] , wherein the antigen-binding molecule comprises an amino acid substitution of a cysteine ​​residue at one or more positions selected from the group consisting of EU number positions 119-123, 131-140, 148-150, 155-167, 174-178, 188-197, 201-214 and 218-219.

[0291] This disclosure also relates to the following inventions:

[0292]

[301] The method according to any one of

[201] to

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

[0293]

[302] The method according to any one of

[201] to

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

[0294]

[303] The method according to any one of

[201] to

[252] ,

[301] and

[302] , wherein at least one disulfide bond is formed between the heavy chain of the first antigen-binding domain and the heavy chain of the second antigen-binding domain, between the light chain of the first antigen-binding domain and the light chain of 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.

[0295]

[304] According to the method of

[303] , 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.

[0296]

[305] According to the method of

[304] , 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 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219.

[0297]

[306] According to the method of

[304] , 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 The numbers are 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.

[0298]

[307] According to the method of

[304] , 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, 196 and 197.

[0299]

[308] According to the method of

[304] , 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.

[0300]

[309] According to the method of

[304] , 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.

[0301]

[310] According to the method of

[304] , 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 the following: EU number positions 131, 132, 133, 134, 135, 136, 137, 138, 139 and 140.

[0302]

[311] According to the method of

[304] , 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.

[0303]

[312] According to the method of

[304] , 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 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and 167.

[0304]

[313] According to the method of

[304] , 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 174, 175, 176, 177 and 178.

[0305]

[314] According to the method of

[304] , 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 the following: EU number positions 188, 189, 190, 191, 192, 193, 194, 195, 196 and 197.

[0306]

[315] According to the method of

[304] , 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 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 and 214.

[0307]

[316] According to the method of

[304] , 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 218 and 219.

[0308]

[317] The method according to any one of

[304] to

[316] , 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.

[0309]

[318] According to the method of

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

[0310]

[319] According to the method of

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

[0311]

[320] According to the method of

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

[0312]

[321] According to the method of

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

[0313]

[322] According to the method of

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

[0314]

[323] According to the method of

[304] , wherein

[0315] (i) The amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU position 191, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU position 191.

[0316] (ii) The amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU position 195, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU position 195, or

[0317] (iii) The amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU position 197, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU position 197.

[0318]

[324] The method according to any one of

[304] to

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

[0319]

[325] The method according to any one of

[201] to

[252] and

[301] to

[324] , 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, wherein the additional disulfide bonds are formed via amino acid residues at the following positions according to EU numbers in the CH1 regions of the first and second antigen-binding domains:

[0320] (a) Between amino acid residues at positions 131 to 138, 194 and 195 in each of the two antigen-binding domains;

[0321] (b) Between amino acid residues at position 131 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0322] (c) Between amino acid residues at position 132 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0323] (d) Between amino acid residues at position 133 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0324] (e) Between amino acid residues at position 134 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0325] (f) Between amino acid residues at position 135 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0326] (g) Between amino acid residues at position 136 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0327] (h) Between amino acid residues at position 137 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0328] (i) Between amino acid residues at position 138 in each of the two antigen-binding domains and between amino acid residues at position 194 in each of the two antigen-binding domains;

[0329] (j) Between amino acid residues at position 131 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0330] (k) Between amino acid residues at position 132 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0331] (l) Between amino acid residues at position 133 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0332] (m) Between amino acid residues at position 134 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0333] (n) Between amino acid residues at position 135 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0334] (o) Between amino acid residues at position 136 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains;

[0335] (p) Between amino acid residues at position 137 in each of the two antigen-binding domains, and between amino acid residues at position 195 in each of the two antigen-binding domains; and

[0336] (q) Between amino acid residues at position 138 in each of the two antigen-binding domains and between amino acid residues at position 195 in each of the two antigen-binding domains.

[0337]

[326] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more charged 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 amino acid residues with opposite charges at EU number positions 193 to 195 in the CH1 region.

[0338]

[327] The method according to any one of

[201] to

[252] and

[301] to

[324] , 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 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 negatively charged amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0339]

[328] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more negatively charged 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 positively charged amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0340]

[329] The method according to any one of

[201] to

[252] and

[301] to

[324] , 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:

[0341] (a) The amino acid residue at position 136 of the EU code is either glutamic acid (E) or aspartic acid (D);

[0342] (b) The amino acid residue at position 137 of the EU code is either glutamic acid (E) or aspartic acid (D); and

[0343] (c) The amino acid residue at position 138 of the EU number is either glutamic acid (E) or aspartic acid (D);

[0344] Furthermore, the other of the first and second antigen-binding domains contains one, two, or more of the following amino acid residues in the CH1 region:

[0345] (d) The amino acid residue at position 193 of the EU number is lysine (K), arginine (R), or histidine (H);

[0346] (e) The amino acid residue at position 194 of the EU number is lysine (K), arginine (R), or histidine (H); and

[0347] (f) The amino acid residue at position 195 of the EU number is lysine (K), arginine (R) or histidine (H).

[0348]

[330] The method according to any one of

[201] to

[252] and

[301] to

[324] , 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:

[0349] (a) The amino acid residue at position 136 of the EU number is lysine (K), arginine (R), or histidine (H);

[0350] (b) The amino acid residue at position 137 of the EU number is lysine (K), arginine (R), or histidine (H); and

[0351] (c) The amino acid residue at position 138 of the EU number is lysine (K), arginine (R) or histidine (H);

[0352] Furthermore, the other of the first and second antigen-binding domains contains one, two, or more of the following amino acid residues in the CH1 region:

[0353] (d) The amino acid residue at position 193 of the EU code is either glutamic acid (E) or aspartic acid (D);

[0354] (e) The amino acid residue at position 194 of the EU code is either glutamic acid (E) or aspartic acid (D); and

[0355] (f) The amino acid residue at position 195 of the EU number is either glutamic acid (E) or aspartic acid (D).

[0356]

[331] The method according to any one of

[201] to

[252] ,

[301] to

[324] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises any one of a combination of specific charged amino acids (according to EU numbers) in the CH1 region as shown in Tables 1, 2 or 3.

[0357]

[332] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or 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.

[0358]

[333] According to the method of

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

[0359]

[334] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein each of the first antigen-binding domain and the second antigen-binding domain comprises any one of a combination of specific hydrophobic amino acids (according to EU numbers) in the CH1 region as shown in Table 4.

[0360]

[335] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or the second antigen-binding domain contains a "pepper" amino acid residue 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 "mortar" amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0361]

[336] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more "mortar" 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 "palm" amino acid residue at EU number positions 193 to 195 in the CH1 region.

[0362]

[337] According to the method of

[336] , the "pound" 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).

[0363]

[338] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein either the first antigen-binding domain or the second antigen-binding domain contains one, two or more aromatic 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 positively charged amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0364]

[339] The method according to any one of

[201] to

[252] and

[301] to

[324] , 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 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 aromatic amino acid residues at EU number positions 193 to 195 in the CH1 region.

[0365]

[340] The method according to any one of

[201] to

[252] and

[301] to

[324] , wherein 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).

[0366]

[341] According to the method of

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

[0367]

[342] According to the method of

[341] , 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 number positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213.

[0368]

[343] According to the method of

[341] , 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 number 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.

[0369]

[344] According to the method of

[341] , 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 the following: Kabat number positions 108, 109, 110, 111 and 112.

[0370]

[345] According to the method of

[341] , 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 the following: Kabat number positions 121, 122, 123, 124, 125, 126, 127 and 128.

[0371]

[346] According to the method of

[341] , 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 the following: Kabat number positions 151, 152, 153, 154, 155 and 156.

[0372]

[347] According to the method of

[341] , 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 the following: Kabat number positions 184, 185, 186, 187, 188, 189 and 190.

[0373]

[348] According to the method of

[341] , 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 number positions 195 and 196.

[0374]

[349] According to the method of

[341] , 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 the following: Kabat number positions 200, 201, 202 and 203.

[0375]

[350] According to the method of

[341] , 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 the following: Kabat number positions 208, 209, 210, 211, 212 and 213.

[0376]

[351] The method according to any one of

[341] to

[350] , 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.

[0377]

[352] According to the method of

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

[0378]

[353] According to the method of

[303] , 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.

[0379]

[354] According to the method of

[353] , the positions of the amino acid residues in the CH1 region are selected from the group consisting of EU number 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 number positions 121, 122, 123, 124, 125, 126, 127 and 128.

[0380]

[355] According to the method of

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

[0381]

[356] The method according to any one of

[201] to

[252] and

[301] to

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

[0382]

[357] According to the method of

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

[0383]

[358] The method according to any one of

[341] to

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

[0384]

[359] According to the method of

[303] , 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.

[0385]

[360] According to the method of

[359] , the positions of the amino acid residues in the VH 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 6, 8, 16, 20, 25, 26, 28, 74 and 82b.

[0386]

[361] According to the method of

[303] , 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.

[0387]

[362] According to the method of

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

[0388]

[363] According to the method of

[361] , the positions of the amino acid residues in the VL region (λ subclass) of the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of the following: Kabat number positions 6, 19, 33 and 34.

[0389]

[364] According to the method of

[303] , 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.

[0390]

[365] According to the method of

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

[0391] This disclosure also relates to the following inventions:

[0392]

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

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

[101] to

[113] ,

[201] to

[252] and

[301] to

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

[0393] A first antigen-binding domain and a second antigen-binding domain capable of binding to CD3 and CD137, but not simultaneously binding to both CD3 and CD137, and

[0394] A third antigen-binding domain capable of binding to DLL3, preferably human DLL3.

[0395]

[402] According to the method of

[401] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise antibody variable regions that may be the same as or different from each other, and each comprises antibody variable regions independently selected from the group consisting of (a1) to (a4):

[0396] (a1) Contains the following antibody variable regions:

[0397] Heavy chain variable region, which includes:

[0398] Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 27,

[0399] Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 28, and

[0400] Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 29; and

[0401] The light chain variable region, which includes:

[0402] Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30,

[0403] The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and

[0404] Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 32;

[0405] (a2) Contains the following antibody variable regions:

[0406] Heavy chain variable region, which includes:

[0407] Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 33,

[0408] Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 34, and

[0409] Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 35; and

[0410] The light chain variable region, which includes:

[0411] Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30,

[0412] The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and

[0413] Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 32;

[0414] (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and

[0415] (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

[0416]

[403] According to the method of

[401] or

[402] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise antibody variable regions that may be the same as or different from each other, and each comprises antibody variable regions independently selected from the group consisting of (a1) to (a4):

[0417] (a1) Contains the following antibody variable regions:

[0418] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and

[0419] The light chain variable region contains the amino acid sequence of SEQ ID NO: 37; and

[0420] (a2) Contains the following antibody variable regions:

[0421] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 38, and

[0422] The light chain variable region contains the amino acid sequence of SEQ ID NO: 37;

[0423] (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and

[0424] (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

[0425]

[404] The method according to any one of

[401] to

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

[0426] (a1) Contains the following antibody variable regions:

[0427] Heavy chain variable region, which includes:

[0428] Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 46,

[0429] Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 47, and

[0430] Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 48; and

[0431] The light chain variable region, which includes:

[0432] Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 49,

[0433] The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 50, and

[0434] Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 51;

[0435] (a2) Contains the following antibody variable regions:

[0436] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 52, and

[0437] The light chain variable region contains the amino acid sequence of SEQ ID NO: 53;

[0438] (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and

[0439] (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

[0440]

[405] The method according to any one of

[401] to

[404] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising:

[0441] Heavy chain variable region, which includes:

[0442] Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 27,

[0443] Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 28, and

[0444] Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 29; and

[0445] The light chain variable region, which includes:

[0446] Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30,

[0447] The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and

[0448] The light chain CDR 3 contains the amino acid sequence of SEQ ID NO: 32.

[0449]

[406] The method according to any one of

[401] to

[405] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising:

[0450] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and

[0451] The light chain variable region contains the amino acid sequence of SEQ ID NO: 37.

[0452]

[407] The method according to any one of

[401] to

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

[0453] Heavy chain variable region, which includes:

[0454] Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 46,

[0455] Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 47, and

[0456] Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 48; and

[0457] The light chain variable region, which includes:

[0458] Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 49,

[0459] The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 50, and

[0460] The light chain CDR 3 contains the amino acid sequence of SEQ ID NO: 51.

[0461]

[408] The method according to any one of

[401] to

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

[0462] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 52, and

[0463] The light chain variable region contains the amino acid sequence of SEQ ID NO: 53.

[0464]

[409] The method according to any one of

[401] to

[408] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising:

[0465] The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and

[0466] The light chain variable region contains the amino acid sequence of SEQ ID NO: 37.

[0467] Furthermore, the third antigen-binding domain comprises an antibody variable region, which includes:

[0468] The heavy chain variable region, which includes SEQ ID NO: 52, and

[0469] The light chain variable region contains SEQ ID NO: 53.

[0470]

[410] The method according to any one of

[401] to

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

[0471]

[411] The method according to any one of

[401] to

[410] , wherein each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a Fab, said Fab comprising:

[0472] Heavy chains containing VH and CH1 regions, and

[0473] Light chains containing VL and CL regions,

[0474] The C-terminus of the CH1 region of the heavy chain of the third antigen-binding domain is fused directly or via a peptide linker to the N-terminus of the VH region of the Fab heavy chain of either the first or second antigen-binding domain.

[0475]

[412] According to the method of

[411] , 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.

[0476]

[413] The method according to any one of

[401] to

[412] , wherein the third antigen-binding domain is a cross-Fab in which the VH region is connected to the CL region and the VL region is connected 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 connected to the CH1 region and the VL region is connected to the CL region.

[0477]

[414] The method according to any one of

[401] to

[413] , wherein:

[0478] In the CL region of each of the first and second antigen-binding domains, the amino acid residues at positions 123 and 124 of Kabat are arginine and lysine, respectively.

[0479] In the CH1 region of each of the first and second antigen-binding domains, the amino acid residues at EU number positions 147 and 213 are both glutamic acid.

[0480]

[415] The method according to any one of

[401] to

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

[0481]

[416] According to the method of

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

[0482] The first Fc subbase is selected from the group consisting of the following items:

[0483] Fc region polypeptide containing alanine at each of positions 234 and 235;

[0484] A polypeptide containing an alanine at each of positions 234, 235, and 297; and

[0485] An Fc region polypeptide containing alanine at each of positions 234, 235, and 297, cysteine ​​at position 354, and tryptophan at position 366, and

[0486] The second Fc subunit is selected from the following groups:

[0487] Fc region polypeptide containing alanine at each of positions 234 and 235;

[0488] A polypeptide containing an alanine at each of positions 234, 235, and 297; and

[0489] An Fc region polypeptide containing 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.

[0490] All locations are based on EU numbers.

[0491]

[417] The method according to any one of

[416] or

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

[0492] (a) A first Fc subunit containing the amino acid sequence of SEQ ID NO: 23 and a second Fc subunit containing the amino acid sequence of SEQ ID NO: 24;

[0493] (b) A first Fc subunit containing the amino acid sequence of SEQ ID NO: 25 and a second Fc subunit containing the amino acid sequence of SEQ ID NO: 26; or

[0494] (c) A first Fc subunit containing the amino acid sequence of SEQ ID NO: 58 and a second Fc subunit containing the amino acid sequence of SEQ ID NO: 59.

[0495]

[418] The method according to any one of

[401] to

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

[0496] (a1) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 39, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 40, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 41, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO: 42.

[0497] (a2) A polypeptide chain containing the amino acid sequence of SEQ ID NO: 43 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO: 40 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO: 44 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO: 42; and

[0498] (a3) A polypeptide chain containing the amino acid sequence of SEQ ID NO: 45 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO: 40 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO: 44 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO: 42.

[0499] Preferably, the five polypeptide chains (chain 1 to chain 5) are connected and / or associated with each other according to the orientation shown in Figure 3.

[0500]

[419] According to the method of

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

[0501] The polypeptide chain containing the amino acid sequence of SEQ ID NO: 54 (chain 1), the polypeptide chain containing the amino acid sequence of SEQ ID NO: 55 (chain 2), the polypeptide chain containing the amino acid sequence of SEQ ID NO: 56 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO: 57.

[0502] Preferably, the five polypeptide chains (chain 1 to chain 5) are connected and / or associated with each other according to the orientation shown in Figure 3.

[0503]

[420] According to the method of

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

[0504] A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 54.

[0505] A polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 55.

[0506] A polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 60, and

[0507] Two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO: 57.

[0508] Preferably, the five polypeptide chains (chain 1 to chain 5) are connected and / or associated with each other according to the orientation shown in Figure 3.

[0509]

[421] According to the method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains, the five polypeptide chains being: a polypeptide chain (A chain) comprising the amino acid sequence shown in SEQ ID NO: 63, a polypeptide chain (L chain) comprising the amino acid sequence shown in SEQ ID NO: 64, a polypeptide chain (P chain) comprising the amino acid sequence shown in SEQ ID NO: 65, and two polypeptide chains (two S chains) each comprising the amino acid sequence shown in SEQ ID NO: 66, and preferably, the five polypeptide chains are connected and / or associated with each other according to the orientation shown in FIG. 6.

[0510]

[422] According to the method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains, the five polypeptide chains being: a polypeptide chain (A chain) comprising the amino acid sequence shown in SEQ ID NO: 67, a polypeptide chain (L chain) comprising the amino acid sequence shown in SEQ ID NO: 68, a polypeptide chain (P chain) comprising the amino acid sequence shown in SEQ ID NO: 69, and two polypeptide chains (two S chains) each comprising the amino acid sequence shown in SEQ ID NO: 70, and preferably, the five polypeptide chains are connected and / or associated with each other according to the orientation shown in FIG. 6.

[0511]

[423] According to the method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains, the five polypeptide chains being: a polypeptide chain (A chain) comprising the amino acid sequence shown in SEQ ID NO: 71, a polypeptide chain (L chain) comprising the amino acid sequence shown in SEQ ID NO: 72, a polypeptide chain (P chain) comprising the amino acid sequence shown in SEQ ID NO: 73, and two polypeptide chains (two S chains) each comprising the amino acid sequence shown in SEQ ID NO: 74, and preferably, the five polypeptide chains are connected and / or associated with each other according to the orientation shown in FIG6.

[0512]

[424] According to the method of

[401] , wherein the antigen-binding molecule is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 79 and the light chain comprising the amino acid sequence shown in SEQ ID NO: 80.

[0513]

[425] According to the method of

[401] , wherein the antigen-binding molecule is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 81 and the light chain comprising the amino acid sequence shown in SEQ ID NO: 82.

[0514]

[426] According to the method of

[401] , wherein the antigen-binding molecule is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 83 and the light chain comprising the amino acid sequence shown in SEQ ID NO: 84.

[0515] This disclosure also relates to the following inventions:

[0516]

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

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

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] further comprises measuring, determining or quantifying the ratio (LINC ratio) of an antigen-binding molecule (in LINC form) having at least one disulfide bond in a region outside the hinge region to the sum of the following:

[0517] (i) An antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and

[0518] (ii) Incorrect disulfide-bonded and / or non-disulfide-bonded (unLINC) forms of antigen-binding molecules.

[0519]

[502] According to the method of

[501] , the steps of measuring, determining or quantifying include electrophoresis or chromatography.

[0520]

[503] According to the method of

[502] , 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).

[0521]

[504] The method according to

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

[0522]

[505] The method according to any one of

[501] to

[504] , wherein the method further comprises adding the protease to the formulation prior to the measurement, determination or quantification step.

[0523]

[506] The method according to

[505] wherein the protease is an enzyme that does not digest the LINC form but can digest the unLINC form.

[0524]

[507] The method according to

[505] or

[506] , wherein the protease is a cysteine ​​protease capable of digesting human IgG1 antibodies.

[0525]

[508] The method according to any one of

[505] to

[507] , wherein the protease is an enzyme capable of cleaving the hinge region of a human IgG1 antibody.

[0526]

[509] The method according to any one of

[505] to

[508] , wherein the protease is a protease that digests human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[0527]

[510] The method according to any one of

[505] to

[509] , wherein the protease is IgdE.

[0528]

[511] The method according to

[510] , wherein IgdE is derived from Streptococcus agalactiae.

[0529]

[512] The method according to

[510] or

[511] , wherein IgdE is a protein selected from the group consisting of the following (a) to (d):

[0530] (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 61;

[0531] (b) A protein encoded by the base sequence shown in SEQ ID NO: 62;

[0532] (c) A protein comprising one or more amino acids substituted, deleted, added, and / or inserted relative to the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP; and

[0533] (d) A protein comprising an amino acid sequence having at least 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[0534]

[513] The method according to any one of

[505] to

[512] , wherein the protease is FabALACTICA (registered trademark).

[0535]

[514] The method according to any one of

[505] to

[513] , wherein the steps of measuring, determining or quantifying include non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC).

[0536]

[515] The method according to

[514] , wherein the steps of measuring, determining or quantifying include non-reducing capillary SDS gel electrophoresis (CE-SDS), and wherein the method further includes performing non-reducing capillary SDS gel electrophoresis (CE-SDS) on formulations without added protease and samples containing only protease.

[0537]

[516] The method according to

[515] further includes obtaining electrophoretic images of a formulation with added protease, a formulation without added protease, and a sample containing only protease.

[0538] The electrophoresis diagram of the formulation without added protease includes: peaks originating from the LINC form and from the unLINC form that has not been digested by protease, as well as a peak (peak Z) that is neither originating from the LINC form nor from the unLINC form that has not been digested by protease.

[0539] The electrophoresis diagram of the formulation with added protease includes: a peak originating from the LINC form (peak L), a peak originating from the unLINC form after protease digestion (peak unL), a peak originating from the protease (peak E), and a peak that is neither originating from the LINC form, nor from the unLINC form after protease digestion, nor from the protease (peak Z).

[0540] The electrophoresis pattern of the sample containing the protease only contains the peak (peak E) derived from the protease.

[0541]

[517] According to the method of

[516] , the obtained LINC ratio is the ratio of (area value of peak L) to [(sum of area values ​​of peak L, peak E and peak Z) – (area value of peak Z) – (area value of peak E) + (area value of peak L)].

[0542]

[518] According to the method of

[514] , the steps of measuring, determining or quantifying include performing hydrophobic interaction chromatography (HIC) and obtaining a chromatogram.

[0543]

[519] According to the method of

[518] , the LINC ratio is obtained as the ratio of the area value of the peak derived from the LINC form to the sum of the area values ​​of the peak derived from the LINC form and the area values ​​of all peaks derived from the unLINC form.

[0544]

[520] The method according to any one of

[501] to

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

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

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] is membrane chromatography.

[0545]

[521] The method according to any one of

[505] to

[520] , wherein the method further comprises culturing the preparation after adding the protease.

[0546] This disclosure also relates to the following inventions:

[0547]

[601] A formulation comprising an antigen-binding molecule produced by any one of [1] to

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

[201] to

[252] ,

[301] to

[365] ,

[401] to

[425] and

[501] to

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

[0548]

[602] A pharmaceutical composition comprising an formulation 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 formulation is prepared by any one of the methods described in [1] to

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

[201] to

[252] ,

[301] to

[365] ,

[401] to

[425] , and

[501] to

[521] .

[0549] This disclosure also relates to the following inventions:

[0550]

[701] The method according to any one of

[101] to

[113] , wherein the composition (1) is a formulation produced by any one of [1] to

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

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] .

[0551]

[702] A composition obtained by any one of

[101] to

[113] and

[701] .

[0552] This disclosure also relates to the following inventions:

[0553]

[801] A method for producing a pharmaceutical composition or pharmaceutical preparation comprising an antigen-binding molecule (LINC form) having at least one disulfide bond formed in a region other than the hinge region, the method comprising:

[0554] (a) The ratio (LINC ratio) of the total number of LINC forms and the sum of LINC forms and unLINC forms of antigen-binding molecules (unLINC forms) that do not have disulfide bonds formed in regions other than the hinge region, in a composition comprising LINC and unLINC forms, is measured, determined, or quantified by any of the methods described in

[101] to

[113] .

[0555] (b) Select compositions having a LINC ratio of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and

[0556] (c) Prepare a pharmaceutical composition or pharmaceutical formulation comprising the composition selected in step (b) and a pharmaceutically acceptable carrier.

[0557]

[802] A method for producing a pharmaceutical composition or pharmaceutical preparation comprising an antigen-binding molecule (LINC form) having at least one disulfide bond formed in a region other than the hinge region, the method comprising:

[0558] (a) The ratio (LINC ratio) of the total number of LINC forms and the sum of LINC forms and unLINC forms of antigen-binding molecules (unLINC forms) that do not have disulfide bonds formed in regions other than the hinge region, in a composition comprising LINC and unLINC forms, is measured, determined, or quantified by any of the methods described in

[101] to

[113] .

[0559] (b) Confirm that the LINC ratio of the composition is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and

[0560] (c) Prepare a pharmaceutical composition or pharmaceutical formulation comprising the composition having been demonstrated in step (b) to a LINC ratio and a pharmaceutically acceptable carrier.

[0561]

[803] The composition according to

[801] or

[802] , wherein the antigen-binding molecule is a molecule defined as any one of

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] .

[0562]

[804] The method according to any one of

[801] to

[803] , wherein the composition in step (a) is a formulation produced by any one of [1] to

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

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] .

[0563] This disclosure also relates to the following inventions:

[0564]

[901] A composition comprising an antigen-binding molecule in the form of having at least one disulfide bond formed in a region outside the hinge region (LINC form), an antigen-binding molecule in the form of not having a disulfide bond formed in a region outside the hinge region (unLINC form), and a protease.

[0565]

[902] The composition according to

[901] wherein the protease is an enzyme that does not digest the LINC form but is capable of digesting the unLINC form.

[0566]

[903] The composition according to

[901] or

[902] , wherein the protease is a cysteine ​​protease capable of digesting human IgG1 antibodies.

[0567]

[904] The composition according to any one of

[901] to

[903] , wherein the protease is an enzyme capable of cleaving the hinge region of a human IgG1 antibody.

[0568]

[905] The composition according to any one of

[901] to

[904] , wherein the protease is an enzyme that digests human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[0569]

[906] The method according to any one of

[901] to

[905] , wherein the protease is IgdE.

[0570]

[907] The composition according to

[906] wherein IgdE is derived from Streptococcus agalactiae.

[0571]

[908] The composition according to

[906] or

[907] , wherein IgdE is a protein selected from the group consisting of (a) to (d) below:

[0572] (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 61;

[0573] (b) A protein encoded by the base sequence shown in SEQ ID NO: 62;

[0574] (c) A protein comprising one or more amino acids substituted, deleted, added, and / or inserted relative to the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP; and

[0575] (d) A protein comprising an amino acid sequence having at least 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[0576]

[909] The composition according to any one of

[901] to

[908] , wherein the protease is FabALACTICA (registered trademark).

[0577]

[910] The composition according to any one of

[901] to

[909] , wherein the antigen-binding molecule is a molecule defined as any one of

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] .

[0578]

[911] The composition according to any one of

[901] to

[910] , wherein the composition is a mixture of a protease and a formulation produced by any one of [1] to

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

[201] to

[252] ,

[301] to

[365] and

[401] to

[425] .

[0579] [Invention Effects]

[0580] This disclosure provides an efficient and easy method for producing and purifying antigen-binding molecules (LINC form) with suitable disulfide bonds in regions outside the hinge region. The method in this disclosure enables the easy, rapid, and high-yield production of the LINC-Ig form. The method in this disclosure has the advantage of a wide range of reducing agent types, concentrations, pH values, and reaction times.

[0581] Furthermore, compared to such production and purification in a vessel, production and purification of LINC-Ig forms in a column (such as those in the methods of this disclosure) has advantages such as reduced likelihood of aggregation of antigen-binding molecules, shorter reaction times, ease of operation, the possibility of automation using programmed liquid chromatography (LC), the possibility of maintaining a constant concentration of reducing agent in the reaction, and ease of reproducibility across scales.

[0582] This disclosure also provides a method for measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules having at least one disulfide bond formed in a region outside the hinge region (LINC form) to the sum of said LINC form and antigen-binding molecules not having a disulfide bond formed in a region outside the hinge region (unLINC form). The method of this disclosure allows for the measurement, determination, or quantification of the proportion of LINC-Ig forms contained in a composition. The method of this disclosure has the advantage that the ratio of LINC and unLINC forms of antigen-binding molecules that can be contained in regions outside the hinge region (more specifically, between sites in the CH1 region of one heavy chain and sites in the CH1 region of another heavy chain) can be measured, determined, or quantified. Attached Figure Description

[0583] Figure 1 shows the analytical results for Cys-reduced samples. From left to right, the control (1 sample) with the reduction / oxidation step skipped and affinity chromatography performed is shown, along with 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). A significant increase in the LINC ratio was confirmed at both pH 7.0 and 8.0, particularly in the concentration range of 0.1 to 10 mmol / L. For the 100 mmol / L results, no bands could be confirmed on the gel, and the symbol "-" is used because the accurate LINC ratio could not be calculated.

[0584] Figure 2 shows the analytical results for TCEP-reduced samples. From left to right, the results show a control (1 sample) with the reduction / oxidation step skipped and affinity chromatography performed, 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). For results from 0.1 to 1 mmol / L, bands could not be confirmed on the gel, and the symbol "-" is used because the accurate LINC ratio could not be calculated.

[0585] Figure 3 is a diagram illustrating the design and naming rules for trivalent antibodies in the dual / LINC (1+2) form.

[0586] Figure 4 shows the electrophoresis pattern from CE-SDS.

[0587] Figure 5 shows the electrophoresis results from the HIS analysis, including peak names. A standard electrophoresis plot (top) and a magnified electrophoresis plot (bottom) are shown.

[0588] Figure 6 is a schematic diagram of the ALPS12 molecule. The dashed lines represent LINC-type disulfide bonds formed between the CH1 domains of the heavy chain.

[0589] Figure 7 shows the digestive site of FabALACTICA (registered trademark) using the IgG1 sequence.

[0590] Figure 8 shows the changes in LINC and unLINC forms after digestion with FabALACTICA (registered trademark).

[0591] Figure 9 is a graph showing the raw data corresponding to Table 12. The numbers in Table 12 correspond to the numbers in Figure 9. “Ori.” represents an undigested sample, and “dig.” represents a digested sample.

[0592] Figure 10 is a diagram illustrating the method for calculating the content ratio in LINC form using the data in Figure 4.

[0593] Figure 11-1 shows the entire electrophoresis map of the HIC analysis.

[0594] Figure 11-2 is an enlarged view of Figure 11-1.

[0595] Figure 12 illustrates the use of Figure 11-1 The data calculation in Figure 11-2 is a graph of the formulas for quantities in LINC form. Detailed Implementation Example Description

[0596] Those skilled in the art will readily understand and typically use conventional methods to employ the techniques 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, NY; Current Protocols in Molecular Biology (edited by F.M. Usubel et al., (2003)); these series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Mess, and G.R. Taylor (1995)), Harlow and Lane (edited by R.R. Freshney (1988)); Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.R. 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.C. Ellis, 1998) Academic Press. Press; Animal Cell Culture (RIFreshney), editors, 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, editors, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, editors); GeneTransfer Vectors for Mammalian Cells (JMMiller and MPCalos, editors, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., editors, 1994); Current Protocols in Immunology (JEColigan et al., editors, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: ALLaboratory Manual (E. Harlow and D. Lane (Cold) Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti The widely used methods described in *And JD Capra, ed., Harwood Academic Publishers, 1995* and *Cancer: Principles and Practice of Oncology* (VT DeVita et al., ed., JBLippincott Company, 1993) are relevant.

[0597] The following definitions and detailed descriptions are provided to aid in understanding this disclosure.

[0598] I. Definition

[0599] In this document, when describing sites of amino acid alteration, the term “and / or” means each combination of “and” and “or” appropriately. Specifically, for example, “amino acids at positions 33, 55 and / or 96 are substituted” includes variants of the following amino acid alterations: (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) amino acids at positions 33, 55 and 96.

[0600] Unless otherwise stated, the amino acid residues in the light chain constant region are numbered according to Kabat et al., and the amino acid residues in the heavy chain constant region 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0601] amino acids

[0602] In this article, amino acids are described by one 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.

[0603] Changes in amino acids

[0604] For amino acid alterations in the amino acid sequence of antigen-binding molecules (also described herein as "amino acid substitutions" or "amino acid mutations"), 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 employed. Additionally, several known methods can be used as amino acid alteration methods to replace non-natural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system containing tRNA (Clover Direct (Protein Express)) with a non-natural amino acid that complements one of the stop codons (codon UAG (amber codon)) to inhibit tRNA binding is suitable.

[0605] Furthermore, in this document, as a way to indicate amino acid changes, it is appropriate to use a one- or three-letter code indicating the amino acid before and after the change, respectively, before and after the number indicating a specific position. For example, when replacing an amino acid contained in the variable region of an antibody, the change N100bL or Asn100bLeu indicates that Leu is used to replace Asn at position 100b (according to the Kabat number). That is, the number shows the amino acid position according to the Kabat number, the one- or three-letter amino acid code written before the number shows the amino acid before the substitution, and the one- or three-letter amino acid code written after the number shows the amino acid after the substitution. Similarly, when replacing an amino acid in the Fc region contained in the constant region of an antibody, the change P238D or Pro238Asp indicates that Asp is used to replace Pro at position 238 (according to the EU number). That is, the number shows the amino acid position according to the EU number, the one- or three-letter amino acid code written before the number shows the amino acid before the substitution, and the one- or three-letter amino acid code written after the number shows the amino acid after the substitution.

[0606] polypeptide

[0607] As used herein, the term "peptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "peptide" refers to any chain having two or more amino acids, not a specific length of product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain having two or more amino acids is included within the definition of "peptide," and the term "peptide" may be used in place of any of these terms, or interchangeably with any of these terms. The term "peptide" is also intended to refer to post-expression modified products of peptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Peptides may be derived from natural biological sources or produced through recombinant technologies, and are not necessarily translated from a specified nucleic acid sequence. They can be generated in any manner, including through chemical synthesis. As described herein, peptides can be of 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. Peptides can have a defined three-dimensional structure, but they do not necessarily have such a structure. Peptides with a defined three-dimensional structure are called folded; and peptides that do not have a defined three-dimensional structure but can take on a large number of different conformations are called unfolded.

[0608] Amino acid sequence identity percentage (%)

[0609] The "percentage of 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 those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the value of amino acid sequence identity %. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and / or with respect to a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a percentage of amino acid sequence identity with or with respect to a given amino acid sequence B) is calculated as follows:

[0610] 100 multiplied by the fraction X / Y

[0611] Where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % between A and B will not be equal to the amino acid sequence identity % between B and A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0612] antigen-binding molecules

[0613] 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 antigen-binding activity, and may also refer to molecules such as peptides or proteins of about five amino acids or longer. Peptides and proteins are not limited to those derived from organisms; for example, they can be polypeptides produced by artificially designed sequences. They can also be any of naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc. A scaffold molecule containing a known stable conformational structure such as an α / β barrel as a scaffold, with portions of the molecule serving as antigen-binding sites, is also an example of an antigen-binding molecule described herein. In one embodiment, an antigen-binding molecule may contain two or more (e.g., 2, 3, 4, 5, or more) polypeptide chains. In one embodiment, the polypeptide chains constituting the antigen-binding molecule may 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 trivalent antibody comprising chains 1 to 5, as shown in Figure 3 of this application. In one embodiment, the antigen-binding molecule is a non-antibody protein, a fragment thereof, or a derivative thereof.

[0614] Multispecific antigen-binding molecules

[0615] A "multispecific antigen-binding molecule" is an antigen-binding molecule that binds specifically to more than one antigen. The term "bispecific" means that an antigen-binding molecule can bind specifically to at least two different antigenic determinants. The term "trispecific" means that an antigen-binding molecule can bind specifically to at least three different antigenic determinants.

[0616] In some embodiments, the multispecific antigen-binding molecule of this application is a trispecific antigen-binding molecule, which can bind to one of CD3 or CD137, but not to both antigens at the same time, and can specifically bind to DLL3.

[0617] Antigen-binding domain

[0618] In this document, an "antigen-binding domain" refers to a region that specifically binds to and is complementary to all or part of an antigen. In this document, antigen-binding molecules contain antigen-binding domains. When the molecular weight of the antigen is large, the antigen-binding domain can only bind to a specific portion of the antigen. This specific portion is called an "epitope." In one embodiment, the antigen-binding domain contains an antibody fragment that binds to a specific antigen. The antigen-binding domain may be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain contains 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 contains a non-antibody protein that binds to a specific antigen or a fragment thereof. In a specific embodiment, the antigen-binding domain contains one or two Fabs including a CH1 region. In a specific embodiment, the antigen-binding domain contains a hinge region.

[0619] As used herein, the term "antigen-binding domain" directs the entity to which it is attached to to a target site, such as to a specific type of tumor cell expressing the cancer antigen (DLL3). Antigen-binding domains can activate signal transduction via their target antigens, such as T-cell receptor complex antigens (particularly CD3) and / or co-stimulatory receptors (CD137).

[0620] As used herein, the terms "first," "second," and "third" relating to antigen-binding domains, etc., are used for ease of distinction when there is more than one of each type of part. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or orientation to the antigen-binding molecule.

[0621] In this article, "specific binding" means binding in a state where one of the molecules involved in specific binding does not exhibit any significant binding with molecules other than a single or multiple binding partner molecules. Furthermore, it is also used when the antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen. When the epitope bound to the antigen-binding domain is contained in multiple different antigens, the antigen-binding molecule containing that antigen-binding domain can bind to various antigens having that epitope.

[0622] In this disclosure, the phrase "binds to the same epitope" means that the epitopes bound by the two antigen-binding domains at least partially overlap each other. The degree of overlap is, but is not limited to, at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, and 80%, particularly preferably 90% or more, and most preferably 100%.

[0623] Antibody

[0624] The term “antibody” is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0625] The term "immunoglobulin molecule" refers to a protein that has the structure of naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain (also called a light chain constant region). The heavy chains of immunoglobulins can be classified into one of five types, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or µ (IgM), some of which can be further subdivided 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, referred to as κ and λ. Immunoglobulins essentially consist of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

[0626] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or produced during the production of a monoclonal antibody formulation, such variants are typically present in small quantities), the individual antibodies comprising this population are identical and / or bind to the same epitope. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0627] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0628] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0629] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, p. 6) See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be separated using either the VH or VL domain of the antibody binding that antigen, to screen libraries containing complementary VL or VH domains.

[0630] As used herein, the term "hypervariant region" or "HVR" refers to each of the following: a region of an antibody variable domain that is hypervariable in sequence ("complementarity-determining region" or "CDR") and / or forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact site"). Typically, an antibody contains six HVRs: three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). Exemplary HVRs in this document include:

[0631] (a) Hyperchromatic rings present 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));

[0632] (b) CDRs located 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)).

[0633] (c) Antigen contact sites located 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

[0634] (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).

[0635] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are referenced in this paper to Kabat et al., with the same reference numbering above.

[0636] 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.

[0637] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0638] The “human common framework” is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, this subgroup is subgroup κ I as described in Kabat et al. (ibid.). In one embodiment, for VH, this subgroup is subgroup III as described in Kabat et al. (ibid.).

[0639] The term "hinge region" refers to the portion of the antibody heavy chain polypeptide that connects the CH1 and CH2 domains in the wild-type antibody heavy chain, for example, from approximately position 216 to approximately position 230 according to the EU numbering system, or from approximately position 226 to approximately position 243 according to the Kabat numbering system. It is known that in native IgG antibodies, a cysteine ​​residue at position 220 according to the EU number in the hinge region forms a disulfide bond with a 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 cysteine ​​residues at position 226 and between cysteine ​​residues at position 229 according to the EU number in the hinge region. Generally, the "hinge region" is defined as extending from 216 to 238 (EU number) or from 226 to 251 (Kabat number) in human IgG1. This hinge can be further divided into three distinct regions: the upper hinge, the central hinge, and the lower hinge. In human IgG1 antibodies, these regions are typically defined as follows:

[0640] Upper hinge: 216 to 225 (EU number) or 226 to 238 (Kabat number).

[0641] Central hinge: 226 to 230 (EU number) or 239 to 243 (Kabat number).

[0642] Lower hinge: 231 to 238 (EU number) or 244 to 251 (Kabat number).

[0643] By placing the first and last cysteine ​​residues forming 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 of Today 16: 85-90). The hinge regions in this paper include wild-type hinge regions and variants in which the amino acid residues in the wild-type hinge regions are altered by substitution, addition, or deletion.

[0644] The terms “capable of linking” (amino acid residues) and “capable of forming” (disulfide bond) include cases where a disulfide bond has already formed, as well as cases where a disulfide bond has not yet formed but may subsequently form under appropriate conditions.

[0645] The term "disulfide bond not formed between amino acids 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 located in any antibody region outside the "hinge region" as defined above. For example, such disulfide bonds are formed, connected, or linked by amino acids at any location outside the hinge region of the antibody (e.g., from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system). In some embodiments, such disulfide bonds are formed, connected, or linked by amino acids located in the CH1, CL, VL, VH, and / or VHH regions. In some embodiments, such disulfide bonds are formed, linked, or connected by amino acids located in the CH1 region at positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214 according to EU numbers. In some embodiments, such disulfide bonds are formed, linked, or connected by amino acids located in the CH1 region 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 according to EU numbers. In some embodiments, such disulfide bonds are formed, linked, or connected by amino acids located at positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to EU numbers in the CH1 region. In a preferred embodiment, such disulfide bonds are formed, linked, or connected by amino acids located at position 191 according to EU numbers in the CH1 region.

[0646] The term "incorrect disulfide bond form" refers to an antigen-binding molecule that forms disulfide bonds between amino acid residues that are different from the desired amino acid residues. Such antigen-binding molecules are included in the incorrect disulfide bond forms described herein when they contain both disulfide bonds formed between the desired amino acid residues and disulfide bonds formed between amino acid residues that are different from the desired amino acid residues.

[0647] The term "non-disulfide bonded form" refers to an antigen-binding molecule in which the cysteine ​​residue remains in a free state, or in which the cysteine ​​residue forms a disulfide bond with a molecule (such as an impurity molecule) that is different from the molecule forming the antigen-binding molecule. The "non-disulfide bonded forms" described herein only need to have such characteristics, and for example, they are included in the "non-disulfide bonded forms" herein even if they contain disulfide bonds formed between amino acid residues in regions outside the hinge region (desired disulfide bonds), or disulfide bonds that are different from the desired disulfide bonds (erroneous disulfide bonds), as long as they have the aforementioned characteristics.

[0648] 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. Furthermore, the constant region is preferably a heavy chain constant region, more preferably a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4 type antibodies, and even more preferably a heavy chain constant region of human IgG1, IgG2, IgG3, and IgG4 type antibodies. The amino acid sequences of the human IgG1, human IgG2, human IgG3, and human IgG4 constant regions are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, several allomorphic sequences with genetic polymorphism are described in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, and any of them can be used in this invention. Amino acid-modified constant regions may contain other amino acid mutations or modifications, as long as they include the amino acid mutations of this invention.

[0649] In this document, the term "Fc region" or "Fc domain" refers to a region comprising a segment consisting of a hinge or portion thereof in an antibody molecule, along with CH2 and CH3 domains. For IgG classes, the Fc region refers to, but is not limited to, the region extending from, for example, cysteine ​​226 (EU number (also referred to herein as the EU index)) 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, a monoclonal antibody such as IgG1, IgG2, IgG3, or IgG4 with a proteolytic enzyme such as pepsin, followed by elution of fractions adsorbed onto a protein A column or a protein G column. Such proteolytic enzymes are not particularly limited, provided that the enzyme, under appropriately set reaction conditions (e.g., pH), can digest the entire antibody to restrictively form Fab or F(ab')2. Examples include pepsin and papain.

[0650] The Fc region derived from, for example, naturally occurring IgG can be used as the "Fc region". In this context, naturally occurring IgG means a polypeptide containing the same amino acid sequence as naturally occurring IgG and belonging to the antibody class encoded essentially by the immunoglobulin γ gene. Naturally occurring human IgG means, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. Naturally occurring IgG also includes variants derived spontaneously from it. In the target immunological protein sequence of NIH Publication No. 91-3242, multiple allomorphic sequences based on gene polymorphism are described as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, any of which can be used in this invention. In particular, the sequence of human IgG1 may have a DEL or EEM as the amino acid sequence at EU number positions 356 to 358.

[0651] The Fc domain of an antigen-binding molecule consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the antigen-binding molecule described herein contains no more than one Fc domain.

[0652] In this document, the Fc domain of the antigen-binding molecule is the IgG Fc domain. In a specific embodiment, the Fc domain is the IgG1 Fc domain. In another embodiment, the Fc domain is the IgG1 Fc domain. In a further specific embodiment, the Fc domain is the human IgG1 Fc region.

[0653] chimeric antibodies

[0654] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain originates from a specific source or species, while the remainder of the heavy and / or light chain originates 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 and / or light chain variable region originates from a specific source or species, while the remainder of the heavy and / or light chain variable region originates from a different source or species.

[0655] Humanized antibodies

[0656] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, a humanized antibody may contain at least a portion of the antibody constant region derived from a human antibody. An antibody in a "humanized form," such as a non-human antibody, refers to an antibody that has undergone humanization. A "variable region of a humanized antibody" refers to the variable region of a humanized antibody.

[0657] antibody fragments

[0658] "Antibody fragment" refers to a molecule other than the complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bisomatic antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of some antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, 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 containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. Biantibodies are antibody fragments having two antigen-binding sites, which can be bivalent or bispecific. See, for example, 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). Trisomic and tetrasomic antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1). Antibody fragments can be prepared using a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production from recombinant host cells (e.g., E. coli or bacteriophages), as described herein.

[0659] Fv (variable fragment)

[0660] In this paper, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from E. coli periplasmic fractions by inserting an antibody gene downstream of a bacterial signal sequence and inducing its expression in E. coli (Science (1988) 240(4855), 1038-1041). In Fv prepared from periplasmic fractions, VH associates with VL in a manner that binds to the antigen.

[0661] scFv, single-chain antibodies and sc(Fv)2

[0662] In this document, the terms “scFv,” “single-chain antibody,” and “sc(Fv)2” all refer to antibody fragments containing a single polypeptide chain with variable, rather than constant, regions derived from the heavy and light chains. Generally, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which allows for the formation of desired structures that are thought to permit antigen binding. Pluckthun discusses single-chain antibodies in detail in “The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, 269-315 (1994).” See also International Patent Publication WO 1988 / 001649; U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0663] The scFv is an antigen-binding domain in which the VH and VL that form the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be kept in very close proximity by the peptide linker.

[0664] sc(Fv)2 is a single-chain antibody in which the four variable regions of two VLs and two VHs are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). 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 that 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 linking scFv with a linker such as a peptide linker.

[0665] In this paper, the form in which the antigen-binding domain of sc(Fv)2 is formed includes an antibody in which two VH units and two VL units 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-described form, and they can be arranged in any order. Examples of such arrangements are listed below.

[0666] [VL]-Connector-[VH]-Connector-[VH]-Connector-[VL]

[0667] [VH]-Connector-[VL]-Connector-[VL]-Connector-[VH]

[0668] [VH]-Connector-[VH]-Connector-[VL]-Connector-[VL]

[0669] [VL]-Connector-[VL]-Connector-[VH]-Connector-[VH]

[0670] [VL]-Connector-[VH]-Connector-[VL]-Connector-[VH]

[0671] The molecular form of sc(Fv)2 is also described in detail in WO 2006 / 132352. Based on these descriptions, those skilled in the art can appropriately prepare the desired sc(Fv)2 to produce the polypeptide complexes disclosed herein.

[0672] Furthermore, the antigen-binding molecules or antibodies in this disclosure can be conjugated to carrier polymers (such as PEG) or organic compounds (such as anticancer agents). Alternatively, it is preferable to insert a glycan addition sequence into the antigen-binding molecule or antibody so that the glycan produces the desired effect.

[0673] The adapters for linking the variable region of the antibody include any peptide adapters that can be introduced through genetic engineering, synthetic adapters, and adapters disclosed, for example, in Protein Engineering, 9(3), 299-305, 1996. However, peptide adapters are preferred in this disclosure. The length of the peptide adapter is not particularly limited and can be appropriately selected by those skilled in the art as appropriate for the purpose. The length is preferably five amino acids or more (without particular limitation, the upper limit is generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide adapters, their lengths may all be the same or different.

[0674] For example, such peptide linkers include:

[0675] Ser、

[0676] Gly-Ser

[0677] Gly-Gly-Ser、

[0678] Ser-Gly-Gly、

[0679] Gly-Gly-Gly-Ser (SEQ ID NO: 5),

[0680] Ser-Gly-Gly-Gly (SEQ ID NO: 6),

[0681] Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7),

[0682] Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 8),

[0683] Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9),

[0684] Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 10),

[0685] Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 11),

[0686] Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 12),

[0687] (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7))n and

[0688] (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 8))n,

[0689] Where n is an integer of 1 or greater. Those skilled in the art can select the length or sequence of the peptide linker accordingly, depending on the purpose.

[0690] Synthetic linkers (chemical crosslinking agents) are commonly used for crosslinking peptides, and examples include:

[0691] N-hydroxysuccinimide (NHS)

[0692] Disuccinimide (DSS)

[0693] bis(sulfosuccinimino) octanoate (BS3)

[0694] Dithiobis(succiniminopropionate) (DSP)

[0695] Dithiobis(sulfosucciniminopropionate) (DTSSP)

[0696] Ethylene glycol bis(succiniminosuccinate) (EGS)

[0697] Ethylene glycol bis(sulfosucciniminosuccinate) (sulfon-EGS)

[0698] Disuccinimide tartrate (DST) and disulfonylsuccinimide tartrate (sulfon-DST).

[0699] bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES), and

[0700] Bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfon-BSOCOES). These crosslinking agents are commercially available.

[0701] Generally, three adapters are needed to link the four antibody variable regions together. The adapters used can be of the same type or different types.

[0702] Fab, F(ab')2 and Fab'

[0703] A “Fab molecule” refers to a protein composed of the VH and CH1 domains of the heavy chain (“Fab heavy chain”) and the VL and CL domains of the light chain (“Fab light chain”) of an immunoglobulin. The heavy chain of a wild-type Fab molecule cannot form disulfide bonds with another heavy chain molecule. In this document, in addition to wild-type Fab molecules, Fab variants are also included where the amino acid residues in the wild-type Fab molecule are altered through substitution, addition, or deletion. In specific embodiments, mutated amino acid residues contained in Fab variants (e.g., substituted, added, or inserted cysteine ​​or lysine residues) can form disulfide bonds with another heavy chain molecule or a portion thereof (e.g., the Fab molecule).

[0704] The scFab is an antigen-binding domain in which a single light chain forming the Fab and a CH1 region and variable region from a single heavy chain are linked together via peptide linkers. The light chain, the CH1 region from the heavy chain, and the variable region are kept in close proximity by the peptide linkers.

[0705] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin and papain, and specifically refers to the antibody fragment generated by digesting immunoglobulins (monoclonal antibodies) near disulfide bonds present between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds present between the hinge regions of each of the two H chains to generate two homologous antibody fragments, in which the L chain containing the VL (variable region of the L chain) and CL (constant region of the L chain) is linked via disulfide bonds at its C-terminal region to an H chain fragment containing the VH (variable region of the H chain) and CHγ1 (γ1 region in the constant region of the H chain). Each of these two homologous antibody fragments is called Fab'.

[0706] “F(ab')2” consists of two light chains and two heavy chains, the heavy chains containing a constant region of the CH1 domain and a portion 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 onto a protein A column. The protease is not particularly limited, as long as it can selectively cleave the entire antibody to produce F(ab')2 under suitable enzymatic reaction conditions such as pH. Such proteases include, for example, pepsin and figokinase.

[0707] Fusion

[0708] "Fusion" means that components (e.g., Fab molecules and Fc domain subunits) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0709] "Cross" Fab

[0710] A “crossfab” molecule (also known as a “cross-fab”) refers to a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged. Specifically, a crossfab molecule comprises a peptide chain consisting of a variable region of the light chain and a constant region of the heavy chain, and a peptide chain consisting of a variable region of the heavy chain and a constant region of the light chain. For clarity, in a crossfab molecule in which the variable regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the constant region of the heavy chain is referred to herein as the “heavy chain” of the crossfab molecule. Conversely, in a crossfab molecule in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the variable region of the heavy chain is referred to herein as the “heavy chain” of the crossfab molecule.

[0711] "Regular" Fab

[0712] In contrast, "conventional" Fab molecules refer to Fab molecules in their natural form, namely, heavy chains consisting of variable and constant regions of heavy chains (VH-CH1) and light chains consisting of variable and constant regions of light chains (VL-CL).

[0713] Single domain antibodies

[0714] In this paper, the term "single-domain antibody" refers to an antibody whose structure is not particularly limited, as long as the domain itself can exert antigen-binding activity. A common antibody, such as an IgG antibody, exerts antigen-binding activity by forming a variable region through VH and VL pairing. In contrast, known single-domain antibodies can exert antigen-binding activity solely through their own domain structure, without pairing with another domain. Single-domain antibodies typically have a relatively low molecular weight and exist in monomeric form.

[0715] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that naturally lack a light chain, such as the VHH of camels and the V of sharks. NARAnd antibody fragments comprising all or a portion of the antibody VH domain or all or a portion of the antibody VL domain. Examples of single-domain antibodies comprising all or a portion of the antibody VH / VL domains 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).

[0716] 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, camels and transgenic animals in which genes capable of producing single-domain antibodies have been introduced. Camels include camels, llamas, alpacas, dromedary camels, guanacos, etc. Examples of transgenic animals in 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 US Patent Publication No. US2011 / 0123527 A1. Humanized single-chain antibodies can also be obtained by replacing the frame sequence of a single-domain antibody obtained from an animal with a human germline sequence or a similar sequence. Humanized single-domain antibodies (e.g., humanized VHH) are one example of single-domain antibodies.

[0717] Alternatively, single-domain antibodies can be obtained from peptide libraries containing single-domain antibodies using methods such as ELISA and panning. Examples of peptide libraries containing single-domain antibodies include, but are not limited to, initial 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 from 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)).

[0718] As used in this article, an "agonist" antigen-binding molecule or "agonist" antibody is an antigen-binding molecule or antibody that significantly enhances the biological activity of the antigen it binds to.

[0719] As used herein, a “blocking” antigen-binding molecule or “blocking” antibody, or an “antagonistic” antigen-binding molecule or “antagonistic” antibody, is an antigen-binding molecule or antibody that significantly inhibits (partially or completely) the biological activity of the antigen it binds to.

[0720] antigen

[0721] As used herein, the term "antigen" refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-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 serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins referred to herein as antigens (e.g., CD3, CD137, DLL3) can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In specific embodiments, the antigen is human CD3, human CD137, or human DLL3. When referring to a specific protein herein, the term covers "full-length," unprocessed proteins, as well as any form of protein produced by intracellular processing. The term also covers naturally occurring protein variants, such as splice variants or allelic variants.

[0722] Fc region with reduced Fcγ receptor binding activity

[0723] In this document, "reduced Fcγ receptor binding activity" means, for example, that, based on the above analytical methods, the competitive activity of the test antigen-binding molecule or antibody is 50% or less compared to the competitive activity of the control antigen-binding molecule or antibody, 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.

[0724] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies may be appropriately 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). Furthermore, when using antigen-binding molecules or antibodies containing Fc domain mutants of a specific isotype of antibody as test material, antigen-binding molecules or antibodies containing the same isotype of Fc domain are used as controls to assess the effect of the mutation on Fcγ receptor binding activity. As described above, antigen-binding molecules or antibodies containing Fc domain mutants that are determined to have reduced Fc γ receptor binding activity are appropriately prepared.

[0725] Known mutants of this kind include, for example, mutants with deletions of amino acids 231A to 238S (EU number) (WO 2009 / 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).

[0726] Specifically, preferred antigen-binding molecules or antibodies include antigen-binding molecules or antibodies comprising an Fc domain, wherein the Fc domain of the antibody forming a specific isotype has 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 number). There are no particular restrictions on the isotype of the antibody from which the Fc domain is derived, and appropriate Fc domains derived from monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be used. Fc domains derived from IgG1 antibodies are preferred.

[0727] 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, wherein the position of the substitution is specified in the amino acids of the Fc domain forming the IgG1 antibody according to EU numbers (each number represents the position of an amino acid residue in the EU number; 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):

[0728] (a) L234F, L235E, P331S;

[0729] (b) C226S, C229S, P238S;

[0730] (c) C226S, C229S; or

[0731] (d) C226S, C229S, E233P, L234V, L235A;

[0732] (e) L439M, A445N, R449Q, E451S

[0733] (f) M428L, N434A, Q438R, S440E,

[0734] And antigen-binding molecules or antibodies having an Fc domain with a deletion of amino acid sequence at positions 231 to 238.

[0735] Furthermore, preferred antigen-binding molecules or antibodies also include Fc domains comprising any of the substitutions shown below, wherein the substitutions are located in the amino acids of the Fc domain forming the IgG2 antibody according to EU numbers:

[0736] (e) H268Q, V309L, A330S and P331S;

[0737] (f) V234A;

[0738] (g) G237A;

[0739] (h) V234A and G237A;

[0740] (i) A235E and G237A; or

[0741] (j) V234A, A235E, and G237A. Each number represents the position of an amino acid residue in the EU number; 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.

[0742] Furthermore, preferred antigen-binding molecules or antibodies also include Fc domains comprising any of the substitutions shown below, the positions of which are specified according to EU numbers in the amino acids of the Fc domain forming the IgG3 antibody:

[0743] (k) F241A;

[0744] (l) D265A; or

[0745] (m) V264A. Each number represents the position of an amino acid residue in the EU number; 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.

[0746] Furthermore, preferred antigen-binding molecules or antibodies also include Fc domains comprising any of the substitutions shown below, the positions of which are specified according to EU numbers in the amino acids of the Fc domain forming the IgG4 antibody:

[0747] (n) L235A, G237A, and E318A;

[0748] (o) L235E; or

[0749] (p) F234A and L235A. Each number represents the position of an amino acid residue in the EU number; 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.

[0750] Other preferred antigen-binding molecules or antibodies include, for example, antigen-binding molecules or antibodies containing an Fc domain, wherein any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU number) of the amino acid in the Fc domain forming the IgG1 antibody is replaced by the corresponding amino acid at the corresponding position in the EU number of the corresponding IgG2 or IgG4.

[0751] Preferred antigen-binding molecules or antibodies also include, for example, antigen-binding molecules or antibodies comprising an Fc domain, wherein any or more of the amino acids at positions 234, 235, and 297 (EU number) of the Fc domain forming the IgG1 antibody are substituted with other amino acids. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies comprising an Fc domain in which any or more of the amino acids at positions 234, 235, and 297 are substituted with alanine are particularly preferred.

[0752] 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 number) of the Fc domain forming the IgG1 antibody is substituted with another amino acid. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies comprising an Fc domain in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0753] (Preferred) enrichment (or increase)

[0754] The term “(preferred) 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 according to EU number in the respective CH1 region of the first antigen-binding domain and the second antigen-binding domain. In some embodiments, the methods produce homogeneous antibody formulations 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.

[0755] homogeneous

[0756] A “homogeneous” population of antibodies refers to a population of antibodies that primarily comprises a single form of antibody, for example, at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99% or 100% of the antibody in a solution or composition is in its correctly folded form. Similarly, a “homogeneous” population of antibodies having at least one disulfide bond formed outside the hinge region refers to a population of antibodies that primarily 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 said antibody having at least one disulfide bond formed outside the hinge region). In a preferred embodiment, the “homogeneous” population of the antibody contains at least one disulfide bond formed between amino acid residues at position 191 according to EU number in the corresponding CH1 region of the first antigen-binding domain and the second antigen-binding domain (i.e., “paired cysteine” at position 191 according to EU number in the CH1 region).

[0757] A variety of analytical and / or qualitative techniques can be used to determine whether antibody populations are homogeneous and the relative abundance or proportion of protein / antibody conformations in a mixture. If two conformations are dissociated differently during separation techniques such as chromatography, electrophoresis, filtration, or other purification techniques, such purification techniques can be used to determine the relative proportions of conformations in a mixture. For example, at least two different conformations of recombinant IgG can be dissociated by hydrophobic interaction chromatography. Furthermore, since far-UV circular dichroism has been used to estimate the secondary structural composition of proteins (Perczel et al., 1991, Protein Engrg. 4:669-679), such techniques can determine the presence of alternative conformations of proteins. Another technique for determining conformations is fluorescence spectroscopy, which can be used to determine complementary differences in the tertiary structure that can be partitioned by tryptophan and tyrosine fluorescence. Other techniques that can be used to determine differences in conformations and thus the relative proportions of conformations include: online SEC for measuring aggregation states, differential scanning calorimetry for measuring melting transition (Tm) and component enthalpy, and ionizing agent defolding. Another technique that can be used to determine conformational differences and thus the relative proportions of conformations is LC / MS detection to determine protein heterogeneity.

[0758] Alternatively, if there are activity differences between antibody / protein conformations, the relative proportions of the conformations in the mixture can be determined by activity assays (e.g., ligand binding, enzyme activity, biological activity, etc.). Protein biological activity can also be used. Alternatively, binding assays can be used, where activity is expressed as active units / mg protein.

[0759] To determine antibody / protein heterogeneity, IEC chromatography is used. In this case, the antibody is purified or considered "homogeneous," meaning that no peptide peaks or fractions corresponding to other peptides are detected upon analysis by IEC chromatography. In some embodiments, the antibody is purified or considered "homogeneous" such that no peptide bands corresponding to other peptides are detected upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the art will recognize that multiple bands corresponding to a peptide can be visualized by SDS-PAGE due to differential glycosylation, differential post-translational processing, etc. Most preferably, the peptide is purified to be substantially homogeneous, as indicated by a single peptide band after analysis by SDS-PAGE. Peptide bands can be visualized by silver staining, Coomassie brilliant blue staining, and / or (if the peptide is radiolabeled) by autoradiography.

[0760] Examples of SDS-PAGE analysis conditions are provided below. 4% to 20% Mini-PROTEAN (registered trademark) TGX Stain-Free was used. TM Non-reducing SDS-PAGE was performed on a pre-prepared gel (Bio-Rad) with 1x Tris / glycine / SDS run buffer (Bio-Rad). Monoclonal antibody samples were heated at 70°C for 10 min. 0.2 μg of sample was loaded, and electrophoresis was performed at 200 V for 90 min. Protein visualization was performed using a Chemidoc imaging system (Bio-Rad). The percentage of individual bands was analyzed using Image Lab software version 6.0 (Bio-Rad), where the intensity % of a single band (e.g., faster migration (lower band) and slower migration (upper band)) was calculated by dividing the band intensity by the sum of the two bands. The gel was then stained with CBB, and gel images were captured, allowing for band quantification using the imaging device. In the gel images, several bands may be observed for antibody variant samples, such as two bands, namely the "upper band" and the "lower band." In this case, the molecular weight of the upper band corresponds to the molecular weight of the parent antibody (before modification). Structural changes (such as disulfide crosslinking via Fab) can be caused by cysteine ​​substitutions, which can lead to changes in electrophoretic mobility. In this case, the lower band can be considered to correspond to an antibody with one or more engineered disulfide bonds formed between the CH1 regions. Antibody variants with additional cysteine ​​substitutions can show a higher lower band to upper band ratio compared to control samples. Additional cysteine ​​substitutions can enhance / promote disulfide crosslinking of Fab; and can increase the percentage or structural homogeneity of antibody formulations with engineered disulfide bonds formed at the mutation site; and can decrease the percentage of antibody formulations that do not form engineered disulfide bonds at the mutation site. In this paper, the term "lower band to upper band ratio" refers to the ratio between the number / intensity of the lower band and the upper band, which can be quantified during the above SDS-PAGE experiments.

[0761] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a formulation in which the bioactive ingredient contained therein is in a form in which the active ingredient is permitted to be contained and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0762] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0763] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0764] II. For producing a compound comprising at least one amino acid residue having formed between amino acid residues in a region outside the hinge region. Methods for antigen-binding molecules in the form of disulfide bonds (LINC form), and methods for comparing LINC form with LINC form. Antigen binding components that do not have disulfide bonds formed between amino acid residues in regions outside the hinge region and those that do not have such bonds. Methods for measuring, determining, or quantifying the ratio of the sum of sub-sub ...

[0765] This disclosure relates to a method for producing 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. The method of this disclosure includes a step of chromatographically processing a mixture in the presence of a reducing agent, wherein the mixture comprises the antigen-binding molecule and mis-disulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule, the antigen-binding molecule having 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 containing an antigen-binding molecule," "mixture of antigen-binding molecules," etc.). In the method of this disclosure, the mixture containing the antigen-binding molecule may undergo one or more chromatography of one type. Alternatively, the mixture containing the antigen-binding molecule may be subjected to multiple types of chromatography (e.g., two, three, or more types) each, one or more times (e.g., two, three, or more times). When multiple chromatographic processes are performed, these processes may be performed sequentially or non-sequentially.

[0766] In the methods disclosed herein, chromatography techniques 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.

[0767] In the production method of this disclosure, the step of chromatographically processing the mixture containing antigen-binding molecules is carried out in the presence of a reducing agent. There are no limitations on the embodiments of the step of chromatographically processing the mixture containing antigen-binding molecules in the production method of this disclosure, as long as a reducing agent is present. For example, the antigen-binding molecules can be pre-loaded onto a chromatographic matrix and then contacted with a reducing agent, or the mixture containing antigen-binding molecules can be mixed with a reducing agent to obtain a substance that is passed through a chromatograph.

[0768] In one aspect, when using column chromatography, the method of this disclosure may include the step of contacting a chromatographic matrix loaded onto a column for column chromatography with a mixture comprising an antigen-binding molecule and an incorrectly disulfide-bonded and / or non-disulfide-bonded form of the antigen-binding molecule, 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 aspect, when using membrane chromatography, the method of this disclosure may include the step of contacting a chromatographic matrix applied to a membrane for membrane chromatography with a mixture comprising an antigen-binding molecule and an incorrectly disulfide-bonded and / or non-disulfide-bonded form of the antigen-binding molecule, the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. The mixture comprising the antigen-binding molecule may be pre-contaminated with a reducing agent.

[0769] In this disclosure, the step of chromatographically reacting an antigen-binding molecule with a mixture of misdisulfide-bonded and / or non-disulfide-bonded forms of the antigen-binding molecule may include the step of the antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region:

[0770] (a) Contacting a solution containing an antigen-binding molecule and a mixture of the antigen-binding molecule in an incorrectly disulfide-bonded form and / or a non-disulfide-bonded form with a reducing agent, wherein the antigen-binding molecule has at least one disulfide bond formed between amino acid residues in a region outside the hinge region; and

[0771] (b) Remove the reducing agent.

[0772] In one aspect, the methods of this disclosure are used to produce or purify populations of homologous antigen-binding molecules or formulations or concentrates of homologous antigen-binding molecules by the steps described herein.

[0773] In one aspect, the method of this disclosure can increase the proportion of antigen-binding molecules having at least one disulfide bond between amino acid residues in regions outside the hinge region, relative to antigen-binding molecules loaded onto a matrix having amino acid residues that can form at least one disulfide bond between amino acid residues in regions outside the hinge region (such antigen-binding molecules include antigen-binding molecules that properly form at least one disulfide bond between amino acid residues in regions outside the hinge region, and antigen-binding molecules that do not properly form at least one disulfide bond between amino acid residues in regions outside the hinge region (i.e., incorrect disulfide bonding forms and / or non-disulfide bonding forms of antigen-binding molecules)).

[0774] In one aspect, relative to an antigen-binding molecule having at least one disulfide bond between amino acid residues in a region outside the hinge region that is loaded onto a matrix (such antigen-binding molecules include antigen-binding molecules that suitably form at least one disulfide bond between amino acid residues in a region outside the hinge region, and antigen-binding molecules that do not suitably form at least one disulfide bond between amino acid residues in a region outside the hinge region (or in other words, erroneous disulfide bonded forms and / or non-disulfide bonded forms of antigen-binding molecules)), the method of this disclosure produces antigen-binding molecules having at least one disulfide bond between amino acid residues in a region outside 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%.

[0775] Therefore, in one embodiment, this disclosure relates to a method for producing a formulation comprising an increased concentration of an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region. In one embodiment, the formulation produced by the method of this disclosure may be a concentrate of an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region. Furthermore, in one embodiment, this disclosure relates to a method for increasing the concentration of an antigen-binding molecule in a mixture of antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region outside the hinge region. In another embodiment, this disclosure relates to a method for purifying a formulation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region from a mixture comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and mis-disulfide bonded forms and / or non-disulfide bonded forms of the antigen-binding molecule. In this disclosure, antigen-binding molecules that do not have disulfide bonds formed between amino acid residues in regions outside the hinge region include antigen-binding molecules in regions outside the hinge region where cysteine ​​residues are capped, or which exist only as cysteine.

[0776] In the methods of this disclosure, a mixture of antigen-binding molecules is contacted with a solution containing a reducing agent. In specific embodiments, the mixture of antigen-binding molecules loaded onto a matrix is ​​contacted with a solution containing a reducing agent in chromatography. In one aspect, examples of chromatographic matrices of this disclosure include, but are not limited to, affinity chromatography matrices, ion exchange chromatography matrices, hydrophobic interaction chromatography matrices, matrices for multimode chromatography incorporating both ion exchange chromatography and hydrophobic interaction chromatography, and hydroxyapatite matrices. In this disclosure, the terms "matrix," "resin," "ligand," and "substrate" are used interchangeably.

[0777] In this disclosure, the affinity chromatography matrix is ​​not limited, as long as it exhibits affinity chromatography activity. In one aspect, examples of affinity chromatography matrices include, but are not limited to, protein A matrices, protein G matrices, protein L matrices, sequence-selective peptide matrices, and matrices that selectively bind to antigen-binding molecules.

[0778] In the methods disclosed herein, commercially available products can be used as affinity chromatography matrices. In specific aspects, the methods are not limited to the following:

[0779] MabSelect SuRe (registered trademark) (Cytiva Corporation)

[0780] MabSelect Xtra (registered trademark) (Cytiva Corporation)

[0781] MabSelect SuRe (registered trademark) LX (Cytiva Corporation)

[0782] MabSelect SuRe (registered trademark) pcc (Cytiva Corporation)

[0783] MabSelect PrismA (registered trademark) (Cytiva Corporation)

[0784] MabSpeed ​​(registered trademark) rP202 (Cytiva Corporation)

[0785] TOYOPEARL (registered trademark) Protein A HC-650F (TOSOH Corporation)

[0786] Praesto (registered trademark) Jetted A50 (Purolite Corporation)

[0787] Amsphere (registered trademark) A3 (JSR Corporation)

[0788] Amsphere (registered trademark) Protein A JWT203 (JSR Corporation)

[0789] Eshmuno A (registered trademark) (Merck Millipore Corporation)

[0790] Capto L (registered trademark) (Cytiva Corporation) and

[0791] MabSelect (registered trademark) VL (Cytiva Corporation)

[0792] It can serve as an example of an affinity chromatography matrix.

[0793] In one aspect, the ion exchange chromatography matrix of this disclosure includes anion exchange resins (anion exchange ligands) or cation exchange resins (cation exchange ligands). The anion exchange resins and cation exchange resins of this disclosure are not limited, as long as they exhibit anion exchange activity and cation exchange activity, respectively.

[0794] In the methods disclosed herein, commercially available products can be used as anion exchange chromatography matrix. In specific aspects, the methods are not limited to the following:

[0795] YMC-BioPro (YMC Corporation),

[0796] Q Sepharose (registered trademark) High performance (Cytiva Corporation)

[0797] Q Sepharose (registered trademark) Rapid Flow (Cytiva Corporation)

[0798] Q Sepharose (registered trademark) XL (Cytiva Corporation)

[0799] Q Sepharose (registered trademark) large pearl (Cytiva Corporation),

[0800] Capto (registered trademark) Q ImpRes (Cytiva Corporation)

[0801] Capto (registered trademark) Q (Cytiva Corporation)

[0802] Capto (registered trademark) Q XP (Cytiva Corporation)

[0803] Capto (registered trademark) DEAE (Cytiva Corporation)

[0804] SOURCE (registered trademark) 30Q (Cytiva Corporation)

[0805] SOURCE (registered trademark) 15Q (Cytiva Corporation)

[0806] DEAE Sepharose (registered trademark) - Rapid Flow (Cytiva Corporation)

[0807] ANX Sepharose (registered trademark) 4. Rapid Flow (Cytiva Corporation)

[0808] POROS (registered trademark) 50 PI (Thermo Fisher Corporation)

[0809] POROS (registered trademark) 50 HQ (Thermo Fisher Corporation)

[0810] POROS (registered trademark) HQ (Thermo Fisher Corporation)

[0811] POROS (registered trademark) D (Thermo Fisher Corporation)

[0812] POROS (registered trademark) PI (Thermo Fisher Corporation)

[0813] Eshumuno (registered trademark) Q (Merck Millipore Corporation)

[0814] Fractogel (registered trademark) TMAE (Merck Millipore Corporation)

[0815] Fractogel (registered trademark) DEAE (Merck Millipore Corporation),

[0816] Macro-Prep (registered trademark) Q (Bio-Rad Laboratories Corporation)

[0817] Macro-Prep (registered trademark) DEAE (Bio-Rad Laboratories Corporation)

[0818] Giga Cap (registered trademark) Q-650M (TOSOH Corporation)

[0819] Giga Cap (registered trademark) DEAE-650M (TOSOH Corporation) and

[0820] Q HyperCel (registered trademark) (PALL Corporation)

[0821] It can be used as an example of anion exchange chromatography matrix.

[0822] Commercially available products can also be used as cation exchange chromatography matrices. Specifically, this includes, but is not limited to, the following:

[0823] Capto (registered trademark) S (Cytiva Corporation)

[0824] Capto (registered trademark) SP ImpRes (Cytiva Corporation)

[0825] Capto (registered trademark) S ImpaAct (Cytiva Corporation)

[0826] SP Sepharose (registered trademark) Rapid Flow (Cytiva Corporation)

[0827] CM Sepharose (registered trademark) - Rapid Flow (Cytiva Corporation)

[0828] SP Sepharose (registered trademark) High Performance (Cytiva Corporation)

[0829] CM Sepharose (registered trademark) High Performance (Cytiva Corporation)

[0830] SP Sepharose (registered trademark) XL (Cytiva Corporation)

[0831] SP Sepharose (registered trademark) Large Beads (Cytiva Corporation)

[0832] Eshumuno (registered trademark) CPX (Merck Millipore Corporation)

[0833] Eshumuno (registered trademark) CP-FT (Merck Millipore Corporation)

[0834] POROS (registered trademark) 50HS (Thermo Fisher Corporation) and

[0835] POROS (registered trademark) XS (Thermo Fisher Corporation)

[0836] It can serve as an example of a cation exchange chromatography matrix.

[0837] In this disclosure, the matrix used for hydrophobic interaction chromatography is not limited, as long as it exhibits hydrophobic interaction chromatographic activity. In one aspect, the hydrophobic interaction chromatographic matrix includes hydrophobic ligands.

[0838] In the methods disclosed herein, commercially available products can be used as hydrophobic interaction chromatographic matrices. In specific aspects, the methods are not limited to the following:

[0839] Phenyl Sepharose (registered trademark) High Performance (Cytiva Corporation)

[0840] Butyl Sepharose (registered trademark) High Performance (Cytiva Corporation)

[0841] Phenyle Sepharose (registered trademark) 6 Fast Flow (Cytiva Corporation)

[0842] Butyl-S Sepharose (registered trademark) 6 Fast Flow (Cytiva Corporation)

[0843] Butyl Sepharose (registered trademark) 4 Fast Flow (Cytiva Corporation)

[0844] Sepharose (registered trademark) 4. Rapid Flow (Cytiva Corporation)

[0845] Capto (registered trademark) Phenyl ImpRes (Cytiva Corporation)

[0846] Capto (registered trademark) phenyl (Cytiva Corporation)

[0847] Capto (registered trademark) Phenylene (High Sub) (Cytiva Corporation)

[0848] Capto (registered trademark) butyl (Cytiva Corporation)

[0849] Capto (registered trademark) Butyl ImpRes (Cytiva Corporation)

[0850] Capto (registered trademark) (Cytiva Corporation)

[0851] Phenylacetylene Sepharose (registered trademark) 6 Fast Flow (Low Sub) (Cytiva Corporation)

[0852] Phenylacetylene Sepharose (registered trademark) 6 Fast Flow (High Sub) (Cytiva Corporation)

[0853] POROS (registered trademark) Ethyl (Thermo Fisher Corporation)

[0854] Fractogel (registered trademark) phenyl (Merck Millipore Corporation)

[0855] Fractogel (registered trademark) propyl (Merck Millipore Corporation),

[0856] TOYOPEARL (registered trademark) Butyl (TOSOH Corporation)

[0857] TOYOPEARL (registered trademark) Ether (TOSOH Corporation)

[0858] TOYOPEARL (registered trademark) by TOSOH Corporation

[0859] TOYOPEARL (registered trademark) phenyl (TOSOH Corporation)

[0860] TOYOPEARL (registered trademark) PPG (TOSOH Corporation)

[0861] TOYOPEARL (registered trademark) Butyl (TOSOH Corporation)

[0862] TOYOPEARL (registered trademark) Butyl-600 (TOSOH Corporation)

[0863] TOYOPEARL (registered trademark) Phenylen-650C (TOSOH Corporation)

[0864] TOYOPEARL (registered trademark) Phenylenol-650M (TOSOH Corporation)

[0865] TOYOPEARL (registered trademark) Phenylenol-650S (TOSOH Corporation)

[0866] TOYOPEARL (registered trademark) Phenylenol-600M (TOSOH Corporation) and

[0867] Macro-Prep (registered trademark) HIC (Bio-Rad Laboratories Corporation)

[0868] It can serve as an example of a hydrophobic interaction chromatographic matrix.

[0869] In one aspect, the multimode chromatographic matrix of this disclosure may include a matrix combining cation exchange ligands and hydrophobic ligands, or a matrix combining anion exchange ligands and hydrophobic ligands. In the methods of this disclosure, commercially available products can be used in the multimode chromatographic matrix. For example, not limited to, the following...

[0870] Capto (registered trademark) adhere (Cytiva Corporation)

[0871] Capto (registered trademark) adhere ImpRes (Cytiva Corporation)

[0872] Capto (registered trademark) MMC (Cytiva Corporation)

[0873] Capto (registered trademark), MMC ImpRes (Cytiva Corporation), and

[0874] Eshmuno (registered trademark) CMX (Merck Millipore Corporation)

[0875] It can serve as an example of a matrix for use in multimode chromatography.

[0876] In one aspect, the hydroxyapatite chromatographic matrix may include hydroxyapatite or its derivatives (such as fluorapatite). In the methods of this disclosure, commercially available products may also be used in the hydroxyapatite chromatographic matrix. In specific embodiments, the following are not limited to:

[0877] Ceramic hydroxyapatite (registered trademark) Type I (Bio-Rad Laboratories Corporation)

[0878] Ceramic hydroxyapatite (registered trademark) Type II (Bio-Rad Laboratories Corporation)

[0879] Ceramic Fluoroapatite (Registered Trademark) (Bio-Rad Laboratories Corporation)

[0880] MPC (registered trademark) ceramic hydroxyfluoroapatite (Bio-Rad Laboratories Corporation)

[0881] HA Ultrogel (registered trademark) (PALL Corporation) and

[0882] Ca++Pure-HA (Registered Trademark) (TOSOH Corporation)

[0883] This can serve as an example of a matrix for use in hydroxyapatite chromatography.

[0884] In the methods disclosed herein, commercially available products can also be used for membrane chromatography. In specific embodiments, the following are not limited to:

[0885] Mustang (registered trademark) Q (PALL Corporation)

[0886] Mustang (registered trademark) S (PALL Corporation),

[0887] Sartobind (registered trademark) Q (Sartorius Corporation)

[0888] Sartobind (registered trademark) S (Sartorius Corporation)

[0889] Sartobind (registered trademark) STIC (Sartorius Corporation)

[0890] Sartobind (registered trademark) Protein A (Sartorius Corporation)

[0891] Fibro (Cytiva Corporation)

[0892] Protein capture (GORE Corporation)

[0893] Natrix (registered trademark) Q (Merck Millipore Corporation),

[0894] Purexa (registered trademark) - MQ (Purilogics Corporation)

[0895] Purexa (registered trademark) - A (Purilogics Corporation) and

[0896] Purexa (registered trademark) - DMAE (Purilogics Corporation)

[0897] This can serve as an example of a product used in membrane chromatography.

[0898] In this disclosure, the terms "reducing reagent," "reducing agent," and "solution containing a reducing agent" are used interchangeably. In some embodiments, the reducing agent described above is a free thiol. Furthermore, in one aspect, the reducing agent in this disclosure can be a monothiol, a dithiol, a phosphine, or an inorganic reagent. Examples of monothiols in this disclosure include, but are not limited to, glutathione, cysteine, lipoic acid, 2-mercaptoethanol, and 2-MEA. Examples of dithiols in this disclosure include, but are not limited to, dithiothreitol (DTT), DTE, and DTBA. Examples of phosphine in this disclosure include, but are not limited to, tris(2-carboxyethyl)phosphine (TCEP) and THPP. Examples of inorganic reagents in this disclosure include, but are not limited to, sodium sulfite and sodium metabisulfite.

[0899] The reducing agent is preferably composed of compounds selected from the group consisting of glutathione (GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-aminoethanethiol (2-MEA), tris(2-carboxyethyl)phosphine (TCEP), dithionitrobenzene, cysteine, sodium sulfite, sodium metabisulfite, and Na₂SO₃. In some embodiments, TCEP, 2-MEA, DTT, cysteine, GSH, or Na₂SO₃ may be used. In some preferred embodiments, cysteine ​​may be used. In some preferred embodiments, TCEP may be used. In the methods of this disclosure, one or more types (e.g., two, three, or more types) of reducing agents may be used.

[0900] In one embodiment, the reducing agent may be a solution, or preferably in the form of a buffer solution. Examples of buffer solutions include, but are not limited to, buffers used in chromatographic techniques, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer. Furthermore, examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris.

[0901] In this disclosure, "contact" means subjecting or exposing a mixture of antigen-binding molecules to a solution containing a reducing agent, or mixing the mixture of antigen-binding molecules with a solution containing a reducing agent. In the methods of this disclosure, the mixture of antigen-binding molecules may be contacted with a solution containing a reducing agent while the molecules are in a bound state to a solid matrix (e.g., a column chromatography column or a membrane chromatography membrane).

[0902] While not bound by the following theory, it is thought that the presence of unLINC forms (i.e., divalent or trivalent antigen-binding molecules without engineered disulfide bonds or "paired cysteines") may be due to the frequent formation of disulfide bonds between unpaired Cys residues and molecules containing free thiol groups, such as cysteine ​​and glutathioneization, which prevent LINC formation (the formation of engineered disulfide bonds in regions outside the hinge region) by "capping" unpaired Cys residues. To remove molecules with unpaired capped cysteines, reducing agents can facilitate the decapping of surface cysteines, and further re-oxidation of the decapped antigen-binding molecule (e.g., by removing the reducing agent) can promote disulfide bond formation between the decapped cysteines for LINC formation. Therefore, removing cysteine ​​from unpaired thiol groups in unLINC forms through reduction and re-oxidation can eliminate the unLINC form and improve antibody homogeneity.

[0903] In one aspect, in the method of this disclosure, contacting a mixture of antigen-binding molecules with a solution containing a reducing agent causes the cleavage of disulfide bonds formed between amino acid residues that can form disulfide bonds in the antigen-binding molecules. In another aspect, contacting a mixture of antigen-binding molecules with a solution containing a reducing agent causes the decapping of capped cysteine ​​residues in the antigen-binding molecules.

[0904] In one aspect, in the method of this disclosure, the removal of the reducing agent results in the formation of disulfide bonds between amino acid residues that can form disulfide bonds in antigen-binding molecules.

[0905] The mixture of antigen-binding molecules is contacted with a solution containing a reducing agent for a sufficient time to increase the relative proportion of the desired conformation. Any relative increase in 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 the undesired conformation being converted to the antigen-binding molecules having the desired conformation.

[0906] In one aspect, in the method of this disclosure, a solution containing a reducing agent is passed through a chromatographic system containing a matrix carrying antigen-binding molecules. In a specific embodiment, the antigen-binding molecules constitute a stationary phase immobilized to the chromatographic matrix, and the solution containing the reducing agent is part of the mobile phase. In this case, contact can be performed as part of a chromatographic purification procedure.

[0907] The reducing agent is present at a concentration sufficient to increase the desired conformation (e.g., an antigen-binding molecule in the form of "paired cysteine" molecules, which have one or more engineered disulfide bonds between the two Fabs of the antibody (e.g., between regions outside the hinge region). The optimal absolute concentration and molar ratio of the reducing agent depend on the total concentration of the antigen-binding molecules, and in some cases also on the concentration of the specific antigen-binding molecule. Furthermore, the reducing agent concentration also depends on the number and availability of unpaired cysteines in the antigen-binding molecule.

[0908] In one aspect, examples of the concentration of the reducing agent include a range that can be specified by any combination of the following: 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, the concentration of the reducing agent may range from 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. In other embodiments, the concentration of the reducing agent may range from 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. In other embodiments, the concentration of the reducing agent may range from 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. In specific embodiments, the concentration of the reducing agent may 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.

[0909] 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.

[0910] In specific embodiments, 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 specific embodiments, 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.

[0911] To maximize the yield of antigen-binding molecules with 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 bond exchange. In one aspect, the pH of the solution containing the reducing agent of this 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 invention, optimal pH has been found to be from about 7.0, about 7.5, or about 8.0. However, the optimal pH in specific embodiments of the invention can be readily determined experimentally by those skilled in the art.

[0912] In the method of this disclosure, the contact between the chromatographic matrix, the antigen-binding molecule, and the reducing agent occurs in chromatography. In one embodiment, in the method of this disclosure, the contact between the antigen-binding molecule loaded onto the matrix and the reducing agent occurs in chromatography. In other words, in the method of this disclosure, the contact between the reducing agent and the antigen-binding molecule can be achieved by passing a solution containing the reducing agent through a column chromatography column or membrane chromatography apparatus containing antigen-binding molecules loaded onto the matrix.

[0913] In some embodiments, the solution containing the reducing agent is passed through a column chromatography column or membrane chromatography apparatus for a residence time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes. In a specific embodiment, the solution containing the reducing agent is passed through a column chromatography column or membrane chromatography apparatus for a residence time of about 12 minutes or about 30 seconds. In some embodiments, flow may be temporarily stopped while the column or apparatus is filled with the solution containing the reducing agent.

[0914] In one aspect, when using column chromatography, the solution containing the reducing agent is passed 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, the solution containing the reducing agent is passed through the column for a residence time of about 12 minutes. In one aspect, flow can be temporarily stopped when 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 this disclosure can be passed through the column at a rate of about 25 cm / h to about 500 cm / h, about 50 cm / h to about 400 cm / h, about 75 cm / h to about 350 cm / h, or about 100 cm / h to about 300 cm / h. In some embodiments, the solution containing the reducing agent of this disclosure can be passed through the column at a rate of about 100 cm / h. In one aspect, flow can be temporarily stopped when the column is filled with the solution containing the reducing agent.

[0915] In one aspect, when using membrane chromatography, 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 about 3 seconds to about 6 minutes, for example, about 30 seconds. In some embodiments, flow may be temporarily stopped once the membrane device is filled with a solution containing a reducing agent. In one aspect, when using membrane chromatography, a solution containing the reducing agent of the present disclosure may be passed through the membrane device at a rate of about 0.017 MV / min to about 30 MV / min, about 0.17 MV / min to about 20 MV / min, about 1 MV / min to about 10 MV / min, or about 2 MV / min to about 5 MV / min. In a specific embodiment, a solution containing the reducing agent of the present disclosure may be passed through the membrane device at a rate of about 2 MV / min. Flow may be temporarily stopped once the membrane device is filled with solution.

[0916] There is no particular limitation on the amount of the solution containing the reducing agent flowing through the column, but for example, when using column chromatography, the flow volume of the solution containing the reducing agent can be from about 0.1 times (about 0.1 column volume (CV)) to about 100 times (about 100 CV), from about 1 times (about 1 CV) to about 20 times (about 20 CV), or from about 3 times (about 3 CV) to about 10 times (about 10 CV) of the column volume. In some embodiments, the amount of the solution containing the reducing agent flowing through the column can be about 10 times the column volume (about 10 CV). Furthermore, when using membrane chromatography, the flow volume of the solution containing the reducing agent can be from about 1 time (about 1 membrane volume (MV)) to about 500 times (about 500 MV), or from about 2 times (about 2 MV) to about 100 times (about 100 MV) of the membrane device volume. In some embodiments, the amount of the solution containing the reducing agent flowing through the column can be about 15 times the column volume (about 15 MV).

[0917] There is no particular limitation on the reaction time (contact time) between the mixture of antigen-binding molecules and the solution containing the reducing agent, 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 a particular embodiment, the contact time between the mixture of antigen-binding molecules and the solution containing the reducing agent may be about 120 minutes or about 7.5 minutes.

[0918] In one aspect, when using column chromatography, the contact time between the mixture of antigen-binding molecules and the solution containing the reducing agent can be from about 4 minutes to about 1440 minutes, from about 8 minutes to about 300 minutes, from about 30 minutes to about 240 minutes, from about 60 minutes to about 180 minutes, or about 120 minutes. In a specific embodiment, the contact time between the mixture of antigen-binding molecules and the solution containing the reducing agent can be about 120 minutes.

[0919] In one respect, when membrane chromatography is used, the contact time between the mixture of antigen-binding molecules and the solution containing the reducing agent can be from about 6 seconds to about 600 minutes or from about 18 seconds to about 120 minutes, for example, about 7.5 minutes.

[0920] In the methods disclosed herein, antigen-binding molecules can be contacted with a reducing agent in appropriate volumes, such as at analytical scales (1 mL to 50 mL), preparative scales (50 mL to 10 L), and manufacturing scales (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, for example about 10 g or about 25 g per 1 L of chromatographic matrix containing the substrate.

[0921] In one respect, when using column chromatography, the amount of antigen-binding molecules 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 per 1 L of chromatographic matrix containing the substrate, for example, about 10 g.

[0922] In one respect, when using membrane chromatography, the amount of antigen-binding molecules 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, for example about 25 g, per 1 L membrane device volume.

[0923] The method disclosed herein includes a step of removing a reducing agent. In this 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 the re-oxidation of disulfide bonds in regions outside the hinge region between the two Fabs constituting the antigen-binding molecule.

[0924] In one respect, the reducing agent can be removed by contacting the antigen-binding molecule with a solution that does not contain the reducing agent. In another respect, the reducing agent can be removed by passing the solution that does not contain the reducing agent through a column chromatography column or a membrane chromatography apparatus.

[0925] In some embodiments, a solution without a reducing agent is passed through a column chromatography column or a membrane chromatography apparatus 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 without a reducing agent is passed through a column chromatography column or a membrane chromatography apparatus for a residence time of about 4 minutes. In some embodiments, flow may be temporarily stopped when the column or apparatus is filled with solution.

[0926] In some embodiments, when using column chromatography, a solution without a reducing agent is passed 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 without a reducing agent is passed through the column for a residence time of about 4 minutes. In some embodiments, flow may be temporarily stopped when the column is filled with solution.

[0927] In some embodiments, when using column chromatography, a solution not containing a reducing agent can be flowed through the column at a rate of about 25 cm / h to about 500 cm / h, about 50 cm / h to about 450 cm / h, about 200 cm / h to about 400 cm / h, or about 250 cm / h to about 350 cm / h. Furthermore, in a specific embodiment, a solution not containing the reducing agent of this disclosure can be flowed through the column at a rate of about 300 cm / h. Flow can be temporarily stopped when the column is filled with solution.

[0928] In some embodiments, when using membrane chromatography, a solution without a reducing agent is passed through the membrane device for a residence time of about 2 seconds to about 60 minutes or about 3 seconds to about 6 minutes, for example, about 30 seconds. In some embodiments, the flow may be temporarily stopped while the device is filled with solution.

[0929] In some embodiments, when using membrane chromatography, a solution without a reducing agent can be flowed through the membrane device at a rate of about 0.017 MV / min to about 30 MV / min, about 0.17 MV / min to about 20 MV / min, about 1 MV / min to about 10 MV / min, or about 2 MV / min to about 5 MV / min. Furthermore, in a specific embodiment, a solution without the reducing agent of this disclosure can be flowed through the membrane device at a rate of about 2 MV / min. Flow can be temporarily stopped when the membrane device is filled with solution.

[0930] There are no particular limitations on the solution that does not contain a reducing agent, but in one instance, a buffer solution may be used, and more specifically, buffer solutions commonly used in chromatographic techniques, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer. Furthermore, examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris. In one aspect, in the methods of this disclosure, for solutions that do not contain a reducing agent, a solution of the same type as the solution containing a reducing agent used in the contact step may be used, except that a reducing agent is absent.

[0931] In one aspect, when using column chromatography, the flow volume of the solution without reducing agent can be from about 0.1 times (about 0.1 CV) to about 100 times (about 100 CV), from about 1 times (about 1 CV) to about 20 times (about 20 CV), or from about 3 times (about 3 CV) to about 10 times (about 10 CV) of the column volume. In some embodiments, the flow volume of the solution without reducing agent can be about 5.0 times the column volume (about 5.0 CV).

[0932] In one respect, when using membrane chromatography, the flow volume of the solution without reducing agent can be from about 1 MV to about 500 MV, or from about 2 MV to about 100 MV of the membrane device, for example about 15 MV of the membrane device.

[0933] In one aspect, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution without 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 a particular embodiment, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution without a reducing agent can be about 20 minutes.

[0934] In one aspect, when using column chromatography, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution without a reducing agent can be from about 4 minutes to about 1440 minutes or from about 8 minutes to about 300 minutes. In a particular embodiment, when using column chromatography, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution without a reducing agent can be about 20 minutes.

[0935] In one respect, when membrane chromatography is used, the reaction time (contact time) between the mixture of antigen-binding molecules and the 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, for example, about 7.5 minutes.

[0936] The method of the present invention can be carried out over a wide temperature range. For example, the method of the present invention can be carried out at about 4°C to about 37°C or about 15°C to about 37°C. Typical temperatures for formulations used to contact partially or completely purified antigen-binding molecules are about 4°C to about 25°C (ambient temperature), or preferably 23°C, but this contact can also be carried out at lower and higher temperatures. In some embodiments, the method of the present disclosure can be carried out at temperatures from about 20°C to 37°C, preferably 23°C, 25°C, or 37°C, and more preferably 23°C.

[0937] Furthermore, for solutions that do not contain a reducing agent, the optimal pH can be readily determined experimentally by those skilled in the art. Examples include pH 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 particular, in specific embodiments, the optimal pH may 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.

[0938] In one aspect, the method of this disclosure may include, prior to the step of contacting the mixture of antigen-binding molecules with a solution containing a reducing agent, contacting the mixture with a chromatographic matrix to fill the interior of the column with antigen-binding molecules or immobilizing the antigen-binding molecules onto a membrane. In a specific embodiment, when the mixture is a cell culture medium containing antigen-binding molecules (harvested cell culture medium: HCCF), prior to the step of contacting the cell culture medium with a solution containing a reducing agent, contacting the cell culture medium with a chromatographic matrix to fill the interior of the column with antigen-binding molecules or immobilizing the antigen-binding molecules onto a membrane may include. In one aspect, the mixture of antigen-binding molecules includes antigen-binding molecules having at least one disulfide bond in a region outside the hinge region (i.e., LINC form) and antigen-binding molecules not having disulfide bonds in a region outside the hinge region (i.e., unLINC form).

[0939] In some aspects, the antigen-binding molecule may be at least partially purified prior to the contact step with the solution containing the reducing agent. In some embodiments, the production method of this disclosure may include an affinity chromatography (preferably protein A chromatography) step on the cell culture medium containing the antigen-binding molecule prior to the contact step.

[0940] In one aspect, the methods of this disclosure may include a step of removing impurities from the chromatography prior to the contact step. As described in the example, 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.

[0941] In one aspect, the method of this disclosure may further include, before or after the step of removing the reducing agent, the steps of collecting or eluting antigen-binding molecules having at least one disulfide bond in a region outside the hinge region, and / or purifying the antigen-binding molecules. In one embodiment, the collection and elution of antigen-binding molecules can be performed by eluting the antigen-binding molecules from a chromatographic column using 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.

[0942] In one embodiment, formulations of antigen-binding molecules produced as described herein can be further purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The specific conditions used to purify 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, antibodies, ligands, receptors, or antigens bound to the antigen-binding molecule can be used. For example, for affinity chromatography purification of antigen-binding molecules, a matrix containing protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate antigen-binding molecules. 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.

[0943] In some embodiments, antigen-binding molecules having at least one disulfide bond in a region outside the hinge region, produced or purified by the methods described herein, are further processed in a separate processing step using a dissociative denaturing agent, such as sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride (GuHCl). A large amount of dissociative agent is required to observe perceptible unfolding. In some embodiments, the processing step uses a dissociative agent between 0.1 M and 2 M, which produces an effect equivalent to using 0.1 M to 2 M guanidine hydrochloride. In specific embodiments, oxidative refolding is achieved in the presence of about 1.0 M guanidine hydrochloride or a similar amount of other dissociative agent that produces the same or similar amount of refolding as 1 M guanidine hydrochloride. In some embodiments, the method uses a dissociative agent between about 1.5 M and 0.5 M. The amount of dissociative agent used is based on the structural stability of the antigen-binding molecule in the presence of said dissociative agent. Sufficient dissociative agent is required to disrupt the local tertiary and / or quaternary structures of the domain interactions of the antigen-binding molecule, but less than the amount needed to completely unfold the molecule and / or the secondary structures of the individual domains. To determine the point at which the antigen-binding molecule will begin to unfold via equilibrium denaturation, those skilled in the art titrate a dissociative agent into a solution containing the antigen-binding molecule and monitor the structure using techniques such as circular dichroism or fluorescence. Other parameters can be used to unfold or slightly disrupt the structure of the antigen-binding molecule, which can be used in place of a dissociative agent. Temperature and pressure are two fundamental parameters previously used to alter the structure of antigen-binding molecules and can be used in place of a dissociative agent when in contact with oxidizing and / or reducing agents. The inventors anticipate that those skilled in the art can use any parameter proven to denature or disrupt the structure of the antigen-binding molecule in place of a dissociative agent.

[0944] The method disclosed herein improves the "LINC ratio," which is the ratio of LINC-form antigen-binding molecules to antigen-binding molecules loaded onto the matrix (i.e., the sum of LINC-form antigen-binding molecules having at least one disulfide bond in a region outside the hinge region, and unLINC-form antigen-binding molecules). For example, as described in WO2021 / 157679, the LINC ratio can be calculated by separating the cross-linked LINC form from the uncross-linked unLINC form (open form) by non-reducing SDS-PAGE and comparing their respective abundance ratios.

[0945] In one embodiment, the formulation of the antigen-binding molecule produced as described herein may be further subjected to techniques such as electrophoresis and chromatography to determine the LINC ratio. More specifically, after the purification step by chromatography, the production method of this disclosure may include the step of measuring, determining, or quantifying the LINC ratio (i.e., the ratio of antigen-binding molecules having at least one disulfide bond in a region outside the hinge region (LINC form) to (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region outside the hinge region (LINC form) and (ii) the sum of incorrectly disulfide-bonded forms and / or non-disulfide-bonded forms (unLINC forms) of antigen-binding molecules) in the formulation obtained from the purification step. In one embodiment, the production method of this disclosure may include the steps of measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules (LINC form) having at least one disulfide bond in a region outside the hinge region to the sum of (i) antigen-binding molecules (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region and (ii) antigen-binding molecules in a non-disulfide bonded form (unLINC form).

[0946] This disclosure also relates to methods for measuring, determining, or quantifying the ratio (LINC ratio) of LINC form to the sum of LINC and unLINC forms in a composition comprising an antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and mis-disulfide-bonded and / or non-disulfide-bonded forms (unLINC forms) of the antigen-binding molecule. In some embodiments, this disclosure relates to methods for measuring, determining, or quantifying the ratio (LINC ratio) of LINC form to the sum of LINC and unLINC forms in a composition comprising an antigen-binding molecule (LINC form) having at least one disulfide bond formed between amino acid residues in a region outside the hinge region, and non-disulfide-bonded forms (unLINC forms) of the antigen-binding molecule.

[0947] In another embodiment, this disclosure relates to a method for measuring, determining, or quantifying the ratio (LINC ratio) of the LINC form to the sum of the LINC and unLINC forms in a composition comprising an antigen-binding molecule (LINC form) having at least one disulfide bond formed in a region outside the hinge region and an antigen-binding molecule (unLINC form) not having a disulfide bond formed in a region outside the hinge region.

[0948] The methods for measuring, determining, or quantifying in this disclosure include the steps (a) and (b):

[0949] (a) Composition (2) was prepared by adding a protease to composition (1) comprising both LINC and unLINC forms; and

[0950] (b) Perform electrophoresis or chromatography on composition (2).

[0951] The composition (1) comprising both LINC and unLINC forms disclosed herein is not particularly limited, as long as it comprises both LINC and unLINC forms. The ratio of LINC to unLINC forms or the degree of purification are also not limited. In one embodiment, the composition (1) comprising both LINC and unLINC forms disclosed herein may comprise the LINC form and its corresponding unLINC form disclosed herein. In one embodiment, the composition (1) comprising both LINC and unLINC forms disclosed herein may be a formulation obtained by the methods disclosed herein for production, or may be a formulation disclosed herein. In one embodiment, the composition (1) comprising both LINC and unLINC forms disclosed herein may be in the form of a crude purified product. In another embodiment, the composition (1) comprising both LINC and unLINC forms disclosed herein may be in the form of a drug substance or a standard product.

[0952] In one embodiment, the steps for measuring, determining, or quantifying the LINC ratio include electrophoresis, chromatography, etc. In one embodiment, the method for measuring, determining, or quantifying the LINC ratio in this disclosure includes the step of performing electrophoresis or chromatography on the compositions (1) and / or (2) and / or composition (3) described later. In one embodiment, the method for measuring, determining, or quantifying the LINC ratio in this disclosure includes the step of performing electrophoresis or chromatography on the compositions (1) and (2) and composition (3) described later. 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. Furthermore, in one embodiment, hydrophobic interaction chromatography (HIC), etc., can be examples of chromatography. These techniques can be employed using apparatus well known to those skilled in the art. LabChip TMExamples of devices for non-reducing capillary SDS gel electrophoresis (CE-SDS) include PerkinElmer and PA800 (SCIEX). Examples of devices for hydrophobic interaction chromatography include Proteomix (registered trademark) (Sepax Technologies, Inc.), the Shim-pack Bio HIC series (Shimadzu), and MabPAC HIC-10 (Thermo Fisher Scientific).

[0953] In one embodiment, the method of production according to this disclosure may include the step of adding a protease to a formulation of an antigen-binding molecule produced as described herein prior to the step of measuring, determining, or quantifying the LINC ratio. In one embodiment, the method of measuring, determining, or quantifying the LINC ratio according to this disclosure includes the step of adding a protease to a composition (1) comprising both LINC and unLINC forms to prepare composition (2). The protease in this disclosure is not particularly limited, as long as it is an enzyme that does not digest the LINC form but is capable of digesting the unLINC form. In one embodiment, the protease may be a cysteine ​​protease capable of digesting the hinge region of an antibody. Such an enzyme can break down the antibody to produce Fab and Fc regions. In one embodiment, preferably, the antibody is human IgG1.

[0954] In other embodiments, the protease in this disclosure may be IgdE derived from Streptococcus bacteria. Examples of Streptococcus bacteria include *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, *Streptococcus equi*, *Streptococcus suis*, *Streptococcus porcinus*, *Streptococcus pseudoporcinus*, *Streptococcus canis*, *Streptococcus castoreus*, or *Streptococcus merionis*, but they are not limited to these examples.

[0955] In one embodiment, the protease of this disclosure is preferably a protease that digests human IgG1 antibodies between the T and H of the amino acid sequence KSCDKT / HTCPPCP located in the hinge region of the human IgG1 antibody. In another aspect, the protease of this disclosure may be IgdE derived from Streptococcus agalactiae. IgdE derived from Streptococcus agalactiae is an enzyme disclosed in the literature of Spoerry C et al. (PLoS ONE 11(10): e0164809. doi:10.1371 / journal.pone.0164809(2016)) or elsewhere. Those skilled in the art will be able to obtain the enzyme without difficulty using well-known techniques.

[0956] In another specific embodiment, proteins selected from the group consisting of (a) to (d) below can serve as examples of IgdE derived from Streptococcus agalactiae of this disclosure:

[0957] (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 61;

[0958] (b) A protein encoded by the base sequence shown in SEQ ID NO: 62;

[0959] (c) A protein comprising one or more amino acids substituted, deleted, added, and / or inserted relative to the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP; and

[0960] (d) A protein comprising an amino acid sequence having at least 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

[0961] The addition, deletion, substitution, and / or insertion of amino acids can be performed using methods known in the relevant technical fields. For example, site-specific mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)) or overlap extension PCR can be used on nucleic acids encoding amino acid sequences. Such techniques can be used alone or in combination as appropriate.

[0962] Generally, it is known that one or more amino acid modifications (e.g., conserved substitution, deletion, insertion, and / or addition) in a protein do not affect the protein's function or may even enhance it. Amino acids are classified based on the characteristics of their side chains into hydrophobic (A, I, L, M, F, P, W, Y, V) amino acids and hydrophilic (R, D, N, C, E, Q, G, H, K, S, T) amino acids. Amino acid side chains can also be classified as aliphatic side chains (G, A, V, L, I, P), side chains containing hydroxyl groups (S, T, Y), side chains containing sulfur atoms (C, M), side chains containing carboxylic acids and amides (D, N, E, Q), side chains containing bases (R, K, H), and side chains containing aromatic groups (H, F, Y, W). Proteins obtained by modifying the amino acids of a protein containing the amino acid sequence represented in SEQ ID NO: 61 to different amino acids but classified into the same phenotypic group are also included in this disclosure as IgdEs. Note that IgdEs in this disclosure may also contain non-conservative modifications, provided that they are functionally equivalent to proteins containing the amino acid sequence shown in SEQ ID NO: 61.

[0963] In one embodiment, a protein comprising an amino acid sequence having high sequence identity with the amino acid sequence represented in SEQ ID NO: 61 is also included in this disclosure as IgdE. High sequence identity in this disclosure means sequence identity of at least 50% or more, more preferably 70% or more, 75% or more, 80% or more, 85% or more, or further preferably 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) across the entire amino acid or nucleic acid sequence.

[0964] IgdE derived from *Streptococcus agalactiae* is commercially available from Genovis under the trademark "FabALACTICA (registered trademark) (IgdE)". That is, in one embodiment, the protease in this disclosure may be FabALACTICA (registered trademark) (IgdE). The inventors have discovered that by adding IgdE derived from *Streptococcus agalactiae*, more specifically FabALACTICA (registered trademark) (IgdE), to a composition comprising both LINC and unLINC forms, for example, by preparing an antigen-binding molecule as disclosed herein, and then subjecting the IgdE-containing formulation to electrophoresis, chromatography, etc., the LINC ratio of the composition or formulation can be obtained.

[0965] Therefore, this disclosure also relates to compositions comprising an antigen-binding molecule in the form of having at least one disulfide bond formed in a region outside the hinge region (LINC form), an antigen-binding molecule in the form of not having a disulfide bond formed in a region outside the hinge region (unLINC form), and a protease. These compositions can be used to determine a LINC ratio. In one embodiment, the LINC-form antigen-binding molecule, its corresponding unLINC form, and the protease contained in the composition can be those disclosed herein. These compositions may include other components, as long as they can be used to obtain a LINC ratio.

[0966] There are no particular limitations on the concentration of the protease added to the formulation, but in one embodiment, examples include 0.6 to 1.8 units of enzyme / μg of protein. In a particular embodiment where the LINC ratio is measured, determined, or quantified using CE-SDS, a protease of 1.25 units of enzyme / μg of protein may be used. In a particular embodiment where the LINC ratio is measured, determined, or quantified using HIC, a protease of 1.2 units of enzyme / μg of protein may be used.

[0967] In one embodiment, when the method of production in this invention includes the step of adding a protease to a formulation of an antigen-binding molecule produced as described herein, non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC) may be used in the step of measuring, determining, or quantifying the LINC ratio. Furthermore, in one embodiment, the chromatography used to obtain the formulation of the antigen-binding molecule may be membrane chromatography.

[0968] In one embodiment, in the step of measuring, determining, or quantifying the LINC ratio in the method for production disclosed herein, the LINC ratio may be calculated as follows. For example, when using non-reducing capillary SDS gel electrophoresis (CE-SDS) in the measurement, determination, or quantification step, it includes a step of preparing a "protease-added formulation" by adding a protease to a formulation obtained through a purification step. Furthermore, it includes a step of obtaining a "sample containing only the protease." Electrophoretic images of each of the "protease-added formulation," "protease-free formulation," and "protease-free sample" are obtained. The electrophoretic image obtained from the "protease-free formulation" includes peaks originating from the LINC form and peaks originating from the unLINC form (which has not been digested by the protease), as well as peaks (peak Z) that are neither originating from the LINC form nor from the unLINC form (which has not been digested by the protease). Electrophoresis patterns obtained from "preparations with added protease" include: peaks originating from the LINC form (peak L), peaks originating from the unLINC form after protease digestion (peak unL), peaks originating from the protease (peak E), and peaks that are not originating from the LINC form, not originating from the unLINC form after protease digestion, or not originating from the protease (peak Z). Electrophoresis patterns obtained from "samples containing only protease" include peaks originating from the protease (peak E).

[0969] When using CE-SDS to measure, determine, or quantify the LINC ratio, the LINC ratio can be obtained as the ratio of the "area value of peak L" to a value calculated by subtracting the "area value of peak Z" and the "area value of peak E" from the "sum of the area values ​​of peak L, peak E, and peak Z" and then adding the "area value of peak L," as shown in Equation (I) described later. Those skilled in the art will be able to obtain these area values ​​(area values) from the analysis manual of the CE-SDS device. Alternatively, the analysis manual can be modified as needed, and the area values ​​can be obtained from the modified manual.

[0970] In the aforementioned "formulation containing added protease", in addition to the LINC form, the unLINC form digested by the protease, and the protease itself, degradation products of target proteins, etc., are also included. Therefore, the aforementioned "peak Z" includes peaks derived from such products. The aforementioned "sample containing only protease" is not particularly limited, as long as it does not contain either the LINC or unLINC form and contains the protease described in this disclosure. FabALACTICA (registered trademark) (Genovis) and the like can be examples of "sample containing only protease" in this disclosure, but are not limited thereto. In one embodiment, the "sample containing only protease" can be added in the same amount to a formulation to which no sample has been added.

[0971] In another embodiment, in the method of this disclosure, the LINC ratio can be calculated as follows. For example, when hydrophobic interaction chromatography (HIC) is used in a measurement, determination, or quantification step, a "protease-containing formulation" is obtained by adding a protease to a formulation obtained through a purification step. This "protease-containing formulation" is subjected to HIC and a chromatogram is obtained. In this case, the LINC ratio is obtained as the ratio of the area value of the peak derived from the LINC form to the sum of the area values ​​of the peak derived from the LINC form and the area values ​​of all peaks derived from the unLINC form, as shown in Equation (II) described later. Those skilled in the art will be able to obtain these area values ​​(area values) according to the analytical manual of the HIC apparatus. Alternatively, the analytical manual can be modified as needed, and the area values ​​can be obtained according to the modified manual. Those skilled in the art can also appropriately prepare mobile phase A and mobile phase B for hydrophobic interaction chromatography (HIC) with reference to common technical knowledge. Those skilled in the art can appropriately select chromatographic carriers and can use carriers such as those disclosed herein.

[0972] When quantifying the LINC ratio using non-reducing capillary SDS gel electrophoresis (CE-SDS), the methods for measuring, determining, or quantifying the LINC ratio in this disclosure further include the step of preparing a composition (3) containing a protease but not in the LINC or unLINC form, in addition to the above-described compositions (1) and (2). These methods further include the step of performing CE-SDS on each of compositions (1) to (3) and obtaining electrophoretic patterns (1) to (3) from compositions (1) to (3). Composition (3) is not particularly limited, as long as it does not contain the LINC or unLINC form and contains the protease of this disclosure, but in one embodiment, the above-described FabALACTICA (registered trademark) (Genovis) and the like may be used as examples.

[0973] In this disclosure, electrophoresis diagram (1) includes peaks originating from the LINC form and from the unLINC form that has not been digested by the protease, as well as peaks that are not originating from the LINC form or from the unLINC form that has not been digested by the protease (peak Z). Electrophoresis diagram (2) includes, from the “formulation with added protease”, peaks originating from the LINC form (peak L), peaks originating from the unLINC form that has been digested by the protease (peak unL), peaks originating from the protease (peak E), and peaks that are not originating from the LINC form, not originating from the unLINC form that has been digested by the protease, or not originating from the protease (peak Z). Electrophoresis diagram (3) includes peaks originating from the protease (peak E).

[0974] When using non-reducing capillary SDS gel electrophoresis (CE-SDS), the LINC ratio is calculated by equation (I) shown below.

[0975]

[0976] In this formula,

[0977] A represents the area of ​​peak L in electrophoresis diagram (2).

[0978] B is the sum of the area values ​​of peaks unL, E, and Z in electrophoresis diagram (2).

[0979] C is the area value of peak Z in electrophoresis diagram (1), and

[0980] D is the area value of peak E in electrophoresis diagram (3).

[0981] Theoretically, when the unLINC form is digested by a protease, three fragments are produced as shown on the right side of Figure 8 (i.e., when the antigen-binding molecule is a trivalent antibody composed of a molecule consisting of five chains as shown in Figure 3, it produces (i) a fusion of the first antigen-binding domain or the second and third antigen-binding domains, (ii) the remaining one of the first and second antigen-binding domains that does not constitute the fusion, and (iii) the Fc region). The inventors have discovered that, in addition to the three fragments mentioned above, when the unLINC form is digested by a protease, sometimes only one of the two hinge regions in the antigen-binding molecule is digested (specifically, the hinge region on chain 3 when the antigen-binding molecule is a trivalent antibody composed of a molecule consisting of five chains as shown in Figure 3), while the hinge region in the other chain (i.e., chain 1) is not digested. Therefore, the "peaks derived from (by) protease digestion" in this disclosure include peaks derived from the three fragments (i) to (iii) above, as well as peaks derived from the fragments generated when only the hinge region on chain 3 is digested due to the presence of a trivalent antibody composed of a molecule consisting of five chains as shown in Figure 3.

[0982] The method for measuring, determining, or quantifying the LINC ratio in this disclosure, for example, when hydrophobic interaction chromatography (HIC) is used to measure, determine, or quantify the LINC ratio, includes the step of performing hydrophobic interaction chromatography (HIC) on the above composition (2) to obtain a chromatogram. In this case, the LINC ratio can be calculated based on formula (II) described below.

[0983]

[0984] In this formula,

[0985] A represents the area of ​​the peak originating from the LINC form in the chromatogram; and

[0986] B represents the area values ​​of all peaks in the chromatogram originating from the unLINC form after protease digestion.

[0987] "All peaks derived from the unLINC form (by protease digestion)" includes peaks derived from all unLINC form fragments produced by protease digestion of the unLINC form, or more specifically, peaks derived from the four fragments mentioned above.

[0988] In this disclosure, the unLINC ratio, i.e. the ratio of unLINC form to the sum of LINC form and unLINC form, can be obtained by subtracting the LINC ratio (%) from 100%.

[0989] In one embodiment, the method of this disclosure may further include the step of culturing the formulation or composition after the protease is added to it and before electrophoresis or chromatography. There are no particular limitations on the culture conditions, but incubation may be carried out, for example, in phosphate buffer (pH 6 to 8), at 37°C for 16 to 20 hours, such as 16 hours, 18 hours, or 20 hours.

[0990] Additionally, in this disclosure, the antigen-binding molecule subjected to chromatography for purification may contain a signal sequence. Specifically, when an antigen-binding molecule having amino acid residues that can form at least one disulfide bond between amino acid residues in regions other than the hinge region is recombinantly expressed in cells, the antigen-binding molecule may sometimes have a signal sequence to promote expression and / or secretion. In one embodiment, when the translation product from the nucleic acid sequence used for recombinant expression of the antigen-binding molecule contains a signal sequence, the method of this disclosure may include the step of cleaving the signal sequence from the antigen-binding molecule. The signal sequence may be cleaved before or after the step of performing chromatography for purification of the antigen-binding molecule, or before or after the step of quantifying the LINC ratio of the substance purified by chromatography. The cleavage of the signal sequence may be performed by methods well known to those skilled in the art.

[0991] In one respect, the amino acid residues that can form disulfide bonds are mutated, substituted, introduced, or engineered cysteine ​​residues.

[0992] In one aspect, at least one disulfide bond is formed between the polypeptides constituting the antigen-binding molecule in a region outside the hinge region. More specifically, in a particular embodiment, at least one disulfide bond in a region outside the hinge region of this 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.

[0993] In one respect, at least one disulfide bond in the region outside the hinge region is an engineered disulfide bond that is not present in wild-type IgG.

[0994] III. Antigen-binding molecules

[0995] In the antigen-binding molecules of this disclosure, disulfide bonds can be formed between regions outside the hinge region of one heavy chain and regions outside the hinge region of another heavy chain. In this disclosure, an antigen-binding molecule having a disulfide bond between regions outside the hinge region of one heavy chain and regions outside the hinge region of another heavy chain is called a LINC form, and an antigen-binding molecule without such a disulfide bond is called an unLINC form. In this disclosure, the region outside the hinge region may refer to the CH1 region. The unLINC form in this disclosure includes mis-disulfide bonded forms and / or non-disulfide bonded forms. In this disclosure, the LINC form may be referred to as the LINC form, and the unLINC form may be referred to as the unLINC form.

[0996] In one aspect, the antigen-binding molecule of this disclosure includes a first antigen-binding domain and a second antigen-binding domain that can be linked to each other via at least one disulfide bond. In another aspect, in an antigen-binding molecule produced or purified by the methods of this disclosure, the first antigen-binding domain and the second antigen-binding domain are linked via at least one disulfide bond. In embodiments of the above aspects, the first antigen-binding domain and the second antigen-binding domain are linked via two, three, four, or more disulfide bonds.

[0997] In some embodiments, at least one of the first antigen-binding domain and the second antigen-binding domain is itself active in binding to an antigen (i.e., a single antigen-binding domain independently has antigen-binding activity). In some embodiments, each of the first antigen-binding domain and the second antigen-binding domain is itself active in binding to an antigen.

[0998] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain in this disclosure may have a Fab, Fab', scFab, Fv, scFv, or VHH structure.

[0999] In one aspect, each of the first and second antigen-binding domains is a Fab molecule, and the antigen-binding molecule contains at least one disulfide bond formed between the first and second antigen-binding domains. Preferably, the at least one disulfide bond is formed between amino acid residues (cysteine ​​residues) not present in the hinge region, or preferably between amino acid residues (cysteine ​​residues) in the CH1 region of each antigen-binding domain.

[1000] In the embodiments described above, both the first antigen-binding domain and the second antigen-binding domain include a Fab region and a hinge region.

[1001] In some embodiments, at least one of the amino acid residues forming disulfide bonds 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, this 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, for example, amino acid substitution.

[1002] Alternatively, in another embodiment, in the antigen-binding molecule of this disclosure, amino acid residues (e.g., cysteine ​​residues) present in the wild-type Fab or hinge region and potentially involved in disulfide bonds between antigen-binding domains may be substituted or deleted by other amino acid residues. Examples of such cysteine ​​residues include cysteine ​​residues at positions 220, 226, and 229 according to EU numbers in the hinge region, and cysteine ​​residue at position 214 in the CL region.

[1003] In one embodiment of the foregoing aspects, 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 some embodiments, the antibody fragment is Fab, Fab', scFab, Fv, scFv, or a single-domain antibody. In some embodiments, the first and / or second antigen-binding domain comprises a hinge region. Amino acid residues forming disulfide bonds are respectively present in the first and second antigen-binding domains, and the bonds between the antigen-binding domains are formed by the linkage of these amino acid residues. In some embodiments, at least one amino acid residue forming a disulfide bond between the antigen-binding domains is present within the antibody fragment.

[1004] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain in this disclosure may or may not include a hinge region. In some embodiments, the first antigen-binding domain and the second antigen-binding domain in this disclosure may each include a Fab region forming an F(ab')2 structure and a hinge region.

[1005] In embodiments described above, both the first antigen-binding domain and the second antigen-binding domain bind to the same antigen. In some embodiments, both the first antigen-binding domain and the second antigen-binding domain bind to the same epitope on the same antigen. In some 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 some embodiments, the antigen-binding molecule of this disclosure is a bicomponent antigen-binding molecule (e.g., a bicomponent antibody) targeting a specific antigen.

[1006] In another embodiment of the above aspects, each of the first antigen-binding domain and the second antigen-binding domain binds to a different antigen.

[1007] In another embodiment of the foregoing aspects, the antigen-binding molecule in this disclosure is a clamp antigen-binding molecule (e.g., a clamp antibody). As used herein, a clamp antigen-binding molecule 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 (binding to antigen A), thereby enhancing the binding activity of the antigen-binding molecule bound to antigen A for antigen A (or alternatively, stabilizing the antigen / antigen-binding molecule complex formed by antigen A and the antigen-binding molecule bound 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 weakened CD3 antibody), thereby enhancing the binding activity of the weakened CD3 antibody to CD3 (or alternatively, stabilizing the antigen-antibody complex formed by CD3 and the weakened CD3 antibody). In some embodiments, the first and / or second antigen-binding domains in the antigen-binding molecule of this disclosure may be antigen-binding domains (clamp antigen-binding domains) derived from clamp antigen-binding molecules.

[1008] In the embodiments described above, 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.

[1009] In another embodiment of the foregoing aspects, the antigen-binding molecule of this disclosure has the activity of modulating the interaction between two antigen molecules. Free from any particular theory, the activity of modulating the interaction is considered to arise from the antigen-binding molecule of this disclosure holding the two antigen molecules in a spatially closer position and reducing their mobility. In some embodiments, the antigen-binding molecule of this disclosure can enhance or weaken the interaction between two antigen molecules compared to a control antigen-binding molecule. The control antigen-binding molecule differs from the antigen-binding molecule of this disclosure only in that the control antigen-binding molecule has one less disulfide bond between the two antigen-binding domains. In another embodiment, the missing disulfide bond may be selected from a mutated amino acid residue (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region) that is absent from the wild-type Fab or hinge region. The mutated amino acid residue is, for example, an artificially mutated, substituted, introduced, or engineered cysteine ​​residue.

[1010] In one embodiment, the antigen molecule is selected from the group consisting of: receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ion channel receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, co-stimulatory molecules, and cell adhesion molecules.

[1011] In some embodiments, the two antigen molecules bound by the antigen-binding molecule of this disclosure may be a ligand and its receptor, respectively. The antigen-binding molecule of this disclosure has the activity of promoting receptor activation through the ligand. In some other embodiments, the two antigen molecules bound by the antigen-binding molecule of this disclosure may be an enzyme and its substrate, respectively. The antigen-binding molecule of this disclosure has the activity of promoting the catalytic reaction between the enzyme and the substrate.

[1012] Furthermore, in some other embodiments, both of the two antigen molecules bound by the antigen-binding molecule of this disclosure may be antigens (e.g., proteins) present on the cell surface. The antigen-binding molecule of this disclosure has the activity of promoting the interaction between a cell expressing a first antigen and a cell expressing a second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen may be a cell with cytotoxic activity and its target cell, respectively. The antigen-binding molecule of this disclosure promotes damage to target cells by cells with cytotoxic activity. Cells with cytotoxic activity are, for example, T cells, NK cells, monocytes, or macrophages.

[1013] In embodiments described above, the antigen-binding molecules of this disclosure are resistant to protease cleavage. In some embodiments, the antigen-binding molecules of this disclosure exhibit increased resistance to protease cleavage compared to control antigen-binding molecules. In some embodiments, the proportion of the full-length molecule (e.g., full-length IgG molecule) remaining after protease treatment is increased in the antigen-binding molecules of this disclosure compared to control antigen-binding molecules. In some embodiments, the proportion of specific fragments (e.g., Fab monomers) generated after protease treatment is reduced in the antigen-binding molecules of this disclosure compared to control antigen-binding molecules.

[1014] In embodiments of the foregoing aspects, when the antigen-binding molecule of this disclosure is treated with a protease, a dimer of the antigen-binding domain or a fragment thereof (e.g., a cross-linked Fab dimer) is cleaved. In one embodiment, the protease may cleave the hinge region of the antigen-binding molecule.

[1015] In one aspect, at least one of the first antigen-binding domain and the second antigen-binding domain of this disclosure can bind to soluble proteins. In another embodiment, at least one of the first antigen-binding domain and the second antigen-binding domain of this disclosure can bind to membrane proteins.

[1016] The first antigen-binding domain and the second antigen-binding domain in this 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 humans, mice, rats, monkeys, rabbits, or dogs. In some embodiments, examples of the first antigen and the second antigen include, but are not limited to, 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 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, co-stimulatory molecules, and cell adhesion molecules.

[1017] In some embodiments, either the first antigen or the second antigen may be a molecule specifically expressed on, for example, T cells, and the other antigen may 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 a molecule specifically expressed on, for example, T cells, and the other antigen is a molecule expressed on immune cells and is different from the initially selected antigen.

[1018] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the site on CD3 to which the antigen-binding molecule of this disclosure binds can be any epitope present in the γ, δ, or ε chain sequence constituting human CD3. Epitopes present in the extracellular region of the ε chain in the human CD3 complex are particularly preferred. The polynucleotide sequences constituting the γ, δ, 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). Other examples of antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[1019] In one embodiment, the first antigen may 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 may 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 proteins 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 may be CD3 and the second antigen may be CD137. In this document, the first antigen and the second antigen are used interchangeably.

[1020] In some embodiments, the antigen-binding molecule of this disclosure specifically binds to all or a portion of a fraction of the peptide of CD3. In particular embodiments, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In particular embodiments, the antigen-binding molecule is cross-reactive (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 CD3 ε subunit of CD3 shown in SEQ ID NO: 13 (NP_000724.1) (RefSeq registration number shown in parentheses). In some embodiments, the antigen-binding molecule is capable of specifically binding to the CD3 ε chain expressed on the surface of eukaryotic cells. In some embodiments, the antigen-binding molecule binds to the CD3 ε chain expressed on the surface of T cells.

[1021] In some embodiments, the antigen-binding molecule of this disclosure specifically binds to all or part of a subset of the peptide of CD137. The term "CD137" as used herein, also known as 4-1BB, 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.

[1022] In some embodiments, the CD137 is human CD137. In some embodiments, advantageous examples of antigen-binding molecules in this disclosure include antigen-binding molecules that bind to an epitope identical to the human CD137 epitope bound by an antibody selected from the group consisting of:

[1023] Antibodies that recognize the region of the human CD137 protein containing the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 14)

[1024] Antibodies that recognize regions containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 15)

[1025] Antibodies that recognize regions containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 16) and

[1026] Antibodies that recognize regions containing the LQDPCSNCPAGTFCDNNRNQIC sequence (SEQ ID NO: 17).

[1027] In embodiments described above, 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 some embodiments, the non-antibody protein is either 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, co-stimulatory molecules, and cell adhesion molecules.

[1028] In one aspect, the antigen-binding molecule of this disclosure may further include a third antigen-binding domain. In another aspect, the third antigen-binding domain may be fused with either the first antigen-binding domain or the second antigen-binding domain.

[1029] In one aspect, the third antigen-binding domain may be Fab or scFv; in this case, the third antigen-binding domain may optionally be fused at its C-terminus to the N-terminus of the Fab heavy chain (VH region) of either the first or second antigen-binding domain via a peptide linker.

[1030] In some embodiments, each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain of this disclosure may be a Fab molecule; in this case, the third antigen-binding domain may optionally be fused via a peptide linker to the N-terminus of the Fab heavy chain (VH region) of either the first or the second antigen-binding domain at the C-terminus of its Fab heavy chain (CH1 region).

[1031] In one aspect, examples of peptide linkers disclosed herein may be sequences 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 are not limited thereto.

[1032] In one aspect, the third antigen-binding domain of this 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.

[1033] In one aspect, the third antigen-binding domain of this disclosure can bind to a third antigen that is different from the first and second antigens described above. The third antigen-binding domain binding to the third antigen can be an antigen-binding domain that recognizes any antigen. The third antigen-binding domain binding to the third antigen in this disclosure can be an antigen-binding domain that recognizes molecules specifically expressed in cancer cells or cancerous tissue.

[1034] In some embodiments, the third antigen is derived from human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule specifically expressed on cells or organs derived from human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule not systematically expressed on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, and also includes antigens expressed in association with malignant changes in cells, as well as abnormal glycans appearing on the cell surface or on protein molecules during malignant transformation of cells. Specific examples include ALK receptor (pleiotropic receptor), pleiotropic factor, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, 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), and 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, carcinoma-fetoprotein antigen (e.g., carcinoma-fetotrophoblast glycoprotein 5T4 and carcinoma-fetobladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neogenic glycoprotein, sphingolipids, breast cancer antigens (e.g., EGFR (epithelial 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 found in fetal erythrocytes) Antigens; Primary endoderm I antigen found in adult erythrocytes; I (Ma) found in pre-implantation 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 type 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 embryonic cancer cells; TL5 (blood type A), EGF receptor found in A431 cells; E1 series (blood type B) found in pancreatic cancer; FC10.2 found in embryonic cancer cells; gastric cancer antigen; CO-514 (blood type Lea) found in adenocarcinoma; NS-10 found in adenocarcinoma; in A431 CO-43 (blood type Leb) and G49 were found in cellular EGF receptors; MH2 (blood type ALeb / Ley) was found in colon cancer; 19.9 was found in colon cancer; gastric cancer mucin; T5A7 was found in bone marrow cells; R24 was found in melanoma; and 4.2, GD3, and D1 were found in embryonic cancer cells.1. OFA-1, GM2, OFA-2, GD2 and M1:22:25:8; SSEA-3 and SSEA-4 found in 4-cell to 8-cell embryos), cutaneous 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 brain-testis-cancer antigens (tumor neuron antigen MA2 and paraneoplastic neuron antigen), ventral neuronoma antigen 2 (NOVA2), hematologic malignancy 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 fragments of these peptides and their modified structures (the modified phosphate groups, glycans, etc.), EpCAM, EREG, CA19-9, CA15-3, sialylated SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[1035] In a preferred embodiment, the third antigen is phosphatidylinositol proteoglycan-3 (GPC3). In another embodiment, the third antigen is DLL3 (δ-sample 3). In some embodiments, the third antigen-binding domain may be a "DLL3 antigen-binding domain" that binds to DLL3.

[1036] Unless otherwise specified, as used herein, the term "DLL3" refers to any naturally occurring DLL3 (δ-like 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" unprocessed DLL3, as well as any form of DLL3 produced through cellular processing. The term also covers naturally occurring variants of DLL3, such as splice variants or allelic variants. The amino acid sequence of exemplary human DLL3 is referred to as the NCBI Reference Sequence (RefSeq) NM_016941.3, and the amino acid sequence of exemplary cynomolgus monkey DLL3 is referred to as the NCBI Reference Sequence XP_005589253.1, and the amino acid sequence of exemplary mouse DLL3 is referred to as the NCBI Reference Sequence NM_007866.2.

[1037] The human DLL3 protein contains a transmembrane (TM) region and an intracellular domain at the C-terminus, and a DSL (Notch) domain at the N-terminus. Additionally, DLL3 has an EGF domain comprising six regions (EGF1 through EGF6 from the N-terminus to the C-terminus).

[1038] 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 DLL3 protein expressed on the surface of eukaryotic cells. In some embodiments, the DLL3 antigen-binding domain binds to DLL3 protein expressed on the surface of cancer cells.

[1039] In some embodiments, the DLL3 antigen-binding domain of this disclosure binds to epitopes within the extracellular domain (ECD) (i.e., the domain extending from the N-terminus to immediately preceding the TM region), but not to the intracellular domain of the TM region or the C-terminus. The DLL3 antigen-binding domain of this disclosure may bind to epitopes within any of the aforementioned domains / regions within the ECD. In a preferred embodiment, the DLL3 antigen-binding domain of this disclosure binds to epitopes within the region extending from EGF6 to immediately preceding the TM region. More specifically, the DLL3 antigen-binding domain of this disclosure may bind to epitopes within the region defined in SEQ ID NO: 21 of human DLL3. In some embodiments, the DLL3 antigen-binding domain of this disclosure binds to the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to immediately preceding the TM region, or to an epitope in the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to immediately preceding the TM region. In some embodiments, the antigen-binding molecule or the DLL3 antigen-binding domain may be derived from previously reported anti-DLL3 antibodies (e.g., WO2019131988 and WO2011093097) in which the bound DLL3 epitope has been characterized.

[1040] In human DLL3, the above-mentioned domain / region has the following amino acid residues (see, for example, http: / / www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746):

[1041] Extracellular domain (ECD): amino acid residues at positions 1 to 492;

[1042] DSL domain: amino acid residues at positions 176 to 215;

[1043] EGF domain: amino acid residues at positions 216 to 465;

[1044] EGF1 region: amino acid residues at positions 216 to 249;

[1045] EGF2 region: amino acid residues at positions 274 to 310;

[1046] EGF3 region: amino acid residues at positions 312 to 351;

[1047] EGF4 region: amino acid residues at positions 353 to 389;

[1048] EGF5 region: amino acid residues at positions 391 to 427;

[1049] EGF6 region: amino acid residues at positions 429 to 465;

[1050] From EGF6 to the region immediately preceding the TM region: amino acid residues at positions 429 to 492;

[1051] TM region: amino acid residues at positions 493 to 513; and

[1052] C-terminal intracellular domain: amino acid residues at positions 516 to 618 (or 516 to 587 in some isoforms). These amino acid positions also refer to the amino acid positions in the amino acid sequence shown in SEQ ID NO: 22.

[1053] Therefore, the antigen-binding molecule of this disclosure can bind to the aforementioned region / domain of the amino acid residue at the aforementioned position in human DLL3. That is, the antigen-binding molecule of this disclosure can bind to the epitope within the aforementioned region / domain of the amino acid residue at the aforementioned position in human DLL3.

[1054] The DLL3 protein used in this disclosure is not limited to any particular source, but is preferably human or cynomolgus monkey DLL3 protein.

[1055] In some embodiments, for the DLL3 protein, a DLL3 ECD fragment protein (or ECD variant) may be used. Depending on the truncated site, the fragment / variant may include DSL domains to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6, from the N-terminal to the C-terminal side. The fragment / variant may further include a region extending from immediately following the EGF6 region to immediately preceding the TM region. A Flag tag may be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[1056] The CD3, CD137, or DLL3 proteins in this disclosure can be proteins having the above-described sequences, or modified proteins having sequences derived from the above-described sequences through modification of one or more amino acids. Examples of modified proteins having sequences derived from the above-described sequences through modification of one or more amino acids may include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more identity with the above-described amino acid sequences. Alternatively, partial peptides of these CD3, CD137, or DLL3 proteins may be used.

[1057] In some 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 some embodiments, the antigen-binding molecules of this disclosure are trispecific antigen-binding molecules, that is, 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 simultaneously to two antigens, and capable of specifically binding to DLL3.

[1058] On the one hand, the third antigen-binding domain is a conventional Fab molecule, and

[1059] (a) The first polypeptide comprises (from the N-terminus to the C-terminus) a VH region of the third antigen-binding domain, a heavy chain constant region (CH1); a VH region of the first antigen-binding domain, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3).

[1060] (b) The second polypeptide contains a VL of the third antigen-binding domain (from the N-terminus to the C-terminus) and a light chain constant region (CL);

[1061] (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 the hinge region and / or the Fc region (CH2 and CH3).

[1062] (d) The fourth polypeptide comprises (from the N-terminus to the C-terminus) the VL of the second antigen-binding domain and the light chain constant region (CL); and

[1063] (e) The fifth polypeptide comprises (from the N-terminus to the C-terminus) the VL of the first antigen-binding domain and the light chain constant region (CL).

[1064] On one hand, 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

[1065] (a) The first polypeptide comprises (from the N-terminus to the C-terminus) a VL region of the third antigen-binding domain, a heavy chain constant region (CH1); a VH region of the first antigen-binding domain, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3).

[1066] (b) The second polypeptide contains a VH region of the third antigen-binding domain (from the N-terminus to the C-terminus) and a light chain constant region (CL);

[1067] (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 the hinge region and / or the Fc region (CH2 and CH3).

[1068] (d) The fourth polypeptide comprises (from the N-terminus to the C-terminus) the VL of the second antigen-binding domain and the light chain constant region (CL); and

[1069] (e) The fifth polypeptide comprises (from the N-terminus to the C-terminus) the VL of the first antigen-binding domain and the light chain constant region (CL).

[1070] In one aspect, the antigen-binding molecule of this disclosure may further include an Fc region. In another aspect, the Fc region consists of a first Fc region subunit and a second Fc region subunit capable of stable association.

[1071] In one aspect, in the antigen-binding molecule of this disclosure, each of the first antigen-binding domain and the second antigen-binding domain is Fab, wherein the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc region, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc region.

[1072] In one aspect, the Fc region in this disclosure may be derived from human. In some embodiments, the Fc region in this disclosure may be an IgG Fc region, preferably a human IgG Fc region, and more preferably a human IgG1 Fc region.

[1073] In some embodiments, the Fc region of the antigen-binding molecule consists of a first Fc region subunit and a second Fc region subunit capable of stable association, and wherein the Fc region exhibits reduced binding affinity to the human Fc γ receptor compared to the natural human IgG1 Fc region.

[1074] In a particular embodiment, the Fc region of the antigen-binding molecule described herein includes modifications that promote association between the first and second subunits of the Fc region. These modifications are so-called "mortar and pestle" modifications, comprising a "mortar" modification in one of the two subunits of the Fc region and a "pestle" modification in the other of the two subunits of the Fc region, as described in more detail below.

[1075] The pestle-and-mortar technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion (“pestle”) at the interface of a first polypeptide and a corresponding cavity (“mortar”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[1076] Therefore, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the antigen-binding molecule, amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby creating a protrusion within the CH3 domain of the first subunit, the protrusion being localized in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a cavity within the CH3 domain of the second subunit, the protrusion within the CH3 domain of the first subunit being localized within the cavity.

[1077] Protrusions and cavities can be prepared by altering the nucleic acids encoding polypeptides, for example, through site-specific mutagenesis or peptide synthesis.

[1078] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, additionally, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

[1079] In yet another embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by 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)).

[1080] In a specific embodiment, in the Fc domain composed of a first Fc subunit and a second Fc subunit that are capable of stable association and exhibit reduced binding affinity to the human Fc-γ receptor compared to the natural human IgG1 Fc domain, the first Fc subunit is selected from the group consisting of:

[1081] (a1) Fc region polypeptide, which contains mutants L234A and L235A;

[1082] (a2) Fc region polypeptides containing mutants L234A, L235A, and N297A; and

[1083] (a3) Fc region polypeptide, which includes mutants L234A, L235A, N297A, S354C, and T366W; and

[1084] The second Fc region polypeptide is selected from the group consisting of the following:

[1085] (a4) Fc region polypeptide, which includes mutants L234A and L235A;

[1086] (a5) Fc region polypeptides containing mutants L234A, L235A, and N297A; and

[1087] (a6) Fc region polypeptide, which contains mutants L234A, L235A, N297A, Y349C, T366S, L368A, Y407V (amino acid positions are numbered using EU index numbers).

[1088] In some embodiments, the Fc region of the antigen-binding molecule described herein exhibits enhanced FcRn binding activity under acidic pH conditions (e.g., pH 5.8) compared to the Fc region of natural IgG. 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.

[1089] In some embodiments, the Fc domain includes: Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to the EU number.

[1090] In some embodiments, the Fc domain further includes: Ile or Leu at position 428; and / or Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering.

[1091] In the embodiments, the Fc domain comprises a combination of amino acid substitutions selected from the group consisting of:

[1092] (a) N434A / Q438R / S440E;

[1093] (b) N434A / Q438R / S440D;

[1094] (c) N434A / Q438K / S440E;

[1095] (d) N434A / Q438K / S440D;

[1096] (e) N434A / Y436T / Q438R / S440E;

[1097] (f) N434A / Y436T / Q438R / S440D;

[1098] (g) N434A / Y436T / Q438K / S440E;

[1099] (h) N434A / Y436T / Q438K / S440D;

[1100] (i) N434A / Y436V / Q438R / S440E;

[1101] (j) N434A / Y436V / Q438R / S440D;

[1102] (k) N434A / Y436V / Q438K / S440E;

[1103] (l) N434A / Y436V / Q438K / S440D;

[1104] (m) N434A / R435H / F436T / Q438R / S440E;

[1105] (n) N434A / R435H / F436T / Q438R / S440D;

[1106] (o) N434A / R435H / F436T / Q438K / S440E;

[1107] (p) N434A / R435H / F436T / Q438K / S440D;

[1108] (q) N434A / R435H / F436V / Q438R / S440E;

[1109] (r) N434A / R435H / F436V / Q438R / S440D;

[1110] (s) N434A / R435H / F436V / Q438K / S440E;

[1111] (t) N434A / R435H / F436V / Q438K / S440D;

[1112] (u) M428L / N434A / Q438R / S440E;

[1113] (v) M428L / N434A / Q438R / S440D;

[1114] (w) M428L / N434A / Q438K / S440E;

[1115] (x) M428L / N434A / Q438K / S440D;

[1116] (y) M428L / N434A / Y436T / Q438R / S440E;

[1117] (z) M428L / N434A / Y436T / Q438R / S440D;

[1118] (aa) M428L / N434A / Y436T / Q438K / S440E;

[1119] (ab) M428L / N434A / Y436T / Q438K / S440D;

[1120] (ac) M428L / N434A / Y436V / Q438R / S440E;

[1121] (ad) M428L / N434A / Y436V / Q438R / S440D;

[1122] (ae) M428L / N434A / Y436V / Q438K / S440E;

[1123] (af) M428L / N434A / Y436V / Q438K / S440D;

[1124] (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[1125] (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to EU number.

[1126] In several embodiments, the Fc region of the antigen-binding molecule contains a combination of amino acid substitutions of M428L / N434A / Q438R / S440E.

[1127] In one aspect, the Fc region of this disclosure may comprise a combination of one or more amino acid substitutions that promote the polymerization of the Fc region. Examples of amino acid substitutions that promote polymerization 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 numbers (e.g., see WO2016 / 164480). In specific embodiments, examples of polymers include, but are not limited to, dimers, trimers, and tetramers.

[1128] In one aspect, the antigen-binding molecule of this disclosure may have an amino acid residue in its hinge region resulting from the substitution of at least one of the cysteine ​​residues. In some embodiments, the cysteine ​​residue may be present at positions 226 and / or 229 according to the EU number of the hinge region.

[1129] In one aspect, the antigen-binding molecule of this disclosure may have the amino acid sequence KSCDKTHTCPPCP in one or two hinge regions of its heavy chain. In one embodiment, preferably, the antigen-binding molecule of this disclosure has the amino acid sequence KSCDKTHTCPPCP in the corresponding hinge region of its heavy chain. Preferably, the antigen-binding molecule of this disclosure comprises an amino acid substitution producing a cysteine ​​residue at one or more positions selected from the group consisting 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, according to EU numbers.

[1130] In this embodiment, the Fc region includes any of the following:

[1131] (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;

[1132] (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

[1133] (c) A first Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 58 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 59;

[1134] In one aspect, the antigen-binding molecule disclosed herein is a multispecific antigen-binding molecule. In some embodiments, the multispecific antigen-binding molecule is a bispecific antigen-binding molecule or a trispecific antigen-binding molecule.

[1135] In one aspect, the antigen-binding molecule of this disclosure is an antibody. In a specific embodiment, the antibody of this disclosure is an IgG antibody, preferably an IgG1, IgG2, IgG3, or IgG4 antibody.

[1136] IV. Disulfide bonds

[1137] In this disclosure, at least one disulfide bond in the region outside the hinge region can be described using the abbreviation "LINC". Using this abbreviation, in some embodiments, the antigen-binding molecule in this disclosure can be represented, for example, as "double / LINC", "DLL3-double / LINC", "paired cysteine ​​form", etc. An antigen-binding molecule whose first antigen-binding domain and second antigen-binding domain are not connected by at least one disulfide bond can be described using the abbreviation "unLINC" or "double-LINC-Ig with unpaired cysteine ​​(registered trademark)", etc.

[1138] In one aspect of the invention, each of the first antigen-binding domain and the...

Claims

1. A method for measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules (LINC forms) having at least one disulfide bond formed in a region outside the hinge region, said LINC ratio being the ratio of said LINC forms to the sum of said LINC forms and antigen-binding molecules (unLINC forms) not having a disulfide bond formed in a region outside the hinge region, said method comprising the steps of: (a) Adding the protease to composition (1) comprising the LINC form and the unLINC form to prepare composition (2); and (b) Perform electrophoresis or chromatography on composition (2).

2. The method of claim 1, wherein the protease is an enzyme that does not digest the LINC form but is capable of digesting the unLINC form.

3. The method according to claim 1 or 2, wherein the protease is a cysteine ​​protease capable of digesting human IgG1 antibodies.

4. The method according to any one of claims 1 to 3, wherein the protease is an enzyme capable of cleaving the hinge region of a human IgG1 antibody.

5. The method according to any one of claims 1 to 4, wherein the protease is an enzyme that digests human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

6. The method according to any one of claims 1 to 5, wherein the protease is IgdE.

7. The method of claim 6, wherein IgdE is derived from Streptococcus agalactiae.

8. The method according to claim 6 or 7, wherein IgdE is a protein selected from the group consisting of (a) to (d): (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 61; (b) A protein encoded by the base sequence shown in SEQ ID NO: 62; (c) A protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted relative to the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP; and (d) A protein comprising an amino acid sequence having at least 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between T and H in the amino acid sequence KSCDKT / HTCPPCP.

9. The method according to any one of claims 1 to 8, wherein the protease is FabALACTICA (registered trademark).

10. The method according to any one of claims 1 to 9, Step (a) further includes obtaining a composition (3) containing the protease but not the LINC form or the unLINC form. In step (b), each of compositions (1) to (3) is subjected to non-reducing capillary SDS gel electrophoresis (CE-SDS) and electrophoretic images (1) to (3) of compositions (1) to (3) are obtained respectively. Electrophoresis image (1) includes: Peaks containing the LINC form and the unLINC form that has not been digested by the protease, as well as peaks that are neither derived from the unLINC form that has not been digested by the protease nor from the LINC form (peak Z). The electrophoresis diagram (2) includes: a peak originating from the LINC form (peak L), a peak originating from the unLINC form digested by the protease (peak unL), a peak originating from the protease (peak E), and a peak that is neither originating from the unLINC form digested by the protease, nor from the LINC form, nor from the protease (peak Z). The electrophoresis diagram (3) includes a peak (peak E) derived from the protease.

11. The method of claim 10, further comprising: (c) Calculate the ratio (%) using the following formula (I): in, A represents the area of ​​peak L in electrophoresis diagram (2). B represents the sum of the area values ​​of peaks unL, E, and Z in electrophoresis diagram (2). C is the area value of peak Z in electrophoresis diagram (1), and D is the area value of peak E in electrophoresis diagram (3).

12. The method according to any one of claims 1 to 9, wherein step (b) involves performing hydrophobic interaction chromatography (HIC) on the composition (2) and obtaining a chromatogram.

13. The method of claim 112, further comprising: (c) Calculate the ratio (%) using the following formula (II): in, A represents the area value of the peak originating from the LINC form in the chromatogram; and B is the sum of the area values ​​of all peaks in the chromatogram originating from the unLINC form digested by the protease.

14. The method according to any one of claims 1 to 13, 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.

15. The method of claim 14, wherein the at least one disulfide bond is formed between the first antigen-binding domain and the second antigen-binding domain.

16. The method according to claim 14 or 15, wherein the at least one disulfide bond is formed between the heavy chain of the first antigen-binding domain and the heavy chain of the second antigen-binding domain.

17. The method according to any one of claims 14 to 16, wherein the at least one disulfide bond is formed between the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain.

18. The method according to any one of claims 14 to 17, wherein the disulfide bond is formed between an amino acid residue at position 191 according to EU number in the heavy chain of the first antigen-binding domain and an amino acid residue at position 191 according to EU number in the heavy chain of the second antigen-binding domain, between an amino acid residue at position 195 according to EU number in the heavy chain of the first antigen-binding domain and an amino acid residue at position 195 according to EU number in the heavy chain of the second antigen-binding domain, or between an amino acid residue at position 197 according to EU number in the heavy chain of the first antigen-binding domain and an amino acid residue at position 197 according to EU number in the heavy chain of the second antigen-binding domain.

19. The method according to any one of claims 14 to 18, 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 activity that promotes interaction between cells expressing the first antigen and cells expressing the second antigen.

20. The method of claim 19, wherein the cell expressing the first antigen is a cell with cytotoxic activity, and the cell expressing the second antigen is a target cell of the cell with cytotoxic activity, and wherein the antigen-binding molecule promotes damage to the target cell by the cell with cytotoxic activity.

21. The method of claim 20, wherein the cell having cytotoxic activity is a T cell, NK cell, monocyte, or macrophage.

22. The method according to any one of claims 19 to 21, 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, co-stimulatory molecules, and cell adhesion molecules.

23. The method according to any one of claims 14 to 22, wherein the first antigen-binding domain and the second antigen-binding domain are each capable of binding to CD3 and / or CD137.

24. The method according to any one of claims 1 to 23, wherein the antigen-binding molecule further comprises a third antigen-binding domain.

25. The method of claim 24, wherein the third antigen-binding domain is fused with either the first antigen-binding domain or the second antigen-binding domain.

26. The method according to claim 24 or 25, wherein the third antigen-binding domain is Fab or scFv.

27. The method according to any one of claims 24 to 26, wherein the third antigen-binding domain is fused at its C-terminus to the N-terminus of the Fab heavy chain (VH region) of either the first antigen-binding domain or the second antigen-binding domain, optionally via a peptide linker.

28. The method according to any one of claims 24 to 27, wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each Fab molecules, wherein the third antigen-binding domain is fused at the C-terminus of its Fab heavy chain (CH1 region) to the N-terminus of the Fab heavy chain (VH region) of either the first antigen-binding domain or the second antigen-binding domain, optionally via a peptide linker.

29. The method according to claim 27 or 28, 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.

30. The method according to any one of claims 24 to 29, 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.

31. The method according to any one of claims 24 to 30, wherein the third antigen-binding domain is capable of binding to an antigen expressed on cancer cells or cancerous tissue.

32. The method according to any one of claims 24 to 31, wherein the third antigen-binding domain is capable of binding to DLL3, preferably human DLL3.

33. The method according to any one of claims 1 to 32, wherein the antigen-binding molecule further comprises an Fc region.

34. The method according to any one of claims 14 to 33, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region that may be the same as or different from each other, and each comprises an antibody variable region independently selected from the group consisting of (a1) to (a4): (a1) Contains the following antibody variable regions: Heavy chain variable region, which contains: Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 27, Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 28, and Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 29; and The light chain variable region, which includes: Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30, The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 32; (a2) Contains the following antibody variable regions: Heavy chain variable region, which contains: Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 33, Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 34, and Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 35; and The light chain variable region, which includes: Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30, The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 32; (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

35. The method according to any one of claims 14 to 34, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region that may be the same as or different from each other, and each comprises an antibody variable region independently selected from the group consisting of (a1) to (a4): (a1) Contains the following antibody variable regions: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and The light chain variable region contains the amino acid sequence of SEQ ID NO: 37; (a2) Contains the following antibody variable regions: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 38, and The light chain variable region contains the amino acid sequence of SEQ ID NO: 37; (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

36. The method according to any one of claims 24 to 35, wherein the third antigen-binding domain comprises an antibody variable region independently selected from the group consisting of (a1) to (a4): (a1) Contains the following antibody variable regions: Heavy chain variable region, which contains: Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 46, Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 47, and Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 48; and The light chain variable region, which includes: Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 49, The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 50, and Light chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 51; (a2) Contains the following antibody variable regions: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 52, and The light chain variable region contains the amino acid sequence of SEQ ID NO: 53; (a3) An antibody variable region that binds to the same epitope as the antibody variable region bound to (a1) or (a2); and (a4) is an antibody variable region that competes with (a1) or (a2) for binding to the antigen.

37. The method according to any one of claims 14 to 36, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising: Heavy chain variable region, which contains: Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 27, Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 28, and Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 29; and The light chain variable region, which includes: Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 30, The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 31, and The light chain CDR 3 contains the amino acid sequence of SEQ ID NO:

32.

38. The method according to any one of claims 14 to 37, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and The light chain variable region contains the amino acid sequence of SEQ ID NO:

37.

39. The method according to any one of claims 24 to 38, wherein the third antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: Heavy chain variable region, which contains: Heavy chain complementarity-determining region (CDR) 1, which contains the amino acid sequence of SEQ ID NO: 46, Heavy chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 47, and Heavy chain CDR 3, which contains the amino acid sequence of SEQ ID NO: 48; and The light chain variable region, which includes: Light chain CDR 1, which contains the amino acid sequence of SEQ ID NO: 49, The light chain CDR 2, which contains the amino acid sequence of SEQ ID NO: 50, and The light chain CDR 3 contains the amino acid sequence of SEQ ID NO:

51.

40. The method according to any one of claims 24 to 39, wherein the third antigen-binding domain comprises an antibody variable region, the antibody variable region comprising: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 52, and The light chain variable region contains the amino acid sequence of SEQ ID NO:

53.

41. The method according to any one of claims 24 to 40, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, the antibody variable region comprising: The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 36, and The light chain variable region contains the amino acid sequence of SEQ ID NO:

37. Furthermore, the third antigen-binding domain comprises an antibody variable region, which includes: The heavy chain variable region, which includes SEQ ID NO: 52, and The light chain variable region contains SEQ ID NO:

53.

42. The method according to any one of claims 14 to 41, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab having a cysteine ​​residue at EU number positions 191, 195 or 197 in the heavy chain, and having a disulfide bond formed by two cysteine ​​residues.

43. The method according to any one of claims 1 to 42, wherein the antigen-binding molecule comprises five polypeptide chains selected from any combination of the group consisting of: (a1) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:

39. A polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO:

40. A polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 41, and Two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:

42. (a2) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:

43. A polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO:

40. A polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 44, and Two polypeptide chains (chain 4 and chain 5), each containing the amino acid sequence of SEQ ID NO: 42; and (a3) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:

45. A polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO:

40. A polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 44, and Two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:

42. in, Preferably, the five polypeptide chains (chains 1 to 5) are connected and / or associated with each other according to the orientation shown in Figure 3.

Images